microbial enzymes and their applications in industries and

BioMed Research International

Microbial Enzymes and Their Applications in Industries and Medicine 2016

Guest Editors Periasamy Anbu Subash C B Gopinath Bidur P Chaulagain and Thangavel Lakshmipriya

Microbial Enzymes and Their Applications inIndustries and Medicine 2016



Guest Editors Periasamy Anbu Subash C B GopinathBidur P Chaulagain andThangavel Lakshmipriya

Copyright copy 2017 Hindawi Publishing Corporation All rights reserved

This is a special issue published in ldquoBioMed Research Internationalrdquo All articles are open access articles distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the originalwork is properly cited

Contents

Microbial Enzymes andTheir Applications in Industries and Medicine 2016Periasamy Anbu Subash C B Gopinath Bidur Prasad Chaulagain and Thangavel LakshmipriyaVolume 2017 Article ID 2195808 3 pages

Synthesis of L-Ascorbyl Flurbiprofenate by Lipase-Catalyzed Esterification and TransesterificationReactionsJia-ying Xin Li-rui Sun Shu-ming Chen Yan Wang and Chun-gu XiaVolume 2017 Article ID 5751262 6 pages

Biotechnological Processes in Microbial Amylase ProductionSubash C B Gopinath Periasamy Anbu M K Md Arshad Thangavel Lakshmipriya Chun Hong VoonUda Hashim and Suresh V ChinniVolume 2017 Article ID 1272193 9 pages

Extracellular Ribonuclease from Bacillus licheniformis (Balifase) a New Member of the N1T1 RNaseSuperfamilyYulia Sokurenko Alsu Nadyrova Vera Ulyanova and Olga IlinskayaVolume 2016 Article ID 4239375 9 pages

Cloning Expression and Characterization of a NovelThermophilic Monofunctional Catalase fromGeobacillus sp CHB1Xianbo Jia Jichen Chen Chenqiang Lin and Xinjian LinVolume 2016 Article ID 7535604 8 pages

Laboratory Prototype of Bioreactor for Oxidation of Toxic D-Lactate Using Yeast Cells OverproducingD-Lactate Cytochrome cOxidoreductaseMaria Karkovska Oleh Smutok and Mykhailo GoncharVolume 2016 Article ID 4652876 5 pages

Improved Production of Aspergillus usamii endo-120573-14-Xylanase in Pichia pastoris via CombinedStrategiesJianrong Wang Yangyuan Li and Danni LiuVolume 2016 Article ID 3265895 9 pages

EditorialMicrobial Enzymes and Their Applications in Industries andMedicine 2016

Periasamy Anbu1 Subash C B Gopinath23

Bidur Prasad Chaulagain4 and Thangavel Lakshmipriya12

1Department of Biological Engineering College of Engineering Inha University Incheon 402-751 Republic of Korea2Institute of Nano Electronic Engineering (INEE) Universiti Malaysia Perlis 01000 Kangar Perlis Malaysia3School of Bioprocess Engineering Universiti Malaysia Perlis 02600 Arau Perlis Malaysia4Directorate of Research andTrainingHimalayanCollege of Agricultural Sciences andTechnology (HICAST) PO Box 25535 KalankiKathmandu Nepal

Correspondence should be addressed to PeriasamyAnbu anbu25yahoocomand SubashC BGopinath subashunimapedumy

Received 14 December 2016 Accepted 4 January 2017 Published 28 March 2017

Copyright copy 2017 Periasamy Anbu et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Enzymes are biocatalysts that play an important role inmetabolic and biochemical reactions [1] Microorganisms arethe primary source of enzymes because they are cultured inlarge quantities in short span of time and genetic manipula-tions can be done on bacterial cells to enhance the enzymeproduction [2ndash4] In addition the microbial enzymes havebeen paidmore attention due to their active and stable naturethan enzymes from plant and animal [2ndash4] Most of themicroorganisms are unable to grow and produce enzymeunder harsh environments that cause toxicity to microor-ganisms However some microorganisms have undergonevarious adaptations enabling them to grow and produceenzymes under harsh conditions [5 6] Recently several linesof study have been initiated to isolate new bacterial andfungal strains from harsh environments such as extreme pHtemperature salinity heavy metal and organic solvent forthe production of different enzymes having the propertiesto yield higher [6ndash9] This special issue covers six articlesincluding one review article highlighting the importance andapplications of biotechnologically and industrially valuablemicrobial enzymes

There are redundancies in genetic code that amino acidmight be encoded by multiple synonymous codons Thisscenario gives an opportunity to choose a codon other thanthe naturally occurring one in the genome to optimizethe production with heterologous expression system Codonoptimization in another sense is a guided mutagenesis in

the gene expression system which can be utilized for thebenefits of human kind ranging from industrial agricul-ture to medicine J Wang et al have applied a series ofstrategies to improve the expression level of recombinantendo-120573-14-xylanase from Aspergillus usamii in Pichia pas-toris The endo-120573-14-xylanase gene (xynB) from A usamiiwas optimized for expression in P pastoris Their analysisshowed the codon for amino acid residues in P pastoris isdifferent from the original codon of A usamii Thus theyreplaced the codons in endo-120573-14-xylanase gene to fit to thecellular environment of P pastoris Similarly they optimizedthe codons of Vitreoscilla hemoglobin gene (vhb) to fit toheterologous expression system in P pastoris cell systemWhile optimizing codons they replaced the AT-rich stretcheswith GC-rich stretches because G+C content affects thesecondary structure of mRNA and influences the expressionlevel of heterologous gene Totally 105 and 57 amino acidswere optimized in native xynB and vhb respectively Besidesoptimizing the genetic system J Wang et al have also opti-mized the environment to express those recombinant genesThe codon optimized vhb gene has significantly improvedthe oxygen availability for host P pastoris since oxygensupply is one of most critical factors for the cell growth andheterologous protein expression in recombinant P pastorisBy optimizing the temperature effect on the system theyincreased the xylanase production combined with highercell viability Overall this work has supported the notion of

HindawiBioMed Research InternationalVolume 2017 Article ID 2195808 3 pageshttpdxdoiorg10115520172195808

2 BioMed Research International

genetic engineering in code optimization and a piece of novelwork for the recombinant biotechnology

X Jia and his colleagues have produced the recombinantcatalase in Escherichia coli from Geobacillus sp gene (Kat)This Kat gene has 1467 bases and encoded a catalase with488 amino acid residuals with 81 similarity to the previouslystudied Bacillus sp catalase Fermentation broth of therecombinant E coli showed a high catalase activity levelup to 35831UmL The purified recombinant catalase had aspecific activity of 40526Umg and a Km of 511mM Theoptimal reaction temperature of this recombinant enzymeis 60 to 70∘C and it exhibits a high activity over a widerange of reaction temperatures The enzyme retained 947of its residual activity after incubation at 60∘C for 1 hourThe high yield and excellent thermophilic properties of thisrecombinant catalase have valuable features for industrialapplications

In the work performed by M Karkovska et al alaboratory column-type bioreactor for removing a toxicD-lactate on permeabilized thermotolerant methylotrophicyeast (Hansenula polymorpha ldquotr6rdquo) cells and alginate gelwas constructed and tested Using recombinant H poly-morpha ldquotr6rdquo overproduced the D-lactate cytochrome c-oxidoreductase At about 6-fold overexpression of D-lactatecytochrome c-oxidoreductase under a strong constitutivepromoter (prAOX) was demonstrated Overexpression of D-lactate dehydrogenase coupled with the deletion of L-lactate-cytochrome c oxidoreductase activity opens the possibility forusage of the strain as a base for construction of bioreactorfor removing D-lactate from fermented products due to theoxidation to nontoxic pyruvate

RNases are regarded as alternative to classical chemother-apeutic agents due to their selective cytotoxicity towardstumor cells In the work demonstrated by Y Sokurenkoet al extracellular ribonuclease from Bacillus licheniformis(balifase) a new member of the N1T1 RNase superfamily isshown to have antitumor effects The new RNase producedis with a high degree of structural similarity with binase(73) and barnase (74) having a molecular weight of12422 Daltons and pI 89 The physicochemical properties ofbalifase are similar to those of barnaseThe gene organizationand promoter activity of balifase are closer to binase Inthis study similar to the biosynthesis of binase balifasesynthesis was induced under phosphate starvation howeverin contrast to binase balifase does not form dimers undernatural conditions This study also proposed that the higheststability of balifase allows retaining its structure withoutoligomerization

In their review S Gopinath et al focused on impor-tance of microbial amylase in the field of biotechnologyIn this article the authors have discussed isolation andscreening of amylase from bacterial strains They also dis-cussed the improvement of enzyme production by opti-mization and recombinant DNA technology The majoradvantages of microbial enzymes in many industries suchas food detergent pharmaceutical paper and textile indus-tries are discussed The technologies of high-throughputscreening and processing with efficient microbial speciesalong with the ultimate coupling of genetic engineering of

amylase-producing strains will all help in enhancing amylaseproduction for industrial and medicinal applications

Efficient delivery of drug to the target cell is very impor-tant for treatment Drugs with brain tissue related treatmentsare hindered by the blood brain barrier Flurbiprofen is one ofthe potent drugs that may help to prevent Alzheimerrsquos diseasebut it has problem with blood-brain barrier permeability Toovercome this problem J Xin et al have worked to designand modify this drug as L-ascorbyl flurbiprofenate with theaddition of ascorbic acid as a specific carrier system for braindelivery In the process they have tried to optimize lipase-catalyzed esterification and transesterification They foundthat synthesis of L-ascorbyl flurbiprofenate was influenced bythe specific reaction conditions and the most important stepwas the efficient removal of byproducts during the reactionWhile comparing those esterification and transesterificationmethods although the rate of esterification was found slowerthan that of transesterification from the standpoint of pro-ductivity and the amount of steps required lipase-catalyzedesterification was found superior for the synthesis of L-ascorbyl flurbiprofenate

Periasamy AnbuSubash C B Gopinath

Bidur Prasad ChaulagainThangavel Lakshmipriya

References

[1] P Nigam ldquoMicrobial enzymes with special characteristics forbiotechnological applicationsrdquo Biomolecules vol 3 no 3 pp597ndash611 2013

[2] P Anbu S C B Gopinath A C Cihan and B P ChaulagainldquoMicrobial enzymes and their applications in industries andmedicinerdquo BioMed Research International vol 2013 Article ID204014 2 pages 2013

[3] P Anbu S C B Gopinath B P Chaulagain T-H Tangand M Citartan ldquoMicrobial enzymes and their applications inindustries and medicine 2014rdquo BioMed Research Internationalvol 2015 Article ID 816419 3 pages 2015

[4] S C B Gopinath P Anbu T Lakshmipriya and A HildaldquoStrategies to characterize fungal lipases for applications inmedicine and dairy industryrdquo BioMed Research Internationalvol 2013 Article ID 154549 10 pages 2013

[5] Y N Sardessai and S Bhosle ldquoIndustrial potential of organicsolvent tolerant bacteriardquo Biotechnology Progress vol 20 no 3pp 655ndash660 2004

[6] P Anbu ldquoEnhanced production and organic solvent stabilityof a protease from Brevibacillus laterosporus strain PAP04rdquoBrazilian Journal of Medical and Biological Research vol 49 no4 Article ID e5178 2016

[7] P Anbu S C B Gopinath A Hilda T Lakshmipriya andG Annadurai ldquoOptimization of extracellular keratinase pro-duction by poultry farm isolate Scopulariopsis brevicaulisrdquoBioresource Technology vol 98 no 6 pp 1298ndash1303 2007

[8] S C B Gopinath A Hilda T Lakshmi Priya G Annaduraiand P Anbu ldquoPurification of lipase from Geotrichum can-didum conditions optimized for enzymeproduction usingBox-Behnken designrdquoWorld Journal ofMicrobiology and Biotechnol-ogy vol 19 no 7 pp 681ndash689 2003

BioMed Research International 3

[9] S C B Gopinath P Anbu and A Hilda ldquoExtracellular enzy-matic activity profiles in fungi isolated from oil-rich environ-mentsrdquoMycoscience vol 46 no 2 pp 119ndash126 2005

Research ArticleSynthesis of L-Ascorbyl Flurbiprofenate by Lipase-CatalyzedEsterification and Transesterification Reactions

Jia-ying Xin12 Li-rui Sun1 Shu-ming Chen3 YanWang1 and Chun-gu Xia2

1Key Laboratory for Food Science amp Engineering Harbin University of Commerce Harbin 150076 China2State Key Laboratory for Oxo Synthesis amp Selective Oxidation Lanzhou Institute of Chemical Physics Chinese Academy of SciencesLanzhou 730000 China3College of Animal Science and Veterinary Medicine Shanxi Agricultural University Taigu 030801 China

Correspondence should be addressed to Jia-ying Xin xinjiayingvip163com

Received 17 May 2016 Accepted 31 July 2016 Published 21 March 2017

Academic Editor Bidur P Chaulagain

Copyright copy 2017 Jia-ying Xin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The synthesis of L-ascorbyl flurbiprofenate was achieved by esterification and transesterification in nonaqueous organic mediumwith Novozym 435 lipase as biocatalyst The conversion was greatly influenced by the kinds of organic solvents speed of agitationcatalyst loading amount reaction time and molar ratio of acyl donor to L-ascorbic acid A series of solvents were investigatedand tert-butanol was found to be the most suitable from the standpoint of the substrate solubility and the conversion for both theesterification and transesterification When flurbiprofen was used as acyl donor 610 of L-ascorbic acid was converted against464 in the presence of flurbiprofen methyl ester The optimal conversion of L-ascorbic acid was obtained when the initial molarratio of acyl donor to ascorbic acid was 5 1 kinetics parameters were solved by Lineweaver-Burk equation under nonsubstrateinhibition condition Since transesterification has lower conversion from the standpoint of productivity and the amount of stepsrequired esterification is a better method compared to transesterification

1 Introduction

The treatment of Alzheimerrsquos disease is still a major chal-lenge for the medical field The clinical failure of efficientAlzheimerrsquos disease drug deliverymay be largely attributed tothe low permeability of drugs due to the blood-brain barrierand a lack of appropriate drug delivery systems [1] Flur-biprofen is one of the most potent nonsteroidal anti-inflam-matory drugs (NSAIDs) andmay help to prevent Alzheimerrsquosdisease [2 3] However poor brain delivery of flurbiprofenand its serious gastrointestinal side effects have hampered theapplication of flurbiprofen as neuroprotective agents [4]

Localized and controlled delivery of drugs at their desiredsite of action can reduce toxicity and increase treatment effi-ciency L-Ascorbic acid is essential for many enzymatic reac-tions which is transported directly across the blood-brainbarrier via Nathorn-dependent vitaminC transporter SVCT2 andparticularly prevalent in the brain [1] It has been reportedthat L-ascorbic acid could be used as a carrier to promotebrain drug delivery [5ndash7] To overcome the problems of

the low blood-brain barrier permeability of flurbiprofen andto increase its delivery to the brain for the treatment ofAlzheimerrsquos disease one attractive approach is to design andsynthesize flurbiprofen ester prodrug named L-ascorbyl flur-biprofenate containing ascorbate as a specific carrier systemfor brain delivery

So far there are very few reports on the lipase-catalyzedsynthesis of L-ascorbyl flurbiprofenate Wang and Tang stud-ied the synthesis of L-ascorbyl flurbiprofenate by lipase-catalyzed esterification of L-ascorbic acid with flurbipro-fen in tertiary amyl alcohol but no optimum operatingparameters were provided [8] Liu and Tang investigated thekinetics and thermodynamics of the lipase-catalyzed esteri-fication of L-ascorbic acid with flurbiprofen in 2-methy-l2-butanol [9] Only limited information on the lipase-catalyzedesterification was available There were no reports on thelipase-catalyzed synthesis of L-ascorbyl flurbiprofenate bytransesterification However solvent properties quantity ofenzyme and molar ratio of L-ascorbic acid to flurbiprofenmay influence the biocatalytic reaction So far the influence



of these factors for the synthesis of L-ascorbyl flurbiprofenatehas not been investigated in detail To obtain high conversionit is important to determine the optimal reaction conditionsand understand the kinetics parameters

The present study focused on lipase-catalyzed synthesis ofL-ascorbyl flurbiprofenateThe lipase-catalyzed esterificationand transesterification approaches have been compared Itwas worthwhile to compare the merits and demerits of thetwo processes and optimize process conditions

2 Experimental

21 Materials Novozym 435 lipase (Lipase B from Can-dida antarctica immobilized on macroporous acrylic resinspecific activity 10000Ug) was purchased from Novoz-ymes Denmark Porcine pancreas lipase Type II (powder30ndash90Umg) and Candida rugosa lipase (Type VII powder706Umg) were purchased from Sigma(119877 119878)-flurbiprofen (purity gt 99) was purchased from

Shanghai Mei Lan Chemical Co Ltd (Shanghai China)The purity of substrates is over 997 for L-ascorbic acidAll solvents were dehydrated before use with activated 3 Amolecular sieves Thin-layer chromatography (TLC) plateswere purchased fromMerck (KGaA Darmstadt Germany)

22 Synthesis of Ester Themethyl ester of (119877 119878)-flurbiprofenwas prepared by the classical methodology using thionylchloride and methanol Thionyl chloride 15mL (020mol)was added dropwise to cooled stirred suspension of (119877 119878)-flurbiprofen (012mol) in methanol (250mL) The reactionmixture was refluxed for 25 h and then the solvent was evap-orated and the residue purified by column chromatographyusing SiO2 as adsorbent and petroleum ether purified aseluant and its purity was gt 98 using the HPLC methodsdescribed in a later section

23 Reaction Conditions Unless otherwise stated the ester-ification and transesterification reactions were performedin 50mL closed screw-capped glass vials containing 25mLof organic solvent flurbiprofen or flurbiprofen methyl ester(26ndash260mmolL) L-ascorbic acid (26mmolL) and 800mgof molecular sieve 3 A The reaction had been started byadding 40mg of lipase The headspace in the vials wasfilled with nitrogen gas and refilled after each samplingThe reaction mixture was stirred with a magnetic stirrer atdifferent temperature In both cases samples were withdrawnat specified time intervals formeasurement of the conversionControl experiments without enzymes were carried out inparallel

24 Analytical Methods In order to monitor the reac-tion progress samples of 100 120583L were withdrawn at inter-vals and analyzed by thin-layer chromatography (TLC) onsilica gel 60 F

254plates with fluorescent indicators The

TLC migration was carried out with a solvent mixture ofchloroformmethanolglacial acetic acidwater (80 10 8 2VVVV)The TLC plates were visualized under UV Resultswere estimated from intensity of spots on TLC

Quantitative analysis was done by HPLC (ThermoU3000) on a XDB C18 reversed phase column (5120583m 46 times150mm) with acetonitrilewaterformic acid (802002VVV) as mobile phase at 1mLmin flow rate Detection wasachieved usingUVdetection at 294 nm Samples were filteredto remove the enzyme and molecular sieves and 100 120583Lof the solution was diluted with acetonitrilewaterformicacid (802002 VVV) L-Ascorbic acid was a limitingsubstrate throughout this study therefore the conversionwascalculated as the ratio in moles of the product to initial L-ascorbic acid All experiments were performed in triplicateand standard deviations were calculated

The enantiomeric excess values of flurbiprofen and flur-biprofenmethyl ester (ee)were determined byHPLC (Agilent1200) by using a chiral column (Chiralcel OD-H 5 120583m250mm times 46mm) capable of separating the 119877- and 119878-isomers of flurbiprofen and flurbiprofenmethyl ester and themobile phase was hexaneisopropanoltrifluoroacetic acidsolution (98 2 01 VVV) at a flow rate of 10mLmin UVdetection at 294 nm was used for quantification at the 25∘CThe enantiomeric excess (ee) was obtained from peak areas ofthe 119877- and 119878-isomers of flurbiprofen or flurbiprofen methylester by the following equation ee = (119860

119878minus 119860119877)(119860119878+ 119860119877)

where 119860119878is peak areas of the 119878-isomer and 119860

119877is peak areas

of the 119877-isomer

25 Kinetic Study Reactions were carried out no more than5 conversion and the initial rate was determined as the slopeof the reaction curve tangent to the initial stage of the reactionand expressed as mol of products hminus1 Lminus1 Because all exper-iments were performed in triplicate reaction curves wereconstructed using average values of the reaction rate for eachexperimental point A linear portion of the reaction curve atvarious substrate concentrations consisted of 5 experimentalpoints where the number of experimental points includedwas determined by the condition that correlation coefficientsof the initial straight line must be above 095

3 Results and Discussion

31 Esterification and Transesterification It was previouslyreported that esterification of flurbiprofen with L-ascorbicacid by lipase in tert-amyl alcohol produced L-ascorbyl flur-biprofenate regioselectively whichwas only the product iden-tified in the HPLC analysis [9] In this paper lipase-catalyzedacylation of L-ascorbic acid was performed in presence ofracemic flurbiprofen (esterification) or racemic flurbiprofenmethyl ester (transesterification) Both kinds of reaction ledto only one product identified as L-ascorbyl flurbiprofenatein the HPLC analysis Furthermore the chiral HPLC analysisindicated that the lipases also display stereospecificity in theesterification and transesterification The 119877-isomer reactedwith the Novozym 435 lipase the 119878-isomer reacted with theCandida rugosa lipase and porcine pancreas lipase cannotcatalyze the reaction It has been reported that 119878-flurbiprofenand 119877-flurbiprofen have the same physiological activity inprevention of the development of Alzheimers disease but 119877-flurbiprofen has reduced side effects related to inhibition of


Table 1 The effect of solvent on the conversion of esterification and transesterification

Solvent log119875 Conversion of L-ascorbic acid ()Esterification Transesterification

Acetone minus023 46 300Tert-butanol(2-methyl-2-propanol) 060 360 385Ethyl acetate 068 111 98Tert-amyl alcohol 2-methyl-butanol 089 353 365Benzene 200 mdash mdashChloroform 200 35 mdashToluene 250 mdash mdash

N435

F

COOHCOOH

COO OO O

O

OHOH

HOHO

HO HOHO

F

F

H3C

H2O

CH3

(S)-FlurbiprofenL-Ascorbic acid (R)-L-Ascorbyl flurbiprofenate

+

+ +

H3C

-Flurbiprofen(S R)

(a)

F

FO

F

O

(S)-Flurbiprofenmethyl estermethyl ester

O O

OHOH

HOHO

HOHO

HO

COO

CH3

H3C H3C COOCH3COOCH3

CH3OH

L-Ascorbic acid (R)-L-Ascorbyl flurbiprofenate

++ +

N435

-Flurbiprofen(S R)

(b)

Figure 1 Novozym 435 lipase-catalyzed esterification (a) and transesterification (b)

cyclooxygenase (COX) [10ndash12] Thus Novozym 435 lipaseidentified as a 119877-stereospecific catalyst has been employedin further experimentsThe two reactions for the preparationof L-ascorbyl flurbiprofenate are shown in Figure 1

32 Effect of Solvent Different organic solvents had differentability to distort the essential water layer around lipaseand could greatly influence the activity of lipase Moreoverorganic solvent relatively influenced the solubility of thesubstrates and thus it would affect the synthesis of the L-ascorbyl flurbiprofenate The polarity of L-ascorbic acid isvery different from those of flurbiprofen and flurbiprofenmethyl ester Because of this significant difference in polaritysuch a solvent with relatively high solubility of flurbiprofenflurbiprofen methyl ester and L-ascorbic acid needs to befound

Therefore esterification and transesterification strate-gies for the lipase-catalyzed synthesis of L-ascorbyl flur-biprofenate were compared using different organic solventsThe effect of various organic solvents on conversion of L-ascorbic acid was studied under similar conditions using50mg Novozym 435 lipase 800mg of molecular sieve 3 A23mmoles of flurbiprofen or flurbiprofen methyl ester057mmoles of ascorbic acid and 25mL organic solvent at160 rpm at 50∘C for 72 h It was clear from Table 1 that thetype of organic solvent strongly influenced the synthesis ofL-ascorbyl flurbiprofenate In the case of esterification andtransesterification tert-butanol was the best solventwith tert-amyl alcohol as the next best However the reaction productwas not inspected in benzene and toluene

log119875 is widely used to represent the characteristics of theorganic solvent system where 119875 is the partition coefficient of


EsterificationTransesterification

3032343638404244464850

Con

vers

ion

()

170 190 210 230 250 270150Shaken speed (rpm)

Figure 2 The effect of speed of agitation on the conversion ofesterification and transesterification

the solvent between water and octanol [13] It is generallyreported that solvents with log119875 lt 2 are high polaritythat may strip water from enzyme molecules easily and areless suitable for biocatalytic purpose [14] In this case nocorrelation of the lipase activity with log119875 of the solventscould be established This could be attributed that L-ascorbicacid had very low concentration in solvents with high log119875Since tert-butanol was the optimum solvent for esterificationand transesterification it was used as the solvent for transes-terification and esterification in further experiments

33 Effect of Speed of Agitation In the case of immobilizedenzyme external mass transfer limitationsmay be importantThe reactants have to diffuse from the bulk liquid to theexternal surface of the catalyst External mass transfer canbe minimized by carrying out the reaction at an optimumspeed of agitation The effect of speed of agitation wasstudied both for esterification and transesterification over therange of 160ndash260 rpm by taking 50mg Novozym 435 lipase800mg of molecular sieve 3 A 23mmoles of flurbiprofenor flurbiprofen methyl ester 057mmoles of ascorbic and25mL tert-butanol at 50∘C for 72 h The conversions in boththe cases were independent of the speed of agitation atand beyond 180 rpm for esterification and transesterification(Figure 2) This indicated that the influence of external masstransfer limitation was negligible and a speed of agitationof 180 rpm did not limit reaction rate So all subsequentexperiments were carried out at 180 rpm

34 Effect of Catalyst Loading Amount Internal diffusionproblems could happen when the substrate could not reachthe inner parts of the support L-Ascorbyl flurbiprofenatesynthesis as a function of catalyst loading amountwas studiedat 50∘C and 180 rpm for 72 h The conversion of the reactionincreasedwith increasing immobilized lipase loading rangingfrom 15mg to 55mg in both the cases a linear relationshipbetween the conversion and enzyme load demonstratedthat the internal diffusion limitations could be minimizedMaximal conversion was achieved with 55mg lipase foresterification and transesterification after conversion becameconstant and no further increases (Figure 3)


100150200250300350400450500550600

Con

vers

ion

()

10 20 30 40 50 60 70 800Enzyme (mg)

Figure 3The effect of catalyst loading amount on the conversion ofesterification and transesterification


200

250

300

350

400

450

500

Con

vers

ion

()

2 3 4 5 6 71Molar ratio of acy1 donor to ascorbic acid

Figure 4The effect of the molar ratio of acyl donor to ascorbic acidon the conversion of esterification and transesterification

Also in the case of esterification reaction the conversionwas lower than the transesterification reaction under 15mgand 25mg lipase addition and the asymptotes were seen inthe conversion for transesterification and esterification whenthe amount of enzyme added was beyond 35mg Since therewas no significant increase in the conversion with increasedcatalyst loading from 55mg to 75mg further parameterswere studied using 55mg catalyst loading

35 Effect of the Molar Ratio of Acyl Donor to AscorbicAcid The molar ratio of one substrate to another is animportant parameter affecting the conversion The solubilityof L-ascorbic acid in tert-butanol at 50∘C was 26mmolLwhich is saturated in tert-butanol that would be limitingas substrate Therefore the effect of the molar ratio of thereactants was studied by keeping the concentration of L-ascorbic acid and the catalyst quantity constant and varyingthe concentration of acyl donor in both esterification andtransesterification Figure 4 showed the effect of the molarratio on the conversion for the synthesis of L-ascorbylflurbiprofenate Molar ratio of acyl donor to ascorbic acidwas varied from 1 1 to 7 1 and the L-ascorbic acid waskept constant The conversion at a reaction time of 72 h for


TransesterificationEsterification

0

10

20

30

40

50

60

70

Con

vers

ion

()

2 4 6 8 100Reaction time (days)

Figure 5 Time course of the synthesis of L-ascorbyl flurbiprofenate

esterification and transesterification was compared Indeedwithin experimental error at a fixed concentration of L-ascorbic acid and loading amount of Novozym 435 lipasethe conversion increased with increasing acyl donor con-centrations up to a critical value For esterification andtransesterification when using higher ratios of flurbiprofenor flurbiprofen methyl ester over L-ascorbic acid conversionincreased andhighest conversion (610 for esterification and464 for transesterification) occurred at 5 1 molar ratio offlurbiprofen or flurbiprofen methyl ester to L-ascorbic acidAt molar ratios greater than 5 1 a constant conversion wasobserved

36 Effect of Reaction Time Figure 5 showed the changesin the conversion with time for the direct esterification andtransesterification in tert-butanol at 50∘C with molar ratioof acyl donor to ascorbic acid of 5 1 Direct esterificationand transesterification reaction had a different initial rateand maximal conversion In the case of transesterificationreaction the ratewas faster than that of the esterification reac-tion under otherwise similar conditions within the reactiontime of 24 h This could be attributed that lipase had higheractivity for flurbiprofen methyl ester and lower activity withflurbiprofen Also the difference consisted in the maximalconversion of L-ascorbic acid which was 610 occurringafter 144 h of incubation for esterification against 464occurring after 72 h of incubation for transesterificationThisdifference might be due to methanol production duringthe transesterification reaction which was not eliminatedby the 3 A molecular sieve and disadvantageous reactionequilibrium for L-ascorbyl flurbiprofenate production Evenif water was also produced as a coproduct during theesterification reaction the 3 A molecular sieve favors itselimination by adsorption and then contributes to shift thereaction equilibrium towards the synthesis of L-ascorbylflurbiprofenate

37 Kinetic Study The effects of several parameters includingorganic solvent speed of agitation enzyme amount substratemolar ratio and reaction time on conversion of L-ascorbic

Esterification

Transesterification

0 5 10 15 20 25 3002468

1012141618

Lmiddothmiddotm

molminus

1)

1V

(

Lmiddotmolminus1

)1C (

Figure 6 The Lineweaver-Burk plot of 11198810versus 1[119878]

acid were investigated to optimize the conditions Since L-ascorbic acid was dissolved in tert-butanol with a saturatedconcentration of 26mmolL the effect of concentration offlurbiprofen or flurbiprofen methyl ester on the rate of reac-tion was investigated systematically over a wide range witha constant L-ascorbic acid concentration For the determina-tion of initial rates esterification and transesterification wereconducted by using 55mg Novozym 435 lipase with appro-priate quantities of acyl donor (flurbiprofen or flurbiprofenmethyl ester) and L-ascorbic acid In esterification and trans-esterification experiments the amount of flurbiprofen wasvaried from 35 to 568mmolL at a fixed quantity of L-ascorbicacid (26mmolL) The initial rates were determined for eachexperiment From the initial rate determined it showedthat when the concentration of acyl donor (flurbiprofen orflurbiprofen methyl ester) was increased by keeping theconcentration of L-ascorbic acid constant the initial rate ofreaction increased proportionally This presumed that therewas no evidence of inhibition by acyl donor (flurbiprofenor flurbiprofen methyl ester) at all the concentrations testedAccording to the rate equation for the ping-pong bi-bimechanismwithout substrate inhibition [15] the Lineweaver-Burk plot of 1119881

0versus 1[119878] in Figure 6 showed the kinetic

parameter values The apparent Michaelis-Menten kineticsare 119870rn = 029molL and apparent 119881max = 0563mmolLsdothfor flurbiprofen Apparent Michaelis-Menten kinetics are119870rn = 014molL and apparent 119881max = 134mmolLsdoth forflurbiprofen methyl ester This indicated that Novozym 435lipase has higher affinity for flurbiprofen methyl ester andlower affinitywith flurbiprofen Transesterification has higherreaction rate than esterification

4 Conclusion

The L-ascorbyl flurbiprofenate has been synthesized success-fully by lipase-catalyzed transesterification and esterificationin tert-butanol The goal of this work was to compare lipase-catalyzed esterification and transesterification approachesBoth approaches were studied in a systematic way includingthe effect of various parameters We have tried to opti-mize lipase-catalyzed esterification and transesterification toensure meaningful and objective comparison among dif-ferent approaches Synthesis of L-ascorbyl flurbiprofenatewas influenced by reaction conditions The equilibrium shift


towards the L-ascorbyl flurbiprofenate synthesis was limitedin spite of the presence of an excess of acyl donor Themost important strategy seemed to be the efficient removalof by-products such as water or methanol Addition ofmolecular sieves 3 A during reaction could control wateractivity of the system However addition of molecular sieves3 A during reaction could not control methanol contentIt was observed that the rate of the transesterification wasmuch higher than that of esterification within 24 h If theexperiments were conducted for a long time the conversionwould reach asymptotic values and then the conversion ofesterification was higher than that of the transesterificationThis reflected a greater reactivity of flurbiprofen methyl esterin the lipase-catalyzed transesterification reaction as com-pared with esterification However the inherent drawbackassociated with lipase-catalyzed transesterification was theproduction of methanol and the equilibrium was usually notin favor of transesterification Also in the transesterificationflurbiprofen had to be chemically converted to its methylester and then subjected to the transesterification catalyzed bylipase Although the rate of esterification is slower than thatof transesterification if a choice is at all available from thestandpoint of productivity and the amount of steps requiredlipase-catalyzed esterification has been judged to be superiorfor the synthesis of L-ascorbyl flurbiprofenate

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors thank the National Natural Science Foundationof China (21573055) the Scientific Research Fund of Hei-longjiang Province (GC13C111) the Open Project Program ofthe State Key Laboratory forOxo Synthesis and SelectiveOxi-dation and the State Key Laboratory of Chemical ResourceEngineering for support

References

[1] Y Zhao B Y Qu X Y Wu et al ldquoDesign synthesis and bio-logical evaluation of brain targeting L-ascorbic acid prodrugsof ibuprofen with lsquolock-inrsquo functionrdquo European Journal ofMedicinal Chemistry vol 82 pp 314ndash323 2014

[2] B P Imbimbo ldquoThe potential role of non-steroidal anti-inflam-matory drugs in treating Alzheimerrsquos diseaserdquo Expert Opinionon Investigational Drugs vol 13 no 11 pp 1469ndash1481 2004

[3] L Gasparini E Ongini D Wilcock and D Morgan ldquoActivityof flurbiprofen and chemically related anti-inflammatory drugsin models of Alzheimerrsquos diseaserdquo Brain Research Reviews vol48 no 2 pp 400ndash408 2005

[4] S Cote P-H Carmichael R Verreault J Lindsay J Lefebvreand D Laurin ldquoNonsteroidal anti-inflammatory drug use andthe risk of cognitive impairment and Alzheimerrsquos diseaserdquoAlzheimerrsquos amp Dementia vol 8 no 3 pp 219ndash226 2012

[5] X-Y Wu X-C Li J Mi J You and L Hai ldquoDesign synthesisand preliminary biological evaluation of brain targeting l-ascorbic acid prodrugs of ibuprofenrdquo Chinese Chemical Lettersvol 24 no 2 pp 117ndash119 2013

[6] S Manfredini B Pavan S Vertuani et al ldquoDesign synthesisand activity of ascorbic acid prodrugs of nipecotic kynurenicand diclophenamic acids liable to increase neurotropic activ-ityrdquo Journal of Medicinal Chemistry vol 45 no 3 pp 559ndash5622002

[7] Y Laras M Sheha N Pietrancosta and J-L Kraus ldquoThiazola-mide-ascorbic acid conjugate a 120574-secretase inhibitor with en-hanced blood-brain barrier permeationrdquo Australian Journal ofChemistry vol 60 no 2 pp 128ndash132 2007

[8] ZWang andLHTang ldquoDesign synthesis and characterizationof ascorbic acid pro-drugs of flurbiprofenrdquo Strait Pharmaceuti-cal Journal vol 22 no 110 pp 213ndash217 2010

[9] X N Liu and L H Tang ldquoKinetics and thermodynamics of L-ascorbyl profen esters synthesis catalyzed by lipase in 2-methyl-2-butanolrdquo Chinese Journal of Bioprocess Engineering vol 8 no6 pp 33ndash39 2010

[10] J L Eriksen S A Sagi T E Smith et al ldquoNSAIDs andenantiomers of flurbiprofen target 120574-secretase and lower A12057342in vivordquo Journal of Clinical Investigation vol 112 no 3 pp 440ndash449 2003

[11] H Geerts ldquoDrug evaluation (R)-flurbiprofenndashan enantiomerof flurbiprofen for the treatment of Alzheimerrsquos diseaserdquo TheInvestigational Drugs Journal vol 10 no 2 pp 121ndash133 2007

[12] C Hansch A Leo and D Hoekman Exploring QSAR Hydro-phobic Electronic and Steric Constants American ChemicalSociety 1995

[13] C Hansch A Leo and D Hoekman Exploring QSARmdashHydrophobic Electronic and Steric Constants American Chem-ical Society Washington DC USA 1995

[14] Q-X Song and D-Z Wei ldquoStudy of Vitamin C ester synthesisby immobilized lipase from Candida sprdquo Journal of MolecularCatalysis B Enzymatic vol 18 no 4ndash6 pp 261ndash266 2002

[15] G D Yadav and P S Lathi ldquoIntensification of enzymaticsynthesis of propylene glycolmonolaurate from 12-propanedioland lauric acid undermicrowave irradiation kinetics of forwardand reverse reactionsrdquo Enzyme and Microbial Technology vol38 no 6 pp 814ndash820 2006

Review ArticleBiotechnological Processes in Microbial Amylase Production

Subash C B Gopinath12 Periasamy Anbu3 M K Md Arshad1 Thangavel Lakshmipriya1

Chun Hong Voon1 Uda Hashim1 and Suresh V Chinni4

1 Institute of Nano Electronic Engineering Universiti Malaysia Perlis 01000 Kangar Perlis Malaysia2School of Bioprocess Engineering Universiti Malaysia Perlis 02600 Arau Perlis Malaysia3Department of Biological Engineering College of Engineering Inha University Incheon 402-751 Republic of Korea4Department of Biotechnology Faculty of Applied Sciences AIMST University 08100 Bedong Malaysia

Correspondence should be addressed to Subash C B Gopinath subashunimapedumy

Received 29 October 2016 Accepted 27 November 2016 Published 9 February 2017

Academic Editor Nikolai V Ravin

Copyright copy 2017 Subash C B Gopinath et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Amylase is an important and indispensable enzyme that plays a pivotal role in the field of biotechnology It is produced mainlyfrom microbial sources and is used in many industries Industrial sectors with top-down and bottom-up approaches are currentlyfocusing on improving microbial amylase production levels by implementing bioengineering technologies The further supportof energy consumption studies such as those on thermodynamics pinch technology and environment-friendly technologies hashastened the large-scale production of the enzyme Herein the importance of microbial (bacteria and fungi) amylase is discussedalong with its production methods from the laboratory to industrial scales

1 Introduction

The International Enzyme Commission has categorizedsix distinct classes of enzymes according to the reactionsthey catalyze EC1 Oxidoreductases EC2 Transferases EC3Hydrolases EC4Lyases EC5 Isomerases andEC6Ligases [1]In general biologically active enzymes can be obtained fromplants animals and microorganisms Microbial enzymeshave been generally favored for their easier isolation in highamounts low-cost production in a short time and stability atvarious extreme conditions and their cocompounds are alsomore controllable and less harmful Microbially producedenzymes that are secreted into the media are highly reliablefor industrial processes and applications Furthermore theproduction and expression of recombinant enzymes arealso easier with microbes as the host cell Applicationsof these enzymes include chemical production bioconver-sion (biocatalyst) and bioremediation In this aspect thepotential uses of different microbial enzymes have beendemonstrated [2ndash5] With regard to industrial applicationsenzyme purification studies have predominantly focused onproteases lipases and amylases [4ndash12] Furthermore several

microbes have been isolated from different sources for theproduction of extracellular hydrolases [5 13 14] which areeither endohydrolases or exohydrolases In this overview wefocus on the microbial hydrolase enzyme amylase for itsdownstream applications in industries and medicines

2 Amylase and Its Substrates

Amylases are broadly classified into 120572 120573 and 120574 subtypesof which the first two have been the most widely studied(Figures 1(a) and 1(b)) 120572-Amylase is a faster-acting enzymethan 120573-amylase The amylases act on 120572-1-4 glycosidic bondsand are therefore also called glycoside hydrolases The firstamylase was isolated by Anselme Payen in 1833 Amylasesare distributed widely in living systems and have specificsubstrates [15 16] Amylase substrates are widely availablefrom cheap plant sources rendering the potential applica-tions of the enzymemore plentiful in terms of costs Amylasescan be divided into endoamylases and exoamylases Theendoamylases catalyze hydrolysis in a randommannerwithinthe starchmoleculeThis action causes the formation of linearand branched oligosaccharides of various chain lengths The



(a) (b)

Figure 1 Three-dimensional structures of amylases (a) 120572-Amylase (RCSB PDB accession code 1SMD the calcium-binding regions areindicated) (b) 120573-Amylase (RCSB PDB accession code PDB 2xfr)

exoamylases hydrolyze the substrate from the nonreducingend resulting in successively shorter end products [16]All 120572-amylases (EC 3211) act on starch (polysaccharide)as the main substrate and yield small units of glucose(monosaccharide) and maltose (disaccharide) (Figure 2)Starch is made up of two glycose polymers amylose andamylopectin which comprise glucose molecules that areconnected by glyosidic bonds Both polymers have differentstructures and properties A linear polymer of amylose hasa maximum of 6000 glucose units linked by 120572-14 glycosidicbonds whereas amylopectin is composed of 120572-14-linkedchains of 10ndash60 glucose units with 120572-16-linked side chainsof 15ndash45 glucose units Saboury [17] revealed the 120572-amylasesto be metalloenzymes that require metal (calcium) ions tomaintain their stability activity and structural confirmationBased on sequence alignments of 120572-amylases Nielsen andBorchert [18] revealed that these enzymes have four con-served arrangements (IndashIV) which are found as 120573-strands3 4 and 5 in the loop connecting 120573-strand 7 to 120572-helix7 (Figure 3) Despite the fact that amylases are broadlyavailable from different sources past focus has been on onlymicrobial amylases owing to their advantages over plant andanimal amylases as discussed aboveMicrobial amylases havebeen isolated from several stains and explored for amylaseproduction by the methods described below

3 Isolation Methods

The isolation of potential and efficient bacterial or fungalstrains is important before being screened for their produc-tion of enzymes of interest As stated elsewhere microbes areubiquitous and can be obtained from any source However

the most efficient strains are usually obtained from substrate-rich environments from which the microbes can be adoptedto use a particular substrate [5 13] The common methodof strain isolation is through serial dilution whereupon thenumber of colonies is minimized and thus easy to select[13] Another method is through substrate selection whereefficient strains are isolated according to their affinity fora particular substrate [14] Through these methods severalbacteria and fungi have been isolated and studied for amylaseproduction

4 Microbial Amylase

Microbial amylases obtained from bacteria fungi and yeasthave been used predominantly in industrial sectors and sci-entific research The level of amylase production varies fromonemicrobe to another even among the same genus speciesand strain Furthermore the level of amylase productionalso differs depending on the microbersquos origin where strainsisolated from starch- or amylose-rich environments naturallyproduce higher amounts of enzyme Factors such as pHtemperature and carbon and nitrogen sources also play vitalroles in the rate of amylase production particularly in fer-mentation processes Because microorganisms are amenableto genetic engineering strains can be improved for obtaininghigher amylase yields Microbes can also be fine-tuned toproduce efficient amylases that are thermostable and stableat stringent conditions Such improvements can also reducecontamination by background proteins and minimize thereaction time and lead to less energy expenditure in theamylase reaction [20] The selection of halophilic strains isalso beneficial to the production of amylase under extremeconditions (Figure 4)


Digestion

Glucose

Digestion

Starch

Glucose

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

H

O

H

H

O

H OH

OH

H

O

H

H

O

H OH

OH

H

O

H

HO

O

H OH

OH

H

O

H

H

O

Starch

H

OHOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

HH

OHOOOOOOOOOOOOOOOOOOOOOOOOOOO

H

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

CH2

CH2OH

CH2OH CH2OH

CH2OHCH2OHCH2OH

Figure 2 Scheme for the hydrolysis of starch by amylase Starch is a polysaccharide made up of simple sugars (glucose) Upon the action ofamylase either glucose (a monosaccharide) or maltose (a disaccharide with two glucose molecules) is released

41 Bacterial Amylases Among the wide range of microbialspecies that secrete amylase its production from bacteria ischeaper and faster than from other microorganisms Fur-thermore as mentioned above genetic engineering studiesare easier to perform with bacteria and they are also highlyamenable for the production of recombinant enzymes Awide range of bacterial species has been isolated for amylasesecretion Most are Bacillus species (B subtilis B stearother-mophilus B amyloliquefaciens B licheniformis B coagulansB polymyxa B mesentericus B vulgaris B megateriumB cereus B halodurans and Bacillus sp Ferdowsicous)but amylases from Rhodothermus marinus Corynebacteriumgigantea Chromohalobacter sp Caldimonas taiwanensisGeobacillus thermoleovorans Lactobacillus fermentum Lacto-bacillus manihotivorans and Pseudomonas stutzeri have alsobeen isolated [1 12 16 20 21] Halophilic strains that pro-duce amylases includeHaloarcula hispanicaHalobacillus spChromohalobacter sp Bacillus dipsosauri and Halomonasmeridiana [22] More studies involving the isolation andimprovement of novel strains will pave the way to creatingimportant strains For example Dash et al [23] identified anew B subtilis BI19 strain that produces amylase efficientlyand upon optimizing the conditions enhanced the enzymeproduction about 306 folds Three-dimensional structuralanalysis of such amylases helps in improving their efficiency

For example the crystal structure of 120572-amylase from Anoxy-bacillus has provided insight into this enzyme subclass [19]Studies on the three-dimensional structure also aid in thealteration or mutation of particular amino acids to improvethe efficiency and functions of the enzyme or protein [24ndash26]

42 Fungal Amylases Fungal enzymes have the advantage ofbeing secreted extracellularly In addition the ability of fungito penetrate hard substrates facilitates the hydrolysis processIn addition fungal species are highly suitable for solid-based fermentation The first fungal-produced amylase forindustrial application was described several decades ago [27]Efficient amylase-producing species include those of genusAspergillus (A oryzae A niger A awamori A fumigatusA kawachii and A flavus) as well as Penicillium species(P brunneum P fellutanum P expansum P chrysogenumP roqueforti P janthinellum P camemberti and P olsonii)Streptomyces rimosusThermomyces lanuginosus Pycnoporussanguineus Cryptococcus flavus Thermomonospora curvataandMucor sp [12 16 20 21]

5 Recombinant Amylase

Genetic engineering and recombinant DNA technology arethe current molecular techniques used to promote efficient


Region 1

Region 2Region 3Region 4

Domain B

Domain C

N-term

C-term

1205731 1205732 1205733

12057341205735120573612057371205738

Figure 3 Topology of 120572-amylases The positions of four conserved sequence patterns are indicated with dashed boxes [18]

Figure 4 A flowchart for microbial amylase Three-dimensional structure of the 120572-amylase from Anoxybacillus (RCSB PDB accession code5A2C) [19] is shown


DNA sequenceInsert amylase coding

Double-stranded plasmidDNA expression vector

Promotersequence

restriction nucleaseCut DNA with

DNA into cellsIntroduce recombinant

Overexpressed mRNA Overexpressed amylase

Figure 5 Recombinant DNA technology for amylase production The steps involve selection of an efficient amylase gene insertion of thegene into an appropriate vector system transformation into an efficient bacterial system to produce a higher amount of recombinant mRNAand overproduction of amylase from the bacterial system

enzyme production [18 28ndash30] Recombinant DNA tech-nology for amylase production involves the selection of anefficient amylase gene gene insertion into an appropriatevector system transformation in an efficient bacterial systemto produce a high amount of recombinant protein (in thepresence of an expression-vector promoter-inducing agent)and purification of the protein for downstream applications(Figure 5) In this technology high-copy numbers of the genepromote higher yields of amylase [30] On the other handscreening mutant libraries for selection of the best mutantvariants for recombinant amylase production has been moresuccessful (Figure 6) Zhang et al [31] deleted amyR (encod-ing a transcription factor) fromA nigerCICC2462 which ledto the production of enzymeprotein specifically with lowerbackground protein secretion Wang et al [32] generated anew strategy to express the 120572-amylase from Pyrococcus furio-sus in B amyloliquefaciens This extracellular thermostableenzyme is produced in low amount in P furiosus but itsexpression in B amyloliquefacienswas significantly increasedand had good stability at higher temperature (optimum100∘C) and lower pH (optimum pH 5) By mimicking theP furiosus system they obtained a novel amylase with yieldssim3000- and 14-fold higher amylase unitsmilliliter than thatproduced in B subtilis and Escherichia coli respectively

6 Screening Microbial Amylase Production

Production or secretion of amylase can be screened by dif-ferent common methods including solid-based or solution-based techniques The solid-based method is carried out

on nutrient agar plates containing starch as the substratewhereas solution-based methods include the dinitro salicylicacid (DNS) and Nelson-Somogyi (NS) techniques In thesolid-agar method the appropriate strain (fungi or bacteria)is pinpoint-inoculated onto the starch-containing agar at thecenter of the Petri plate After an appropriate incubationperiod the plate is flooded with iodine solution whichreveals a dark bluish color on the substrate region and a clearregion (due to hydrolysis) around the inoculum indicatingthe utilization of starch by themicrobial amylase Gopinath etal [7] applied this method to determine the amylase activityof Aspergillus versicolor as well as that of Penicillium sp intheir preliminary study (Figure 7)

In the solution-based DNS method the appropriatesubstrate and enzyme are mixed in the right proportion andreacted for 5min at 50∘C After cooling to room temperaturethe absorbance of the solution is read at 540 nm Gusakov etal [33] applied this method to detect the release of reducingsugars from substrate hydrolysis by Bacillus sp amylaseTheyfound that the amylase activity could reach up to 075UmLminus1after 24 h of incubation Similarly in the NSmethod amylaseand starch are mixed and incubated for 5min at 50∘C Thena Somogyi copper reagent is added to stop the reactionfollowed by boiling for 40min and a subsequent cool-down period A Nelson arsenomolybdate reagent is thenadded and the mixture is incubated at room temperaturefor 10min Then after diluting with water the solution iscentrifuged at high speed and the supernatant is measuredat 610 nm [34] Apart from these several other methods areavailable for amylase screening but all use the same substrate(starch)


Select amylase gene

Create library of variants

Insert gene library intoDNA expression vector

Insert gene library into

bacteria which produce amylase variants

Isolate improved gene and repeat the process

Mutationrecombination

Figure 6 Mutant library screening Selection of the best variants is a more successful technique for the ultimate application in recombinantamylase production

7 Enhancing Microbial Amylase Production

The primary objective in amylase production enhancementis to perform basic optimization studies This can be doneeither experimentally or by applying design of experiments(DOE) with further confirmation by the suggested experi-ments from the DOE [35 36] Several DOE methods havebeen proposed and with the advancement of software arecapable of better predictions [35ndash38] Gopinath et al [8]performed an optimization study by using a Box-Behnkendesign involving three variables (incubation time pH andstarch as the substrate) for higher amylase production bythe fungus A versicolor The laboratory experiments werein good agreement with the values predicted from DOEwith a correlation coefficient of 09798 confirming the higherproduction Srivastava et al [37] optimized the conditions forimmobilizing amylase covalently using glutaraldehyde as thecrosslinker on graphene sheets In this study Box-Behnken-designed response surface methodology was used with theefficiency of immobilization shown as 84 This kind ofstudy is importantwhenmolecules such as glutaraldehyde areused owing to two aldehyde groups being available at bothends of the molecule By optimization study the chances ofimmobilizing a higher number of glutaraldehyde moleculescan be predicted In another study the enzyme-assisted

extraction and identification of antioxidative and 120572-amylaseinhibitory peptides from Pinto beans were performed usinga factorial design with different variables (extraction timetemperature and pH) [38] Another way to enhance theaction of amylase is by its encapsulation or entrapment onalginate or other beads (Figure 8)This method facilitates theslow and constant release of enzyme and increases its stability

8 Industrial Applications ofMicrobial Amylase

Amylase makes up approximately 25 of the world enzymemarket [1] It is used in foods detergents pharmaceuticalsand the paper and textile industries [12 21] Its applica-tions in the food industry include the production of cornsyrupsmaltose syrups glucose syrups and juices and alcoholfermentation and baking [1] It has been used as a foodadditive and for making detergents Amylases also play animportant role in beer and liquor brewing from sugars (basedon starch) In this fermentation process yeast is used to ingestsugars and alcohol is produced Fermentation is suitable formicrobial amylase production under moisture and propergrowth conditions Two kinds of fermentation processeshave been followed submerged fermentation and solid-state


A versicolor

(a)

(b)

Figure 7 Amylase production on agar plate In this solid-based method the starch-containing agar plate is pinpoint-inoculated with themicroorganism at the center of the Petri plate After an appropriate incubation period flooding the plate with iodine solution reveals adark bluish color on the substrate region The clear region around the inoculum indicates the zone of hydrolysis (a) Amylolytic activity byAspergillus versicolor (b) amylolytic activity by Penicillium sp

fermentationThe former is the one traditionally used and thelatter has been more recently developed In traditional beerbrewing malted barley is mashed and its starch is hydrolyzedinto sugars by amylase at an appropriate temperature Byvarying the temperatures and conditions for 120572- or 120573-amylaseactivities the unfermentable and fermentable sugars aredetermined With these changes the alcohol content andflavor and mouthfeel of the end product can be varied

The potential industrial applications of enzymes aredetermined by the ability to screen new and improvedenzymes their fermentation and purification in large scaleand the formulations of enzymes As stated above differentmethods have been established for enzyme production Inthe case of amylase the crude extract can function well inmost of the cases but for specific industrial applications (egpharmaceuticals) purification of the enzyme is requiredThis can be accomplished by ion-exchange chromatogra-phy hydrophobic interaction chromatography gel filtrationimmunoprecipitation polyethylene glycolSepharose gel sep-aration and aqueous two-phase and gradient systems [2]where the size and charge of the amylase determine the

method chosen Automated programming system with theabove methods has improved the processes greatly

With these developments microbial amylase productionhas successfully replaced its production by chemical pro-cesses especially in industry [39] Production of amylasehas been improved by using genetically modified strainsthat reduce the polymerization of maltose during amylolyticaction [20] For further improvement in the industrial pro-cess the above-mentioned DOE and encapsulation methodscan be implemented

9 Future Perspectives

Among the different enzymes amylase possesses the high-est potential for use in different industrial and medicinalpurposes The involvement of modern technologies suchas white biotechnology pinch technology and green tech-nology will hasten its industrial production on a largescale This will be further facilitated by implementationof established fermentation technologies with appropriatemicrobial species (bacteria or fungi) and complementation


Nor

mal

enzy

mat

ic p

roce

ssEn

zym

e enc

apsu

lated

bea

ds

StarchStarch

Glucose

Figure 8 Efficient application of amylase Differences between the conventional methods of amylase utilization against alginate bead-encapsulated amylase are shown

of other biotechnological aspects The technologies of high-throughput screening and processing with efficient micro-bial species along with the ultimate coupling of geneticengineering of amylase-producing strains will all help inenhancing amylase production for industrial and medicinalapplications

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by an Inha University ResearchGrant from Inha University Republic of Korea

References

[1] K Mojsov ldquoMicrobial 120572-amylases and their industrial applica-tions a reviewrdquo International Journal of Management IT andEngineering vol 2 pp 583ndash609 2012


[3] S C B Gopinath P Anbu T Lakshmipriya et al ldquoBiotech-nological aspects and perspective of microbial Keratinase pro-ductionrdquo BioMed Research International vol 2015 Article ID140726 10 pages 2015

[4] P Anbu ldquoCharacterization of solvent stable extracellular pro-tease from Bacillus koreensis (BK-P21A)rdquo International Journalof Biological Macromolecules vol 56 pp 162ndash168 2013

[5] P Anbu and B K Hur ldquoIsolation of an organic solvent-tolerantbacterium Bacillus licheniformis PAL05 that is able to secretesolvent-stable lipaserdquo Biotechnology and Applied Biochemistryvol 61 no 5 pp 528ndash534 2014

[6] S C B Gopinath A Hilda T L Priya and G Annadu-rai ldquoPurification of lipase from Cunninghamella verticillataand optimization of enzyme activity using response surfacemethodologyrdquo World Journal of Microbiology and Biotechnol-ogy vol 18 no 5 pp 449ndash458 2002

[7] S C B Gopinath A Hilda T Lakshmi Priya G Annaduraiand P Anbu ldquoPurification of lipase from Geotrichum can-didum conditions optimized for enzyme production usingBox-Behnken designrdquo World Journal of Microbiology andBiotechnology vol 19 no 7 pp 681ndash689 2003

[8] S C B Gopinath A Hilda T Lakshmi Priya G Annaduraiand P Anbu ldquoStatistical optimization of amylolytic activity byAspergillus versicolorrdquoAsian Journal ofMicrobiology Biotechnol-ogy and Environmental Sciences vol 5 no 3 pp 327ndash330 2003


[9] T S Kumarevel S C B Gopinath A Hilda N Gautham andM N Ponnusamy ldquoPurification of lipase from Cunninghamellaverticillata by stepwise precipitation and optimized conditionsfor crystallizationrdquo World Journal of Microbiology and Biotech-nology vol 21 no 1 pp 23ndash26 2005

[10] P Anbu S C B Gopinath A Hilda T Lakshmi Priya andG Annadurai ldquoPurification of keratinase from poultry farmisolate-Scopulariopsis brevicaulis and statistical optimization ofenzyme activityrdquo Enzyme and Microbial Technology vol 36 no5-6 pp 639ndash647 2005

[11] P Anbu S C B Gopinath A Hilda N Mathivanan and GAnnadurai ldquoSecretion of keratinolytic enzymes and keratinol-ysis by Scopulariopsis brevicaulis and Trichophyton mentagro-phytes regression analysisrdquo Canadian Journal of Microbiologyvol 52 no 11 pp 1060ndash1069 2006

[12] I Hussain F Siddique M S Mahmood and S I Ahmed ldquoAreview of the microbiological aspect of 120572-amylase productionrdquoInternational Journal of Agriculture and Biology vol 15 no 5pp 1029ndash1034 2013

[13] S C B Gopinath P Anbu and A Hilda ldquoExtracellularenzymatic activity profiles in fungi isolated from oil-rich envi-ronmentsrdquoMycoscience vol 46 no 2 pp 119ndash126 2005

[14] P Anbu A Hilda and S C B Gopinath ldquoKeratinophilic fungiof poultry farm and feather dumping soil in Tamil Nadu IndiardquoMycopathologia vol 158 no 3 pp 303ndash309 2004

[15] H Guzman-Maldonado O Paredes-Lopez and C G Bili-aderis ldquoAmylolytic enzymes and products derived from starcha reviewrdquo Critical Reviews in Food Science and Nutrition vol 35no 5 pp 373ndash403 1995

[16] R Gupta P Gigras H Mohapatra V K Goswami and BChauhan ldquoMicrobial 120572-amylases a biotechnological perspec-tiverdquo Process Biochemistry vol 38 no 11 pp 1599ndash1616 2003

[17] A A Saboury ldquoStability activity and binding properties studyof 120572-amylase upon interaction with Ca2+ and Co2+rdquo BiologiamdashSection Cellular and Molecular Biology vol 57 no 11 pp 221ndash228 2002

[18] J ENielsen andTV Borchert ldquoProtein engineering of bacterial120572-amylasesrdquo Biochimica et Biophysica Acta vol 1543 no 2 pp253ndash274 2000

[19] K P ChaiN F BOthmanA-H Teh et al ldquoCrystal structure ofAnoxybacillus 120572-amylase provides insights intomaltose bindingof a new glycosyl hydrolase subclassrdquo Scientific Reports vol 6Article ID 23126 2016

[20] A Sundarram andT P KrishnaMurthy ldquo120572-amylase productionand applications a reviewrdquo Journal of Applied amp EnvironmentalMicrobiology vol 2 no 4 pp 166ndash175 2014

[21] P M de Souza and P O E Magalhaes ldquoApplication ofmicrobial-amylase in industrymdasha reviewrdquo Brazilian Journal ofMicrobiology vol 41 pp 850ndash861 2010

[22] K Kathiresan and S Manivannan ldquo120572-Amylase productionby Penicillium fellutanum isolated from mangrove rhizospheresoilrdquoAfrican Journal of Biotechnology vol 5 no 10 pp 829ndash8322006

[23] B K Dash M M Rahman and P K Sarker ldquoMolecularidentification of a newly isolated Bacillus subtilis BI19 and opti-mization of production conditions for enhanced production ofextracellular amylaserdquo BioMed Research International vol 2015Article ID 859805 9 pages 2015

[24] T Kumarevel N Nakano K Ponnuraj et al ldquoCrystal structureof glutamine receptor protein from Sulfolobus tokodaii strain 7in complex with its effector l-glutamine implications of effector

binding in molecular association and DNA bindingrdquo NucleicAcids Research vol 36 no 14 pp 4808ndash4820 2008

[25] T Kumarevel K Sakamoto S C B Gopinath A ShinkaiP K R Kumar and S Yokoyama ldquoCrystal structure of anarchaeal specific DNA-binding protein (Ape10b2) fromAeropy-rum pernix K1rdquo Proteins vol 71 no 3 pp 1156ndash1162 2008

[26] T Kumarevel T Tanaka M Nishio et al ldquoCrystal structureof the MarR family regulatory protein ST1710 from Sulfolobustokodaii strain 7rdquo Journal of Structural Biology vol 161 no 1 pp9ndash17 2008

[27] W Crueger and A Crueger Industrial Microbiology SinauerAssociates Sunderland Mass USA 1989

[28] J M Corbin B I Hashimoto K Karuppanan et al ldquoSemi-continuous bioreactor production of recombinant butyryl-cholinesterase in transgenic rice cell suspension culturesrdquoFrontiers in Plant Science vol 7 article 412 2016

[29] J-W Jung N-S Kim S-H Jang Y-J Shin and M-S YangldquoProduction and characterization of recombinant human acid120572-glucosidase in transgenic rice cell suspension culturerdquo Journalof Biotechnology vol 226 pp 44ndash53 2016

[30] Y J Son A J Ryu L Li N S Han and K J JeongldquoDevelopment of a high-copy plasmid for enhanced productionof recombinant proteins in Leuconostoc citreumrdquoMicrobial CellFactories vol 15 no 1 article 12 2016

[31] H Zhang S Wang X X Zhang et al ldquoThe amyR-deletionstrain of Aspergillus niger CICC2462 is a suitable host strain toexpress secreted protein with a low backgroundrdquoMicrobial CellFactories vol 15 article 68 2016

[32] P Wang P Wang J Tian et al ldquoA new strategy to expressthe extracellular 120572-amylase from Pyrococcus furiosus in Bacillusamyloliquefaciensrdquo Scientific Reports vol 6 Article ID 222292016

[33] A V Gusakov E G Kondratyeva andA P Sinitsyn ldquoCompari-son of twomethods for assaying reducing sugars in the determi-nation of carbohydrase activitiesrdquo International Journal ofAnalytical Chemistry vol 2011 Article ID 283658 4 pages 2011

[34] T Kobayashi H Kanai T Hayashi T Akiba R Akaboshi andK Horikoshi ldquoHaloalkaliphilic maltotriose-forming 120572-amylasefrom the Archaebacterium Natronococcus sp strain Ah-36rdquoJournal of Bacteriology vol 174 no 11 pp 3439ndash3444 1992

[35] R Garcıa A Renedo M Martınez and J Aracil ldquoEnzymaticsynthesis of n-octyl (+)-2-methylbutanoate ester from racemic(plusmn)-2-methylbutanoic acid by immobilized lipase optimizationby statistical analysisrdquo Enzyme and Microbial Technology vol30 no 1 pp 110ndash115 2002

[36] Y-N Chang J-C Huang C-C Lee I-L Shih and Y-MTzeng ldquoUse of response surface methodology to optimizeculture medium for production of lovastatin by Monascusruberrdquo Enzyme andMicrobial Technology vol 30 no 7 pp 889ndash894 2002

[37] G Srivastava K Singh M Talat O N Srivastava and A MKayastha ldquoFunctionalized graphene sheets as immobilizationmatrix for fenugreek 120573-amylase enzyme kinetics and stabilitystudiesrdquo PLoS ONE vol 9 no 11 Article ID e113408 2014

[38] Y-Y Ngoh and C-Y Gan ldquoEnzyme-assisted extraction andidentification of antioxidative and 120572-amylase inhibitory pep-tides from Pinto beans (Phaseolus vulgaris cv Pinto)rdquo FoodChemistry vol 190 pp 331ndash337 2016

[39] P Saranraj and D Stella ldquoFungal amylasemdasha reviewrdquo Interna-tional Journal of Microbiological Research vol 4 pp 203ndash2112013

Research ArticleExtracellular Ribonuclease from Bacillus licheniformis(Balifase) a New Member of the N1T1 RNase Superfamily

Yulia Sokurenko Alsu Nadyrova Vera Ulyanova and Olga Ilinskaya

Institute of Fundamental Medicine and Biology Kazan Federal University Kremlevskaya Str 18 Kazan 420008 Russia

Correspondence should be addressed to Yulia Sokurenko sokurenkoyuliagmailcom

Received 2 June 2016 Accepted 25 July 2016

Academic Editor Subash C B Gopinath

Copyright copy 2016 Yulia Sokurenko et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The N1T1 RNase superfamily comprises enzymes with well-established antitumor effects such as ribotoxins secreted by fungiprimarily by Aspergillus and Penicillium species and bacterial RNase secreted by B pumilus (binase) and B amyloliquefaciens(barnase) RNase is regarded as an alternative to classical chemotherapeutic agents due to its selective cytotoxicity towards tumorcells New RNase with a high degree of structural similarity with binase (73) and barnase (74) was isolated and purified fromBacillus licheniformis (balifase calculated molecular weight 124219Da pI 891) The protein sample with enzymatic activity of 15times 106 unitsA

280was obtained The physicochemical properties of balifase are similar to those of barnase However in terms of its

gene organization and promoter activity balifase is closer to binaseThe unique feature of balifase gene organization consists in thefact that genes of RNase and its inhibitor are located in one operon Similarly to biosynthesis of binase balifase synthesis is inducedunder phosphate starvation however in contrast to binase balifase does not form dimers under natural conditions We proposethat the highest stability of balifase among analyzed RNase types allows the protein to retain its structure without oligomerization

1 Introduction

Ribonuclease (RNase) is involved in many cellular processesincluding control of gene expression angiogenesis apop-tosis and cell defense from pathogens [1ndash5] At presentRNase types possessing antitumor and antiviral activitiesare at the peak of experimental investigation due to theirselective toxicity towards cancer or virus-infected cells [36ndash11] Among them are secreted RNase types of bacterialorigin which belong to N1T1 (b 31273) superfamilySuch RNase types are small (sim125 kDa) extracellular basicproteins Upon RNA hydrolysis these enzymes cleave the3101584051015840-phosphodiester bond between guanosine 31015840-phosphateand the 51015840-OH group of the adjacent nucleotide whilegenerating 2101584031015840-cyclic guanosine phosphate in the first stageof a catalytic reaction This stage is reversible and fasterthan the second one wherein the cyclic intermediate isconverted into the corresponding 31015840-phosphate derivative[12] This type of RNase was isolated from the cultural fluidof B amyloliquefaciens (termed barnase P00648) B pumilus(binase P00649) B altitudinis (balnase A0A0J1IDI7) B

circulans (P35078) and B coagulans (P37203) The catalyticactivity molecular structures and some biological propertiesof such RNase were explored and characterized [13ndash17] Themost studied representatives of bacillary RNase are binaseand barnase produced byB pumilus andB amyloliquefaciensrespectively Consisting of sim110 amino acid residues theseenzymes are highly similar in their structure They also showsimilar physicochemical and catalytic properties namelystability over a wide pH range (3ndash10) with an optimum atpH 85 and nonrequirement in metal ions for ribonucleolyticactivity [2]

Bacillus spp are known to produce another type ofextracellular RNase with high molecular weight (sim30 kDa)and low level of catalytic activity while lacking specificitytowards guanyl residues These enzymes are exemplified byB subtilis Bsn and B pumilus binase II [18 19]

B licheniformis the endospore-forming nonpathogenicGram-positive bacteria is used extensively for industrial pro-duction of exoenzymes (proteases 120572-amylases) and peptideantibiotics [20] Despite its widespread industrial applicationthe extracellular RNase from B licheniformis has not yet

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 4239375 9 pageshttpdxdoiorg10115520164239375


been characterized The RNase encoding BLI RS18290 gene(previously known as BLi03719) was found among the mostdominant protein spots in the extracellular proteome of theB licheniformis grown under phosphate deficiency [21]

Here we have isolated purified and characterized theB licheniformis secreted RNase (balifase) Furthermore wehave compared molecular properties of balifase with those ofbinase and barnase We have shown that the level of balifasecatalytic activity as well as its physicochemical characteristicsand structural features is similar to those of the mainrepresentatives of bacillary N1T1 RNaseThe unique featuresof balifase include the formation of an operon together with agene of its intracellular inhibitor as well as high stability andinability to form natural dimers

2 Materials and Methods

21 Strain and Growth Conditions Wild-type strain of BlicheniformisATCC 14580was obtained fromBacillusGeneticStock Center (BGSC USA) B licheniformis was grown onstandard LB medium or on LP medium (low phosphatepeptone 2 glucose 1 Na

2HPO4 004 CaCl

2 001

MgSO4times 7H

2O 003 MnSO

4 001 NaCl 03) Bac-

teria were cultivated at 37∘b using a laboratory shaker withoscillation intensity of 200 rpm (INFORS HT Switzerland)Culture growth was determined spectrophotometrically at120582 = 590 nm and expressed as optical density units (OD

590)

22 RNase Activity Determination of RNase activity wasperformed by the measurement of acid-soluble hydrolysisproducts of high molecular weight yeast RNA as describedearlier [22] The reaction mixture consisting of enzymesolution RNA and 025M Tris-HCl buffer pH 85 wasincubated for 15 minutes at 37∘b One unit was definedas the amount of enzyme that increases the extinction ofacid-soluble products of RNA hydrolysis at 260 nm per minSpecific activity was calculated as the ratio of the total enzymeactivity to the amount of the protein

23 Enzyme Preparation After 24ndash26 hours of cultivation onLP medium B licheniformis cells were acidified with aceticacid to pH 50 centrifuged at 9000timesg for 20 minutes at4∘C The supernatant was diluted twice with sterile distilledwater and was applied onto the DEAE-cellulose (Serva-cel Germany) column (asymp30mL) equilibrated with 001MNa-acetate buffer pH 50 Then the solution was appliedonto the phosphocellulose P-11 (Whatman England) column(asymp50mL) equilibrated with the same buffer After that thecolumn was washed with 001M Na-acetate buffer pH 50until optical density of eluate at 280 nmdecreased below 005Then the column was equilibrated with 001MNa-phosphatebuffer pH 70 The elution was carried out with 02M Na-phosphate buffer pH 70 Fractions corresponding to RNaseactivity peak were combined and desalted using centrifugalfilter units Ultracel-3K (Merck Millipore USA) The addi-tional purification was carried out using Biologic DuoFlowFPLC system (BioRad USA) on the UNOS

6(BioRad USA)

column equilibrated with 20mM Na-acetate buffer pH 50Proteins were eluted using a linear gradient of 0-1M NaCl

24 SDS-PAGE and Immunoblotting Proteins were sepa-rated by SDS-PAGE [23] and transferred to a nitrocellulosemembrane by Mini Trans-Blot cell (BioRad USA) For thedetection of proteins anti-binase antibodies were used [24]Visualization of protein bands corresponding to RNase wasperformed using anti-rabbit IgG-POD secondary antibodies(Sigma-Aldrich USA) and the LumiLight detection system(Roche Diagnostics Switzerland)

25 Zymography To estimate in-gel RNase activity of pro-teins we performed zymography analysis as described in [25]Proteins were separated in 15 polyacrylamide gel with 01SDS (SDS-PAGE) [23]The resolving gel containedRNA fromTorula yeast (Sigma-Aldrich USA) as a substrate at finalconcentration of 7mgmL Then the gel was washed withbuffer I (10mMTris-HCl 20 isopropanol pH75) for 10minto remove SDS and thenproteinswere refolded by consequentincubation for 10min in 10mM Tris-HCl pH 75 and in100mMTris-HCl pH 75The gel was stained for 10min with02 toluidine blue (Sigma-Aldrich USA)

26 Bioinformatic Analysis The RNase sequences wereextracted from the databases of the National Center forBiotechnology Information NCBI (httpwwwncbinlmnihgov) Gene neighborhoods were compared at ldquoMicrobesOnlinerdquo server of the Virtual Institute for Microbial Stressand Survival (httpwwwmicrobesonlineorg)Orthologs ofintracellular RNase inhibitor (barstar) were identified withthe help of ldquoEDGARrdquo server (httpsedgarcomputationalbiouni-giessende) For multiple alignment of amino acidsequences the program ldquoMUSCLErdquo (httpwwwebiacukToolsmuscle)was appliedAlignmentwas carried out on thebasis of standard criteria The software package ldquoMEGA 60rdquowas used for construction of phylogenetic trees [26] Leaderpeptide of the extracellular RNase of B licheniformis ATCC14580 was determined using PRED-TAT tool (httpwwwcompgenorgtoolsPRED-TATsubmit) Virtual Footprinttool was used for analysis of the transcription factor bindingsites [27] Comparison of physicochemical properties ofproteinswas performedusing ProtParam tool [28]The three-dimensional structure of balifase was modeled with the helpof I-TASSER server without specifying the template [29] AFATCAT web server was used for flexible structure compar-ison and structure similarity search (httpfatcatburnhamorg)


31 Balifase Is Similar to Barnase by Molecular PropertiesTo isolate the B licheniformis extracellular RNase which wasfound upon studies of bacterial cell response to starvation[21] we first compared its gene and amino acid sequencewith those of well-studied barnase and binase The RNaseof B licheniformis is encoded by the BLI RS18290 gene amature protein consists of 109 amino acids The analysisof the primary structure of B licheniformis RNase showedthat the main differences of the B licheniformis RNase frombinase and barnase are primarily concentrated in the regionof the signal and propeptides The signal peptide of balifase


1

1

1

61

61

61

bpu

bpu

bli

bli

bam

bam

121

121

121

bpublibam

SP

MP

PP

lowast

lowast

lowastlowast lowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowastlowastlowastlowast lowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast

lowastlowast∙ ∙∙

∙∙∙

∙∙

∙ ∙ ∙ ∙∙

∙∙

∙∙∙∙

∙∙

MKKISSV-------FTMFALIAAILFSGFIPQQAYAETPLTQTATNETATIQLTSDVHTLMKKILST-------LALGFVLALGFLAGNLFTSSA-EGPQAEKGGTVQSN----------MMKMEGIALKKRLSWISVCLLVLVSAAGMLFSTAA----KTETSSHKAHTE---------

-AVINTFDGVADYLIRYKRLPDNYITKSQASALGWVASKGNLAEVAPGKSIGGDVFSNRE-EVINTFEGVADYIVKYGRLPDNFITKAEASKLGWDPQKGNLAEVAPGKSIGGDIFQNREAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNRE

GRLPSASGRTWREADINYVSGFRNADRLVYSSDWLIYKTTDHYATFTRIRKLLPDASGRIWREADINYTSGFRGSDRIVYSNDRLVYKTTDHYKTFTRMRGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR

Sec pathway

∙∙

∙ ∙ ∙ ∙ ∙ ∙∙∙∙

∙∙∙∙

∙∙

∙∙

∙∙∙∙

∙∙∙ ∙ ∙ ∙∙

∙∙

∙∙

∙

(a)

yvdB

yvdA

yvcT RBAM_031940 yvcN

yvcNBLi03719yrdF

ydfFydfE

yvcTyvdA

yvcL

yvcL

yvcK

yvcK

COG391

COG1660COG1481

COG-FruBCOG-NhoA

BPUM_3110

BPUM_3109BPUM_3107

COG_LdhA

COG-CynTCOG-SUL1

yvcJ

crh

crh

Bacillus amyloliquefaciens FZB42 (+strand 3306922 middot middot middot 3313589)

Bacillus licheniformis DSM13 (+strand 3543866 middot middot middot 3550533)

Bacillus pumilus SAFR-032 (+strand 3084727 middot middot middot 3091394)

(b)

1

1

1

42

60

120

119

179

159

239

127

102

59

bpu

bpubli

blibam

bam

bpu

bpu

bpuSD

bli

bli

bli

bam

bam

bam

-GACGCAGGATTCGGAGCTGCGTCTTTTTATTTATTTCATCAGAAGGTTATCAGGAAAAA--GAAGAAATAGTTCTCGATCGTTTTCCGGCATTCCGGGAGACCAAATAGCATGCGGTTA-------------------GATGAAAAATCCCGGCCGTTTCAGCCGGGGTTTATTTTTTT

minus35 minus10

AGCCTCATTTTAGCAAAGAACCTGTTTCTTTACATTTCCTTCATGTTCGGGTGCTATAATGGAACCTGCGGCAAGCCGTTCACGTCCTCGGCTTTCATGGAAAATTTACAATAATATAATCTAGATCAAAAGAACTATTTTTAAATTCTTTCTATTCCTTTCTCTCGATGCTGATACAAT

ATGAGGTAGACAAGCATCAAGAGGACAGCATCCGATTTC---------------------GATTTTTGGGCATGTTTTAAGGTACAATTCAAGATGATTGCAGACGGAATATGGAAATGAGAAAAGGAATCAGCTTCACATGATA-----------------------------------

AGAAATGCGGCTGTTCAACCCCATGAAGGCACATTCTTCATGTTTATGTTATAAAGTCTA------------------------------------------------------------

------------------------------------------------------------

CTTAATAGGAGGATGAAGATGAAGACAGGGAGGACTTTATCCFAAAAATGGGAGGTATTGCTTTG

(c)

Figure 1 Comparison of B licheniformis RNase with the representatives of N1T1 RNase family (a) Sequence alignments of signalpeptide (SP) propeptide (PP) and mature peptide (MP) of B licheniformis (bli) B amyloliquefaciens (bam) and B pumilus (bpu) RNaseIdentical amino acid residues are marked (lowast) Amino acid residues that incorporate to the active site of enzyme are red colored (b) Geneneighborhood of balifase gene in comparison to barnase and binase genes The data were adopted from the MicrobesOnline Database(httpwwwmicrobesonlineorg) (c) Promoter regions of guanyl-preferring RNase genes from B pumilus (bpu) B licheniformis (bli) andB amyloliquefaciens (bam) Putative PhoP-binding sites are boxed (+1) regions are red colored A colon ldquordquo indicates conservation betweengroups of strongly similar properties A period ldquordquo indicates conservation between groups of weakly similar properties

and binase are similar whereas the propeptide resemblesbarnase by length Responsible for transport maturationand activation of enzymes signal peptide may have an effecton spatial organization of proteins The mature protein ismore conserved and differs by 30 and 28 amino acid residuesfrom barnase and binase respectively (Figure 1(a)) Thus theRNase of B licheniformis has the 73 and 74 degree ofsimilarity with barnase and binase respectively The overallresemblance of the RNase composing the N1T1 family is

reflected in a phylogenetic tree which was reconstructedbased on the primary sequences of the mature RNase (Fig-ure 2(d)) It is shown that the RNase of B licheniformis isequidistant from both B amyloliquefaciens and B pumilusRNase without forming a single cluster with the latter withinthe genus

The three-dimensional structure of balifase was predictedby using the I-TASSER server (Figure 2(a)) The C-score of172 corresponds to a model with high confidence According


N

R86

E72 R82

C

D53H101 K26

(a)

N

R86E72 R82

C

D53H101

K26

(b)

N

R86E72

R82

C

D53

H101

K26

(c)

92

61

41

56

81

9042

9981

100

96

02

98100

100

61

95

Penicillium brevicompactum Penicillium chrysogenum

Aspergillus saitoiAspergillus pallidusAspergillus clavatus

Aspergillus oryzaeNeurospora crassa

Trichoderma harzianumGibberella fujikuroiGibberella baccata

Ustilago sphaerogenaPleurotus ostreatus

Streptomyces aureofaciensSaccharopolyspora erythraea

Bacillus licheniformisBacillus amyloliquefaciens

Bacillus circulansBacillus coagulansBacillus pumilus

(d)

Figure 2 (a) Top model of balifase three-dimensional structure predicted by I-TASSER server without specifying the template (b)Superimposed three-dimensional structures of binase (PDB ID 1buj) and balifase (c) Superimposed three-dimensional structures of barnase(PDB ID 1bnr) and balifaseThe alignment was performed using the FATCAT server with flexiblemode (d)The phylogenetic tree constructedon the basis of amino acid sequences of RNase fromN1T1 familyThe scale bar indicates the average number of amino acid substitutions persite

to FATCAT pairwise alignment balifase is very similar toboth binase (PDB ID 1buj) and barnase (PDB ID 1bnr) withsome minor differences (Figures 2(b) and 2(c)) The 119875 valueis lt005 (with the raw score of 29854 and 28220 resp)which means that the structure pairs are significantly similarThe structure alignment has 109 equivalent positions with anRMSD of 130 and 153 respectively without twists

A set of physicochemical parameters of the B licheni-formisRNase such asmolecular weight and pI was predictedby using the ProtParam tool (Table 1) It is shown thatdespite some variations in these parameters balifase binaseand barnase proteins are found to have similar propertiesHowever balifase is closer to barnase than binase It is amore acidic protein compared to barnase Furthermore thealiphatic index of balifase and barnase is lower than thatof binase which indicates their lower thermostability Theinstability index is significantly higher for balifase than forbinase and barnase which points to the high stability ofbalifase (Table 1) Generally a protein with an instabilityindex lower than 40 is predicted as stable [30] Calculatedfrom themolar extinction coefficient of tyrosine tryptophan

Table 1 Physicochemical properties of balifase as compared withbinase and barnase

RNasecharacteristics Balifase Binase BarnaseNumber of aminoacids 109 109 110

Molecular weight 124219 122116 123827Theoretical pI 891 952 888Extinction coefficient 19940 26930 26930Abs 01 (=1 gL) 1605 2205 2175Grand average ofhydropathicity minus0666 minus0416 minus0643

Instability index 892 2725 2427Aliphatic index 7248 7881 7100

and cystine [31] the extinction coefficient of balifase allowsus to detail the calculation of the protein amount duringpurification


Therefore based on the similarities between barnaseand balifase proteins we have found that the protocol forbarnase purification is also suitable for purification of balifase[13]

32 Gene Context of B licheniformis RNase Differs fromThat of Barnase and Binase Gene neighborhood informa-tion supports a better understanding of putative functionsof a protein encoded by a gene of interest Typically theneighborhoods combine several genes that are involved insimilar process however some of these genes can differ Theexploration of the balifase gene BLI RS18290 neighborhoodshowed that it is organized differently from barnase andbinase (Figure 1(b)) The RNase gene forms an operonwith an intracellular inhibitor gene downstream of ydfEand ydfF genes responsible for metal resistance Yet thesefeatures are not characteristic of binase (BPUM 3110) andbarnase (RBAM 031940) At the 51015840-end they are precededby the yvcT gene encoding gluconate 2-dehydrogenase aswell as the prospective bicistronic operon formed by yvdAand yvdB genes which encodes carbonate dehydrogenaseand permease of the SuIP family At the 31015840-end the genesfor balifase and its inhibitor are followed by yvcN (encod-ing N-acetyltransferase) crh (encoding histidine-containingphosphorus-carrying Hpr-like protein) yvcL (encoding aDNA-binding protein WhiA which controls the process ofsporulation in spore-forming bacteria) yvcK (encoding thefactor of gluconeogenesis) and yvcJ (encoding a nucleotide-binding protein which hydrolyzes nucleoside triphosphates)The gene context of balifase reflects its participation inphosphorus and carbon metabolism

The B licheniformis RNase gene context differs from thegene context of other closely related species of bacilli giventhe fact that balifase gene forms an operon with the gene ofintracellular inhibitor YrdF (Figure 1(b))Our further analysisusing the EDGAR server has revealed that the YrdF inhibitorof balifase represents the ortholog of a well-known barnaseinhibitor barstar

The ydfF gene encodes a transcriptional regulatorbelonging to the family of ArsR-like repressors that activatethe transcription of proteins involved in the efflux of metalsandor detoxification by dissociation from operators [32]The YdfE gene product contains flavin reductase domain ofvarious oxidoreductases and monooxygenases The proteinperforms an antioxidant function in the oxidation of lipidmembrane caused by heavymetalsThe expression of the ydfEgene is controlled by the ydfF gene

33 Regulation of Balifase Gene Expression Is Similar toBinase One To understand how balifase gene expressionis regulated we analyzed its promoter structure Usingcomputational approach it was impossible to identify (minus10)and (minus35) regions clearly Two possible (+1) positions werepredicted (Figure 1(c))Therefore we compared balifase genepromoter to promoters of binase and barnase The RNase ofBacillus with low molecular weight can be divided into twogroups binase-like and barnase-like RNase [33] In contrastto barnase the promoters of binase and balifase genes possess(minus10) and (minus35) regions

Besides that the detection of the transcription factorbinding sites was performed (Table 2) It was found thatexpression of balifase binase and barnase could be regulatedby AbrB GerE and PucR transcription factorsThis indicatesthe involvement of the RNase in scavenge of purines (PucRregulation) as well as in general transition from exponentialgrowth to stationary phase (AbrB regulation) Expressionof RNase genes during sporulation could be inhibited byGerE Binding sites for ComK which is required for thetranscription of late competence genes were not detectedin balifase gene promoter in contrast to binase and barnaseones Genes for barnase and balifase are potentially con-trolled by DegU the regulator of extracellular degradativeenzymes biosynthesis in response to nitrogen starvationFurthermore we identified the potential binding sites forthe PhoP transcription factor which controls cell responseto phosphate deficiency in Bacillus in the B licheniformisRNase gene promoter and in the binase promoter (Fig-ure 1(c)) Potential binding sites for Hpr which providesthe link between phosphorous and carbon metabolism andSpoIIID which regulates gene expression during sporulationwere found in binase and balifase gene promoters as wellThus analysis of balifase promoter structure and activityrevealed its higher similarity to promoter of binase than ofbarnase

34 Purification of Balifase To identify the most suitablemedia for high-level production of the B licheniformisRNase we have grown bacteria in LB and low phosphatepeptone media (LP) differing by the amount of phosphorus(275 120583gmL and 120120583gmL resp) It was found that the cellsproduce twice as much amount of RNase when grown onLP medium during 24 h at 37∘C Balifase appears in theculture fluid after 6 h of cultivation and reaches themaximumafter 22ndash24 h corresponding to the early stationary phase(Figure 3(a))

The purification protocol for barnase [13] was appliedfor balifase However this procedure failed to generate largeamounts of pure balifase and therefore was modified Forprotein purification samples were collected at 22ndash24 h and allsteps of purification shown in Table 3 were performed Afterelution from phosphocellulose a protein sample with theyield of 75 by activity was obtained The final purificationstep was made using the FPLC BioLogic Duo Flow systemProtein was eluted by 035M NaCl (Figure 3(b)) The elutionprofile was characterized by two additional peaks whichhad no RNase activity It was detected via SDS-PAGE thatthe first fraction contained the protein with the molecularweight of 12 kDa (Figure 4(a)) and RNase activity in the gel(Figure 4(b)) The purity of the sample was shown by massspectrometry analysis which also proved the presence of theB licheniformisRNase only (AAU251681) Finally the proteinsample with enzymatic activity of 15 times 106 unitsA

280was

obtainedIt was observed that the oligomeric forms of balifase

with catalytic activity in the gel (Figure 4(c)) appear afterconcentrating freezing and storage The immunoblot anal-ysis showed that anti-binase antibodies interact with loworder oligomers of balifase too (Figure 4(d)) Therefore we


Table 2 Putative binding sites for transcription factors in barnase binase and balifase promoters

Transcription factors Barnase Binase Balifase

AbrB (controls the expressionof genes involved instarvation-induced processes)

TAAAAAAT(125ndash132) GAAAAAAG (54ndash61)

CAAAAATC(119ndash126 noncoding strand)

CATAAACA(219ndash226 noncoding strand)

ComK (is required for thetranscription of the latecompetence genes)

AAAGAACTATTTT (50ndash62) AAAGCCTCATTTT (58ndash70) Not detected

DegU (regulates thedegradative enzymeexpression geneticcompetence biofilmformation and capsulebiosynthesis)

GAAAAATCCCGGCCGTTTCAG(4ndash24) Not detected

ATAGTTCTCGATCGTTTTCCG(7ndash27)

ACAATAATATAATGATTTTTG(106ndash126)

GerE (regulates genetranscription in the terminallydifferentiated mother-cellcompartment during latestages of sporulation)

AAATGGGAGGTA (129ndash140) AAATAAATAAAA (25ndash36noncoding strand)

AAATAGTTCTCG (5ndash16)ATATAATGATTT (112ndash123)

Hpr (provides the linkbetween phosphorous andcarbon metabolism)

Not detected GGTGCTATAATATGAGGTA(109ndash127)

ATGTTTATGTTATAAAGTC(218ndash236)

PucR (regulation of purineutilization) ATACAATGAAA (95ndash105) ATTCGGAGCTG (9ndash19) TTTCATGGAAA (91ndash101)

GTCCTCGGCTT (82ndash92)ResD (regulation of aerobicand anaerobic respiration) AACTATTTTTAAA (54ndash66) TCATTTTAGCAAA (64ndash76)

CTCATTTTAGCAA (63ndash75) AAATTTACAATAA (100ndash112)

SpoIIID (key regulator oftranscription during thesporulation process)

Not detected AGGACAGCAT (141ndash150)TAGACAAGCA (126ndash135)

GGCACATTCT (206ndash215)GGTACAATTC (139ndash148)

18

16

14

12

10

8

6

4

2

0

RNas

e act

ivity

(U

mL)

302826242220181614121086420

Time (h)

Gro

wth

(OD590

) sp

ecifi

cra

te o

f bac

teria

l gro

wth

(sca

le18

1)

spec

ific r

ate o

f RN

ase

secr

etio

n (s

cale18

1)

GrowthSpecific rate of bacterial growthRNase activitySpecific rate of RNase secretion

7000

6000

5000

4000

3000

2000

1000

0

(a)

100

075

050

025

000

minus025

000 1000 2000 3000 4000

Time (min)

50 NaCl Balifase 2000

1000

00

minus1000

(AU

)

(mSmiddot

cmminus1)

(b)

Figure 3 (a) The time-course of B licheniformis ATCC 14580 growth and RNase production on LP medium at 37∘C (b) Elution of balifasefraction in a linear gradient of 00ndash10M NaCl using FPLC Biologic DuoFlow system AU absorbance units mS times cmminus1 conductivity

can conclude that balifase can exist in oligomeric forms insolution but unlike binase not under natural conditions ofbiosynthesis [25]

4 Conclusions

Thenew lowmolecular weight RNase isolated fromB licheni-formis (named balifase) has been characterized according toits physicochemical properties gene context and promoter

organization Balifase combines physicochemical propertiesof barnase (pI grand average of hydropathicity and aliphaticindex) with the regulatory mode of biosynthesis typical forbinase Both balifase gene and gene for its inhibitor arelocated in one operon which makes the gene organizationof balifase unique Balifase synthesis is induced under phos-phate starvation similarly to biosynthesis of binase howeverin contrast to binase balifase does not form dimers undernatural conditions We propose that the highest stability of


Table 3 The isolation and purification of B licheniformis RNase

Stage of purification Vol (V) mL A280

units Specific activityunitsA

280

Degree ofpurification Yield (by activity)

Culture fluid after 24 h of cultivation 1200 8 763 1 100After DEAE-cellulose pH 50 1200 78 780 102 997After elution from phosphocellulose in200mM sodium phosphate buffer pH 70 20 07 225000 353 75

After chromatography on UNOS6

column using FPLC system 4 045 15 times 106 2353 50

10

15

25

35

40

55

70

100

130

180

M 1 2 3 4 5(kDa)

(a)

25

15

10

35

M 1 2 3(kDa)

(b)

10

15

25

35

40

55

70

100

130

180

M 1 2(kDa)

(c)

25

15

10

35

M 1 2(kDa)

(d)

Figure 4 Electrophoretic analysis of purity enzymatic activity and antibody specificity of balifase (a) SDSPAGE of balifase samples atdifferent stages of purification 1 before purification in culture fluid 2 after chromatography on DEAE-cellulose 3 after chromatographyon phosphocellulose P-11 4 after chromatography on UNOS

6column 5 binase (b) Zymography analysis of balifase sample 1 after

chromatography on phosphocellulose P-11 2 after chromatography on UNOS6column 3 binase (c) Zymography analysis of balifase sample

after concentrating freezing and storage 1 balifase 2 binase (d)Western Blot analysis of balifase after chromatography on UNOS6column

1 binase 2 balifase


balifase among analyzed RNase types allows the protein toretain its structure without oligomerization

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this article

Acknowledgments

The research was performed within the Russian GovernmentProgram of Competitive Growth of Kazan Federal Univer-sity and supported by the Russian Research FoundationGrant no 14-14-00522 Bioinformatics analysis was donewith support of the Russian Foundation for Basic ResearchGrant no 15-04-07864 mass spectrometry was performedat Interdisciplinary Center of Kazan Federal University (IDRFMEFI59414X0003)

References

[1] CM Arraiano JM Andrade S Domingues et al ldquoThe criticalrole of RNA processing and degradation in the control of geneexpressionrdquo FEMSMicrobiology Reviews vol 34 no 5 pp 883ndash923 2010

[2] K Takahashi and S Moore The Enzymes Volume 15 NucleicAcids Part B Academic Press New York NY USA 1982

[3] A A Makarov A Kolchinsky and O N Ilinskaya ldquoBinaseand other microbial RNases as potential anticancer agentsrdquoBioEssays vol 30 no 8 pp 781ndash790 2008

[4] A Chakrabarti B K Jha and R H Silverman ldquoNew insightsinto the role of RNase L in innate immunityrdquo Journal ofInterferon amp Cytokine Research vol 31 no 1 pp 49ndash57 2011

[5] H F Rosenberg ldquoRNase A ribonucleases and host defense anevolving storyrdquo Journal of Leukocyte Biology vol 83 no 5 pp1079ndash1087 2008

[6] H A Cabrera-Fuentes M Aslam M Saffarzadeh et alldquoInternalization of Bacillus intermedius ribonuclease (BINASE)induces human alveolar adenocarcinoma cell deathrdquo Toxiconvol 69 pp 219ndash226 2013

[7] V A Mitkevich I Y Petrushanko P V Spirin et al ldquoSensitivityof acute myeloid leukemia Kasumi-1 cells to binase toxic actiondepends on the expression of KIT and AML1-ETO oncogenesrdquoCell Cycle vol 10 no 23 pp 4090ndash4097 2011

[8] V AMitkevich O N Ilinskaya andA AMakarov ldquoAntitumorRNases killerrsquos secretsrdquo Cell Cycle vol 14 no 7 pp 931ndash9322015

[9] O N Ilinskaya I Singh E Dudkina V Ulyanova A Kayumovand G Barreto ldquoDirect inhibition of oncogenic KRAS by Bacil-lus pumilus ribonuclease (binase)rdquo Biochimica et BiophysicaActa (BBA)mdashMolecular Cell Research vol 1863 no 7 pp 1559ndash1567 2016

[10] R S Mahmud and O N Ilinskaya ldquoAntiviral activity of binaseagainst the pandemic influenza A (H1N1) virusrdquo Acta Naturaevol 5 no 19 pp 44ndash51 2013

[11] O N Ilinskaya and R S Mahmud ldquoRibonucleases as antiviralagentsrdquoMolecular Biology vol 48 no 5 pp 615ndash623 2014

[12] H Yoshida ldquoThe ribonuclease T1 familyrdquoMethods in Enzymol-ogy vol 341 pp 28ndash41 2001

[13] R W Hartley and D L Rogerson Jr ldquoProduction and purifi-cation of the extracellular ribonuclease of Bacillus amyloliq-uefaciens (barnase) and its intracellular inhibitor (barstar) IBarnaserdquo Preparative Biochemistry vol 2 no 3 pp 229ndash2421972

[14] I AGolubenkoN P Balaban I B Leshchinskaia T I Volkovaand G I Kleıner ldquoRibonuclease of Bacillus intermedius 7 Ppurification by chromatography on phosphocellulose and sev-eral characteristics of the homogeneous enzymerdquo Biokhimiyavol 44 no 4 pp 640ndash648 1979

[15] E Dudkina V Ulyanova R Shah Mahmud et al ldquoThree-step procedure for preparation of pure Bacillus altitudinisribonucleaserdquo FEBS Open Bio vol 6 no 1 pp 24ndash32 2016

[16] A A Dementiev G P Moiseyev and S V ShlyapnikovldquoPrimary structure and catalytic properties of extracellularribonuclease of Bacillus circulansrdquo FEBS Letters vol 334 no 2pp 247ndash249 1993

[17] S V Shliapnikov and A A Dementrsquoev ldquoAmino acid sequenceand catalytic properties of the Bacillus coagulans extracellularribonucleaserdquo Doklady Akademii Nauk vol 332 no 3 pp 382ndash384 1993

[18] A Nakamura Y Koide H Miyazaki et al ldquoGene cloning andcharacterization of a novel extracellular ribonuclease of Bacillussubtilisrdquo European Journal of Biochemistry vol 209 no 1 pp121ndash127 1992

[19] M A Skvortsova A L Bocharov G I Yakovlev and L VZnamenskaya ldquoNovel extracellular ribonuclease from BacillusintermediusmdashBinase II purification and some properties of theenzymerdquo Biochemistry vol 67 no 7 pp 802ndash806 2002

[20] M Schallmey A Singh and O P Ward ldquoDevelopments inthe use of Bacillus species for industrial productionrdquo CanadianJournal of Microbiology vol 50 no 1 pp 1ndash17 2004

[21] L T Hoi B Voigt B Jurgen et al ldquoThe phosphate-starvationresponse of Bacillus licheniformisrdquo Proteomics vol 6 no 12 pp3582ndash3601 2006

[22] O N Ilinskaya N S Karamova O B Ivanchenko and LV Kipenskaya ldquoSOS-inducing ability of native and mutantmicrobial ribonucleasesrdquo Mutation ResearchmdashFundamentaland Molecular Mechanisms of Mutagenesis vol 354 no 2 pp203ndash209 1996

[23] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[24] V V Ulyanova V S Khodzhaeva E V Dudkina A V LaikovV I Vershinina and O N Ilinskaya ldquoPreparations of Bacilluspumilus secretedRNase one enzyme or twordquoMicrobiology vol84 no 4 pp 491ndash497 2015

[25] E Dudkina A Kayumov V Ulyanova and O Ilinskaya ldquoNewinsight into secreted ribonuclease structure binase is a naturaldimerrdquo PLoS ONE vol 9 no 12 article e115818 2014

[26] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[27] R Munch K Hiller A Grote et al ldquoVirtual footprint andPRODORIC an integrative framework for regulon predictionin prokaryotesrdquo Bioinformatics vol 21 no 22 pp 4187ndash41892005

[28] E Gasteiger C Hoogland A Gattiker et al ldquoProtein identifica-tion and analysis tools on the ExPASy serverrdquo inThe ProteomicsProtocols Handbook pp 571ndash607 Humana Press 2005


[29] J Yang R Yan A Roy D Xu J Poisson and Y Zhang ldquoTheI-TASSER suite protein structure and function predictionrdquoNature Methods vol 12 no 1 pp 7ndash8 2014

[30] K Guruprasad B V B Reddy and M W Pandit ldquoCorrelationbetween stability of a protein and its dipeptide composition anovel approach for predicting in vivo stability of a protein fromits primary sequencerdquo Protein Engineering vol 4 no 2 pp 155ndash161 1990

[31] S C Gill and P H von Hippel ldquoCalculation of protein extinc-tion coefficients from amino acid sequence datardquo AnalyticalBiochemistry vol 182 no 2 pp 319ndash326 1989

[32] D I Osman and J S Cavet ldquoBacterial metal-sensing proteinsexemplified by ArsR-SmtB family repressorsrdquo Natural ProductReports vol 27 no 5 pp 668ndash680 2010

[33] V Ulyanova V Vershinina O Ilinskaya and C R HarwoodldquoBinase-like guanyl-preferring ribonucleases are new membersof Bacillus PhoP regulonrdquoMicrobiological Research vol 170 pp131ndash138 2015

Research ArticleCloning Expression and Characterization ofa Novel Thermophilic Monofunctional Catalase fromGeobacillus sp CHB1

Xianbo Jia Jichen Chen Chenqiang Lin and Xinjian Lin

Soil and Fertilizer Institute Fujian Academy of Agricultural Sciences Fuzhou 350003 China

Correspondence should be addressed to Xinjian Lin xinjianlinviptomcom

Received 18 March 2016 Accepted 3 July 2016

Academic Editor Thangavel Lakshmipriya

Copyright copy 2016 Xianbo Jia et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Catalases are widely used in many scientific areas A catalase gene (Kat) from Geobacillus sp CHB1 encoding a monofunctionalcatalase was cloned and recombinant expressed in Escherichia coli (E coli) which was the first time to clone and express this type ofcatalase of genus Geobacillus strains as far as we knowThis Kat gene was 1467 bp in length and encoded a catalase with 488 aminoacid residuals which is only 81 similar to the previously studiedBacillus sp catalase in terms of amino acid sequence Recombinantcatalase was highly soluble in E coli and made up 30 of the total E coli protein Fermentation broth of the recombinant E colishowed a high catalase activity level up to 35831 UmL which was only lower than recombinant Bacillus sp WSHDZ-01 among thereported catalase production strains The purified recombinant catalase had a specific activity of 40526Umg and 119870

119898of 511mM

The optimal reaction temperature of this recombinant enzyme was 60∘C to 70∘C and it exhibited high activity over a wide range ofreaction temperatures ranging from 10∘C to 90∘CThe enzyme retained 947 of its residual activity after incubation at 60∘C for 1hour High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications

1 Introduction

Catalases (EC11116) are a class of enzymes that specificallycatalyze the decomposition ofH

2O2toH2OandO

2[1] H2O2

has been applied to sterilization and bleaching processes inthe medical food textile and paper-making industries [2 3]Residual H

2O2in products or by-products is harmful to

humanhealth and environmentThus the removal of residualH2O2is necessary in textile production processes food

health pollution prevention and other fields [2] Catalase isan ideal choice for removing H

2O2due to its efficiency and

lack of secondary pollution compared with chemical decom-position methods [4]

Catalase can be divided into four classes monofunctionalheme catalases catalase-peroxidases non-heme catalasesand minor catalases [5] Catalases are widely present inanimals plants fungimost aerobic bacteria and some anaer-obic bacteria [5] Commercial catalase produced by animalsliver plant tissues and microbial fermentation is mainlymesophilic catalase but many industrial applications need

thermostable catalase for example textile bleaching temper-ature is always up to 60∘C [6] so thermophilic catalase ismore comparable on that condition There have been somethermophilic catalases reported [7ndash10] but they were eithercatalase-peroxidase orMn-dependent catalaseThermophilicmonofunctional heme catalases are rarely reported as weknow

High yielding strains are also necessary for enzyme pro-duction in fermentation processes Recombinant expressionis a practical method to increase the yield of a target geneCurrently Pichia pastoris [11] Hansenula polymorpha [12]Lactococcus lactis [13] Bacillus subtilis (B subtilis) [14 15]and Escherichia coli (E coli) [9] are used as host cells to pro-duce recombinant catalase but those recombinant catalasesweremainlymesophilic successfully recombinant expressionprecedents of thermophilic catalases which were few Theadvantages of E coli systems such as their convenience highyields and ease of purification promote their wide applicationin genetic engineering But many thermophilic enzymes can-not be overproduced in active forms in mesophilic host [8]



For example though Thermus thermophilus catalase couldsolubly be expressed in E coli the recombinant catalasewas absolutely inactive [8] recombinant E coli with Bacillusstearothermophilus catalase-peroxidase gene also showed acomparatively lower catalase level (10553UmL) [16] So highsoluble expression with an active form in E coli is importantfor thermophilic catalase

Previously we screened thermophilic bacteria strainsisolated from different high-temperature compost samplesand Geobacillus sp CHB1 was found to be a high catalaseproduction strain So in this work we cloned a novel Katgene encoding a monofunctional catalase from thermophilesGeobacillus sp CHB1 and recombinant expressed this gene inE coli in a highly soluble formThe recombinant catalase wasalso purified and characterized


21 Materials Bacterial Strains Plasmids and Medium Ecoli BL21 (DE3) was cultured in our laboratory and theexpression vector pEASY-E2 was purchased from BeijingTransGen Biotech The Geobacillus sp CHB1 was isolatedin Fuzhou China LB medium (10 gL tryptone 5 gL yeastextract 10 gL NaCl pH 70) was used in culturing E coliBL21 (DE3) Auto-induction medium ZYM-5052 [17] wasused for inducing the expression of recombinant catalaseThemedium for culturing the Geobacillus sp CHB1 consisted ofsoya peptone 09 yeast extract 05 NaCl 01 K

2HPO4

01 and KH2PO40075 at pH 72

22 Expression Vector Construction Geobacillus sp CHB1was incubated at 60∘C for 18 h at 180 rmin then the genomewas extracted according to themethod of Zhou et al [18]TheKat gene was amplified using primers CatalaseF (51015840-GCA-GATACAAAAAAGCTCACAAC-31015840) and CatalaseR (51015840-TGCGTTTGTAATCACATCGTCCG-31015840) Polymerase chainreaction (PCR) was performed with ExTaq DNA polymerase(TaKaRa Dalian China) under the following conditions95∘C initial denaturation for 5min followed by 32 cycles of40 s at 94∘C 40 s at 55∘C and 1min 30 s at 72∘C The PCRproduct was purified using a PCR purification kit (OmegaBio-Tek Inc USA) and sequenced by Sangon Biotech CoLtd (Shanghai China)

Homology search of gene and amino acid sequence wascarried out at BLAST server (httpblastncbinlmnihgov)Program blastp was used to analyze homology of the aminoacid sequences nonredundant protein sequences (Nr) data-base and Swiss Prot database were both used for blastp pro-gram Sequences with high similarity in Swiss Prot databasewere selected to construct phylogenetic tree and do multiplealignment MEGA 402 was used to construct the phyloge-netic tree Multiple alignment of the amino acid sequenceswas carried out using DNAMAN V60399

The purified fragment was ligated with pEASY-E2 usingthe described protocol of the kit The ligated product wastransformed into the competent E coli cell strain Trans-T1(TransGen Biotech Beijing China) A positive clone was

selected using the T7 promoter primer (51015840-TAATAC-GACTCACTATAGGG-31015840) and CatalaseR (51015840-TGCGTT-TGTAATCACATCGTCCG-31015840) and then cultured with LBmedium containing 100 120583gmL of ampicillin The recombi-nant vector pEASY-E2-Kat was extracted using a PlasmidMini Kit (Omega Bio-Tek Inc USA) and then transformedinto competent E coli BL21 (DE3) cells A positive clone forBL21pEASY-E2-Kat was selected using PCR as describedabove

23 Inducing Expression and Purification of RecombinantCatalase Positive clones were cultured in 5mL of LBmedium containing 100 120583gmL of ampicillin at 37∘C at220 rpm for 12 h Next 1mL of the above culture was inoc-ulated into 50mL of ZYM-5052 medium and was culturedat 30∘C and 220 rpm for 16 h to induce the expression of theenzyme To confirm expression 8mL of the induced culturewas centrifuged at 10000timesg for 2min and then suspendedwith 3mL PBS (50mM pH 70) The suspended culturewas subjected to ultrasonication at 400W until clear Then200120583L of ultrasonicated sample was centrifuged at 12000timesgfor 5min at 4∘C and the supernatant was transferred to a newcentrifuge tube The supernatant and sediment sample wereboth subjected to 12 SDS-PAGE to detect the expression ofthe recombinant enzyme a sample containing an empty vec-tor was used as a blank control Electrophoresis parameterswere set as follows 30V for 30min followed by 80V until theendThe coomassie brilliant blue R250 protocol [23] was usedto dye the gel

24 Recombinant Strain Enzyme Production Curve First1mL of seed liquid was inoculated into shake flasks contain-ing 50mL of ZYM-5052 medium and the flasks were placedin shaking tables at 220 rpm and 30∘C Every 4 h sampleswere collected and analyzed for catalytic activityThe sampleswere ultrasonicated before activity measurement collectionEnzyme production curves were then obtained

25 Purification of Recombinant Catalase First 100mLof theinduced recombinant E coli was centrifuged at 10000timesg for5min and washed once with Buffer A (50mM NaH

2PO4

300mMNaCl pH80) After centrifugation the recombinantE coli pellet was resuspended with 3mL of Buffer A Theresuspended sample was ultrasonicated until clearThe ultra-sonicated sample was centrifuged and the supernatant wassubjected to a Nickel-iminodiacetic acid (Ni-IDA) columnThen 10 volumes of Buffer A (50mM NaH

2PO4 300mM

NaCl pH 80) were used to wash proteins that were non-specifically bound to the column Then 10 volumes of BufferB (50mM NaH

2PO4 300mM NaCl 20mM imidazole pH

80) were used to remove other proteins Finally 5 volumes ofBuffer C (50mM NaH

2PO4 300mM NaCl 100mM imida-

zole pH 80) and 5 volumes of Buffer D (50mM NaH2PO4

300mM NaCl 500mM imidazole pH 80) were applied toelute the recombinant catalase The purity of catalase wasconfirmed by SDS-PAGE The purified catalase was placedinto a dialysis bag and dialyzed against Buffer A to remove thehigh concentrations of imidazole BufferAwas replaced everyseveral hours until the imidazole was removed The quantity


of purified catalase was assayed using the Bradford method[24]

26 Recombinant Catalase Characteristics A catalase activityassay was performed according to the method described byBeers Jr and Sizer [25] Purified recombinant catalase wasdiluted several times with PBS (20mM pH 70) to a suitableconcentration and 27mL of 30mM H

2O2(diluted with

20mM PBS pH 70) was then immediately added to 300120583Lof diluted enzyme The absorbance changes at 240 nm weremeasured every 5 s for 1min 1 U of activity was defined asthe amount of enzyme to decompose 1120583mol ofH

2O2permin

Each measurement was performed at least three timesTo assess the optimal reaction temperature 30mMH

2O2

was preincubated at different temperatures from 10∘C to90∘C (in 10∘C increment) and then activities at differenttemperatures were measured with above preincubated H

2O2

To assess enzyme thermostability diluted enzyme wasincubated at 40 50 60 70 80 and 90∘C for 30min and1 hour After the incubation time the heated enzyme wasimmediately transferred onto ice Residual activities weremeasured with method described above

The optimum pH was assayed with 30mM H2O2pre-

pared in the following 50mM buffers sodium citrate 50mM(pH 4ndash6) sodium phosphate 50mM (pH 6ndash8) and glycine-NaOH 50mM (pH 9ndash11) Activities at different pH valueswere measured as above in least three replicates

Kinetic parameters were assayed by calculating the activ-ity of the enzyme under different concentrations of H

2O2

ranging from 25 to 25mM in 20mM PBS (pH 70) at 30∘C119870119898

was obtained by double-reciprocal plots according toLineweaver and Burk [26]


31 Kat Sequence The PCR product (Figure 1) and sequenc-ing results showed that the total length of the Kat gene was1467 bp which encoded a protein with 488 amino acidsNucleotide sequence was submitted to GenBank and Gen-Bank accession number KP202252 was assigned blastp wasrun in the nonredundant protein sequences ofNCBI databasefor homologs of the amino acid sequence The aminosequence had higher similarity with the catalase sequencesof other Geobacillus spp however all of these highly similarKat genes were submitted in the form of sequenced genomesand none had been previously cloned or expressed Amongall studied catalase sequences obtained from Swiss Protdatabase this catalase was most similar to that of a Bacillussp [27] and its identity was 80 Phylogenetic tree (Figure 2)based on the amino acid sequences similarity showed thiscatalase shared closest phylogenetic relationshipwith catalaseof Bacillus subtilis and Lactobacillus sakei in Swiss Protdatabase Multiple alignment of the amino acid sequenceshowed this catalase has seven typically conserved amino acidresiduals for heme binding (Figure 3) All of the reportedthermostable catalases [28 29] from Geobacillus stearother-mophiluswere different from this catalase herein because theybelonged to the second group of catalase family (catalase-peroxidase) and were encoded by Per gene but catalase in

M 1

(bp)

100

250

500

7501000

2000

Figure 1 PCR product showing a 1452-length band that corre-sponds to Kat from aGeobacillus sp CHB1 Lanes M DNAmarkerLane 1 PCR product

this paper belonged to the first group (monofunctional hemecatalase) which was encoded by gene Kat [5] Therefore toour knowledge this study was the first to clone and expressthis type of Geobacillus spp Kat gene

32 Construction of Recombinant Expression Vector pEASY-E2-Kat A positive clone was selected and its sequence wasconfirmed by sequencing The expression vector pEASY-E2-Kat (Figure 4) was transformed into BL21 (DE3) cells ThenSDS-PAGE was applied to confirm whether the catalase wasexpressed and to determine the molecular weight of theenzyme A band was observed between 60 and 70 kDa andthe recombinant catalase was highly soluble (make up to 30of the total E coli protein) with a low inclusion body content(Figure 5) The theoretical molecular weight of the recombi-nant enzyme herein calculated using its amino sequence was56 kDa including the 6timesHis tag which conflicted with themolecular weight of 65 kDa observed via SDS-PAGE Somestudies [30] have found that the 6timesHis tag might result in arecombinant protein with a greater molecular weight how-ever the reason for this phenomenon remains unclear Thespecific activity of the purified catalase was 40526 Umgunder optimal conditions (60∘C pH 70)

33 Production Curve for Recombinant Catalase Productioncurves for the recombinant catalase were constructed at 30∘CWhen cultured at 30∘C for 20 h the total activity of thefermentation broth reached a maximum level of 35831 UmLassayed under optimal conditions (Figure 6) This level wasmuch higher than many catalase production strains and onlylower than recombinant Bacillus sp WSHDZ-01 (Table 1) aswe knowHigh production ensured further application of thiscatalase


Rattus norvegicus (P047623)

Canis lupus familiaris (O974923)


Cavia porcellus (Q644054)

Rugosa rugosa (Q9PWF73)

Ascaris suum (P906822)

Toxoplasma gondii (Q9XZD51)

Dictyostelium discoideum (O772292)

Methanosarcina barkeri (O936621)

Bacillus subtilis (P269015)

Geobacillus sp B1 (AJO270321)

Lactobacillus sakei (P302652)

Streptomyces venezuelae (P33569)

Streptomyces coelicolor (Q9Z5981)

Wolbachia endosymbiont (Q277101)

Pseudomonas putida (Q597141)

100

100

100

85

51

97

91

99

99

100

98

8665

005

Figure 2 Phylogenetic tree of CHB1 catalase amino acid showing the relationship with other strains on catalase amino acid sequence Proteinsequences were selected by running blastp program with CHB1 catalase sequence in Swiss Prot database accession numbers in brackets werethe corresponding accession numbers of the strains in Swiss Prot database Phylogenetic tree was constructed with MEGA 402 with themethod Neighbor-Joining Test of inferred phylogeny was Bootstrap for 1000 replications

Table 1 Catalase production levels of recombinant E coli BL21 andother strains

Strains Activity(UmL) Reference

Recombinant E coli BL21 (DE3) 35831 This studyRecombinant Bacillus spWSHDZ-01 39117 [15]

Bacillus sp WSHDZ-01 28990 [19]Exiguobacterium oxidotoleransT-2-2T 22000 [20]

Serratia marcescens SYBC08 20353 [21]Rhizobium radiobacter strain 2-1 17035 [22]M luteus 6920 [22]

34 Purified Recombinant Catalase Characteristics

341 Effect of Temperature and pH onThis Catalase Temper-ature is a major factor in the application of many enzymesThe activity and stability of our purified catalase at differenttemperatures were assayedThe catalase in this study showeda high activity over awide range of temperatures from 10∘C to90∘C (Figure 7(a)) and showed maximum activity at 60∘C to70∘CThe temperature rangewaswider and optimal tempera-turewas higher thanmany reported catalases such as Serratiamarcescens SYBC08 at temperatures ranging from 0∘C to

70∘C with optimal temperature of 20∘C [31] Psychrobacterpiscatorii T-3 from 10∘C to 60∘C with optimal temperatureof 45∘C [32] and Bacillus altitudinis SYBC hb4 from 20∘Cto 40∘C with optimal temperature of 30∘C [33] This catalasewas stable at temperatures le60∘C (Figure 7(b)) and whenincubated at 70∘C for 1 h the enzyme maintained gt70residual activity however when the incubation temperaturewasgt80∘C the residual activity waslt10 after incubation for1 h Although this enzyme was inactive at ge80∘C it still main-tained high activity when added to reaction systems at 80∘Cand 90∘C Thermophilic catalase isolated from thermophilicThermoascus aurantiacus [34] can retain 100 residual activ-ity after incubation at 60∘C for 1 h whichwas similar to that ofCHB1 catalase This thermophilic property was much betterthan the properties of other catalases such as those fromPsychrobacter piscatorii (P piscatorii) (65∘C 15min 20)[32 35] Vibrio salmonicida (60∘C 20min 0) [36] Vibriorumoiensis S-1T (65∘C 10min 0) [37] and Halomonas spSK1 (55∘C 30min 0) [38] These properties enable theenzyme to be applied in both low and high-temperature pro-cesses in industryThe optimal pH of the enzymewas 6-7 andthe enzyme retained gt50 of its activity between pH 5 and9 (Figure 8)

342 Kinetic Parameters of This Catalase The enzymekinetics of the recombinant catalase were assayed using


96P26901100O9366296P3356997AJO270321

Consensus

M

G

E

KM

MNMA

SSTD

SSQT

NKGK

KVPKl

LLLLt

TTTTt

TTTT

SGES

WFAWg

GGGG

AIAAp

PPPPv

VVVV

GGAGd

DDDD

NDNNq

QQQQn

NNNNs

SSSS

MLEIt

TTTTa

AAAAg

GGGG

SNVN

RRGPg

GGGGp

PPPP

TVVTl

LLLL

IMVIq

QQQQd

DDDD

VVQV

HHLHl

LLLL

LLLI

EDEEk

KKKKl

LLLL

ASAAh

HHHHf

FFFF

NDNN

RHRRe

EEEEr

RRRR

VIIVp

PPPPe

EEEEr

RRRRv

VVVVv

VVVVh

HHHHa

AAAA

KKRKg

GGGGa

AAAAg

GGGGa

AAAA

HGYHg

GGGG

YYTYf

FFFF

EETE

VVLVt

TTTT

NARNd

DDDD

VVVM

TTSS

KKRK

YYWYt

TTTT

KKRKa

AAAA

AKAK

FFFV

LLLF

SSSN

EEEG

VIVVg

GGGGk

KKKKr

RRRRt

TTTT

PEEP

LVTVf

FFFF

IVLVr

RRRRf

FFFFs

SSSSt

TTTTv

VVVV

AGAA

196P26901200O93662196P33569197AJO270321

Consensus g

GGGG

EESE

LKLLg

GGGG

SSASa

AAAAd

DDDD

TSAT

VAVVr

RRRRd

DDDDp

PPPPr

RRRRg

GGGG

FFWFa

AAAA

VVLVk

KKKKf

FFFFy

YYYYt

TTTTe

EEEE

EDEEg

GGGGn

NNNNy

YYYYd

DDDD

ILLIv

VVVVg

GGGGn

NNNNn

NNNNt

TTTTp

PPPP

VVVIf

FFFFf

FFFFi

IIII

RRKRd

DDDD

APAA

ILIIk

KKKKf

FFFFp

PPPPd

DDDDf

FFFFi

IIIIh

HHHHt

TTTTq

QQQQk

KKKKr

RRRR

DNDDp

PPPP

KAYRt

TTTT

HNGH

LCSL

KKQK

NDEN

PPAP

TDDT

AMNA

VFVMw

WWWWd

DDDDf

FFFF

WLWW

SSGSl

LLLL

STSSp

PPPPe

EEEEs

SSSS

LITLh

HHHHq

QQQQv

VVVVt

TTTT

IIWYl

LLLL

MFFF

SSGGd

DDDDr

RRRRg

GGGG

ITIIp

PPPP

AAAL

TTST

LYYYr

RRRR

HNHHm

MMMM

HNNNg

GGGG

FYYY

GSGGs

SSSS

296P26901300O93662296P33569297AJO270321

Consensus h

HHHHt

TTTT

FYYF

KKQKw

WWWW

TYNVn

NNNN

AEEE

EKAKg

GGGGe

EEEE

GYVA

VFFVw

WWWW

IVVV

KQKKy

YYYYh

HHHHf

FFFFk

KKKKt

TTTT

EDDNq

QQQQg

GGGG

VIIVk

KKKKn

NNNN

LLLM

DTTD

VLQP

NEDE

TEELa

AAAA

AENV

KKRK

IILI

AGAAg

GGGG

ESEE

NDDNp

PPPPd

DDDD

YHSY

HAHH

TTQT

ERREd

DDDDl

LLLL

FYRY

NEENa

AAAAi

IIII

EKEE

NKRKg

GGGGd

DDDD

YYFYp

PPPP

ASTSw

WWWW

KTTT

LLVL

YEQY

VMVVq

QQQQi

IIIIm

MMMM

PTPP

LPAL

EEAE

DQDDa

AAAA

NEAK

TDGTy

YYYYr

RRRRf

FFFF

DDNN

PIPP

FRFFd

DDDD

VILVt

TTTTk

KKKKv

VVVVw

WWWW

SPPS

QHHH

KGEKd

DDDD

YFYYp

PPPP

LTPL

IMVI

EKEE

VIIVg

GGGG

RKTR

MLLM

VVEV

395P26901400O93662395P33569396AJO270321

Consensus l

LLLL

DNNNr

RRRRn

NNNNp

PPPP

ETEEn

NNNN

YYIYf

FFFFa

AAAAe

EEEEv

VVVVe

EEEEq

QQQQ

AASA

TAITf

FFFFs

SSSSp

PPPP

GAAG

TNHN

LLFLv

VVVVp

PPPPg

GGGG

IIIV

DGGE

VIPPs

SSSSp

PPPPd

DDDDk

KKKKm

MMMMl

LLLLq

QQQQ

GGGAr

RRRR

LVLLf

FFFF

ASAAy

YYYY

HHGAd

DDDD

ATAAh

HHHH

RIRR

YHYYr

RRRR

VLVVg

GGGG

APIVn

NNNN

HYAH

QNDN

ALHL

LILLp

PPPP

IVVIn

NNNN

RARR

APPP

RKHR

NYAV

KSTE

VPEV

NEAN

NNRN

ST

YYHY

QQSQr

RRRRd

DDDDg

GGGG

QFFF

MMLM

RRR

FVYFd

DDDD

DAGN

NNRN

GGHG

GGKG

GSGG

SGAS

VPKV

YNNNy

YYYY

EWEEp

PPPPn

NNNNs

SSSSf

FFFFg

GGGGg

GGGGp

PPPP

KSVT

EPQE

SDTV

PSDS

EVRE

483P26901497O93662483P33569488AJO270321

Consensus

DYPH

KLLK

QEWT

APQT

APPP

YFTF

PGPPv

VVVV

QSTSg

GGGG

ILVM

AATA

G

DADE

SRHS

VTAV

SLAP

YYPY

DTSD

HHHD

PA

E

YNDDd

DDDD

HDDH

YFFY

TVTTq

QQQQa

AAAAg

GGGG

DNDDl

LLLLy

YYYYr

RRRR

D

LVLLm

MMMM

STSS

EDEE

DYDE

EDEE

RRKK

TEGA

RNRRl

LLLL

VVIV

EGDKn

NNNN

IILI

VVSV

NSGE

AHFS

MLIL

KSAK

PAKQ

VAVV

EQST

KKRK

ERDE

EDEi

IIII

KQAK

LLELr

RRRR

QQAQ

ITII

EAGR

HLNHf

FFFF

YFRY

KKRKa

AAAAd

DDDD

PREP

EDDD

YYFYg

GGGG

KSKRr

RRRR

VVLV

AAEA

EKAE

GGAG

LLVL

GEQG

LLAL

PDLQ

IIRV

KKGP

KED

DVD

SEV

RI

LT

AN

NA

M

T

N

E

483P26901504O93662483P33569488AJO270321

Consensus

E

R

A

R

A

T

E

Figure 3 Multiple alignment of amino acid sequences for CHB1 catalase and catalase from Swiss Prot database The sequences were thosefromBacillus subtilis subsp str 168 (Swiss Prot number P26901)Methanosarcina barkeri str Fusaro (Swiss Prot numberO93662) StreptomycesvenezuelaeATCC 10712 (Swiss Prot number P33569) andGeobacillus sp CHB1 (GenBanknumberAJO270321) Identical amino acid residualswere shaded in black and conserved residuals were shaded in gray Arrows showed the conserved residuals of home binding pocket of thiskind of catalase

6860bppEASY-E2-Kat 6 times His

RBS

f1Ori

Amp

pBR322 ori

LacI

T7Kat

Figure 4 Recombinant expression construction of vector pEASY-E2-kat A schematic diagram of recombinant expression vectorpEASY-E2-kat The kat gene encoding catalase was inserted intoan expression vector with upstream RBS and T7 promoters anddownstream 6timesHis

different H2O2concentrations as substrates a Lineweaverndash

Burk plot was used to calculate 119870119898 511mM and 119881max

1515molminsdotmg (Figure 9) which is similar to previously

M 1 2 3 4

25

70

200

85120

5060

40

30

20

15

(kDa)

Figure 5 SDS-PAGE analyses of recombinant catalase expressionand the purified enzyme Lanes M protein marker Lane 1 totalbacterial protein of BL21 (DE3) cells with empty vector pEASY-E2 Lane 2 insoluble bacterial proteins of BL21 (DE3) with vectorpEASY-E2-kat after induced expression Lane 3 soluble bacterialproteins of BL21 (DE3) with vector pEASY-E2-kat after inducedexpression Lane 4 SDS-PAGE analysis of purified catalase using aNi-IDA column


0 10 20 30

Time (h)

40000

30000

20000

10000

0

Cata

lase

activ

ity (U

mL)

Figure 6 Catalase production curve of the recombinant E coli BL21 (DE3)

100806040200

Temperature (∘C)

70

80

90

100

110

120

Rela

tive a

ctiv

ity (

)

(a)

150

100

50

010050

Time (h)

Resid

ual a

ctiv

ity (

)

40∘C50∘C60∘C

70∘C80∘C90∘C

(b)

Figure 7 Effect of temperature on recombinant catalase activity (a) Relative activity of the enzyme at 10 20 30 40 50 60 70 80 and 90∘C(b) temperature stability of the enzyme Residual activity of the recombinant enzyme by incubation at 40 50 60 70 80 and 90∘C for 05 and1 h

reported values (525mM [23] and 401mM [2]) Many othercatalases from P piscatorii T-3 Micrococcus luteus Bac-teroides fragilis Helicobacter pylori Serratia marcescens andXanthomonas campestris have higher119870

119898values compared to

the purified catalase [39]The relatively low119870119898indicates that

the enzymehas a high affinity forH2O2and that the enzyme is

stable under high H2O2concentrations 119870

119898is an important

characteristic of enzymes and is vital for the assessment oftheir potential applications

4 Conclusions

In this paper we cloned expressed purified and character-ized aKat gene of thermophilic bacteriaGeobacillus sp CHB1

for the first time The recombinant enzyme could maintainits stability and showed a high activity over a wide rangeof temperatures from 10∘C to 90∘C Specific activity of thepurified recombinant catalase was 40526Umg of proteinand recombinant E coli BL21 strain reached a high level ofcatalase production to 35831UmLThis enzymersquos wide rangeof reaction temperatures good thermostability and highproductionmay be suitable for the textile paper-making andother industries

Competing Interests

The authors declare no conflict of interests

BioMed Research International 7Re

lativ

e act

ivity

()

150

100

50

0

4 5 6 7 8 9 10

pH11

Figure 8 Optimal pH of the recombinant catalaseThe buffers usedfor each pH region were 50mM sodium citrate (pH 4ndash6) 50mMsodium phosphate (pH 6ndash8) and 50mM glycine-NaOH (pH 9ndash11)Each pH was assayed at least three times

minus005

0

005

01

015

02

0 01 02 03 04 05minus01

1s (mmolL)minus1

R2 = 09972y = 03393x + 00066

1v

(min

120583m

ol)middot1

03

Figure 9 A LineweaverndashBurk plot of the recombinant catalaseEnzyme activity was assayed in 50mM phosphate buffer (pH 70)and at 30∘C

Acknowledgments

This work was supported by the Argoscientific Research inthe Public Interest (201303094-05) and Fujian Finance [2013]no 1464

References

[1] H-S Ko H Fujiwara Y Yokoyama et al ldquoInducible produc-tion of alcohol oxidase and catalase in a pectin medium byThermoascus aurantiacus IFO 31693rdquo Journal of Bioscience andBioengineering vol 99 no 3 pp 290ndash292 2005

[2] H-W Zeng Y-J Cai X-R Liao S-L Qian F Zhang and D-B Zhang ldquoOptimization of catalase production and purifica-tion and characterization of a novel cold-adapted Cat-2 frommesophilic bacterium Serratia marcescens SYBC-01rdquo Annals ofMicrobiology vol 60 no 4 pp 701ndash708 2010

[3] H D Rowe ldquoBiotechnology in the textileclothing industrymdasha reviewrdquo Journal of Consumer Studies amp Home Economics vol23 no 1 pp 53ndash61 1999

[4] X Fu W Wang J Hao X Zhu and M Sun ldquoPurification andcharacterization of catalase frommarine bacteriumAcinetobac-ter sp YS0810rdquo BioMed Research International vol 2014 ArticleID 409626 7 pages 2014

[5] B S Sooch B S Kauldhar and M Puri ldquoRecent insights intomicrobial catalases isolation production and purificationrdquoBio-technology Advances vol 32 no 8 pp 1429ndash1447 2014

[6] M Spiro and W P Griffith ldquoThe mechanism of hydrogenperoxide bleachingrdquo Textile Chemist and Colorist vol 29 no11 pp 12ndash13 1997

[7] M Kagawa N Murakoshi Y Nishikawa et al ldquoPurificationand cloning of a thermostable manganese catalase from a ther-mophilic bacteriumrdquo Archives of Biochemistry and Biophysicsvol 362 no 2 pp 346ndash355 1999

[8] A Hidalgo L Betancor R Moreno et al ldquoThermus ther-mophilus as a cell factory for the production of a thermophilicMn-dependent catalase which fails to be synthesized in anactive form in Escherichia colirdquo Applied and EnvironmentalMicrobiology vol 70 no 7 pp 3839ndash3844 2004

[9] S Ebara and Y Shigemori ldquoAlkali-tolerant high-activity cata-lase from a thermophilic bacterium and its overexpression inEscherichia colirdquo Protein Expression and Purification vol 57 no2 pp 255ndash260 2008

[10] M Gudelj G O Fruhwirth A Paar et al ldquoA catalase-peroxidase from a newly isolated thermoalkaliphilic Bacillus spwith potential for the treatment of textile bleaching effluentsrdquoExtremophiles vol 5 no 6 pp 423ndash429 2001

[11] X-L Shi M-Q Feng J Shi Z-H Shi J Zhong and P ZhouldquoHigh-level expression and purification of recombinant humancatalase in Pichia pastorisrdquo Protein Expression and Purificationvol 54 no 1 pp 24ndash29 2007

[12] G Gellissen M Piontek U Dahlems et al ldquoRecombinantHansenula polymorpha as a biocatalyst coexpression of thespinach glycolate oxidase (GO) and the S cerevisiae catalase T(CTT1) generdquo Applied Microbiology and Biotechnology vol 46no 1 pp 46ndash54 1996

[13] T Rochat A Miyoshi J J Gratadoux et al ldquoHigh-levelresistance to oxidative stress in Lactococcus lactis conferred byBacillus subtilis catalase KatErdquo Microbiology vol 151 no 9 pp3011ndash3018 2005

[14] X Shi M Feng Y Zhao X Guo and P Zhou ldquoOverexpressionpurification and characterization of a recombinant secretarycatalase from Bacillus subtilisrdquo Biotechnology Letters vol 30 no1 pp 181ndash186 2008

[15] S Xu Y Guo G Du J Zhou and J Chen ldquoSelf-cloningsignificantly enhances the production of catalase in Bacillussubtilis WSHDZ-01rdquo Applied Biochemistry and Biotechnologyvol 173 no 8 pp 2152ndash2162 2014

[16] H Luo Y Zhou Y H Chang L Xiong and L Z Liu ldquoRapidgene cloning overexpression and characterization of a ther-mophilic catalase in E colirdquo Advanced Materials Research vol365 pp 367ndash374 2012

[17] F W Studier ldquoProtein production by auto-induction in highdensity shaking culturesrdquo Protein Expression and Purificationvol 41 no 1 pp 207ndash234 2005

[18] J Zhou M A Bruns and J M Tiedje ldquoDNA recovery fromsoils of diverse compositionrdquoApplied and EnvironmentalMicro-biology vol 62 no 2 pp 316ndash322 1996

[19] Y Deng Z Hua Z Zhao D Yao G Du and J Chen ldquoEffectof nitrogen sources on catalase production by Bacillus subtilisWSHDZ-01rdquo Chinese Journal of Applied and EnvironmentalBiology vol 14 no 4 pp 544ndash547 2008


[20] Y Hanaoka and I Yumoto ldquoManipulation of culture conditionsfor extensive extracellular catalase production by Exiguobac-teriumoxidotoleransT-2-2TrdquoAnnals ofMicrobiology vol 65 pp1183ndash1187 2014

[21] H-W Zeng Y-J Cai X-R Liao et al ldquoSerratia marcescensSYBC08 catalase isolated from sludge containing hydrogenperoxide shows increased catalase production by regulation ofcarbon metabolismrdquo Engineering in Life Sciences vol 11 no 1pp 37ndash43 2011

[22] M Nakayama T Nakajima-Kambe H Katayama K HiguchiY Kawasaki and R Fuji ldquoHigh catalase production by Rhizo-bium radiobacter strain 2-1rdquo Journal of Bioscience and Bioengi-neering vol 106 no 6 pp 554ndash558 2008

[23] V Neuhoff N Arold D Taube and W Ehrhardt ldquoImprovedstaining of proteins in polyacrylamide gels including isoelectricfocusing gels with clear background at nanogram sensitivityusing Coomassie Brilliant Blue G-250 and R-250rdquo Electrophore-sis vol 9 no 6 pp 255ndash262 1988

[24] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[25] R F Beers Jr and I W Sizer ldquoA spectrophotometric methodformeasuring the breakdownof hydrogen peroxide by catalaserdquoThe Journal of Biological Chemistry vol 195 no 1 pp 133ndash1401952

[26] H Lineweaver and D Burk ldquoThe determination of enzyme dis-sociation constantsrdquo Journal of the American Chemical Societyvol 56 no 3 pp 658ndash666 1934

[27] D K Bol and R E Yasbin ldquoThe isolation cloning and identifi-cation of a vegetative catalase gene from Bacillus subtilisrdquo Genevol 109 no 1 pp 31ndash37 1991

[28] F Wang Z Wang W Shao J Liu C Xu and J Zhuge ldquoPurifi-cation and properties of thermostable catalase in engineered EcolirdquoActamicrobiologica Sinica vol 42 no 3 pp 348ndash353 2002

[29] S Loprasert S Negoro and H Okada ldquoCloning nucleotidesequence and expression in Escherichia coli of the Bacillusstearothermophilus peroxidase gene (perA)rdquo Journal of Bacteri-ology vol 171 no 9 pp 4871ndash4875 1989

[30] Y Zhong X-H Zhang J Chen et al ldquoOverexpression purifi-cation characterization and pathogenicity of Vibrio harveyihemolysin VHHrdquo Infection and Immunity vol 74 no 10 pp6001ndash6005 2006

[31] H-W Zeng Y-J Cai X-R Liao F Zhang and D-B ZhangldquoProduction characterization cloning and sequence analysis ofa monofunctional catalase from Serratia marcescens SYBC08rdquoJournal of Basic Microbiology vol 51 no 2 pp 205ndash214 2011

[32] H Kimoto K Yoshimune H Matsuyma and I YumotoldquoCharacterization of catalase from psychrotolerant Psychrobac-ter piscatorii T-3 exhibiting high catalase activityrdquo InternationalJournal of Molecular Sciences vol 13 no 2 pp 1733ndash1746 2012

[33] Y Zhang X Li R Xi Z Guan Y Cai and X Liao ldquoCharac-terization of an acid-stable catalase KatB isolated from Bacillusaltitudinis SYBC hb4rdquo Annals of Microbiology vol 66 no 1 pp131ndash141 2016

[34] H Wang Y Tokusige H Shinoyama T Fujii and T UrakamildquoPurification and characterization of a thermostable catalasefrom culture broth of Thermoascus aurantiacusrdquo Journal ofFermentation and Bioengineering vol 85 no 2 pp 169ndash1731998

[35] W Wang M Sun W Liu and B Zhang ldquoPurification andcharacterization of a psychrophilic catalase from Antarctic

Bacillusrdquo Canadian Journal of Microbiology vol 54 no 10 pp823ndash828 2008

[36] M S Lorentzen EMoeHM Jouve andN PWillassen ldquoColdadapted features ofVibrio salmonicida catalase characterisationand comparison to the mesophilic counterpart from Proteusmirabilisrdquo Extremophiles vol 10 no 5 pp 427ndash440 2006

[37] I Yumoto D Ichihashi H Iwata et al ldquoPurification and char-acterization of a catalase from the facultatively psychrophilicbacterium Vibrio rumoiensis S-1T exhibiting high catalaseactivityrdquo Journal of Bacteriology vol 182 no 7 pp 1903ndash19092000

[38] K Phucharoen K Hoshino Y Takenaka and T ShinozawaldquoPurification characterization and gene sequencing of a cata-lase from an alkali- and halo-tolerant bacteriumHalomonas spSK1rdquo Bioscience Biotechnology and Biochemistry vol 66 no 5pp 955ndash962 2002

[39] J Switala and P C Loewen ldquoDiversity of properties amongcatalasesrdquo Archives of Biochemistry and Biophysics vol 401 no2 pp 145ndash154 2002

Research ArticleLaboratory Prototype of Bioreactor for Oxidation ofToxic D-Lactate Using Yeast Cells Overproducing D-LactateCytochrome c Oxidoreductase

Maria Karkovska Oleh Smutok and Mykhailo Gonchar

Department of Analytical Biotechnology Institute of Cell Biology Drahomanov Street 1416 Lviv 79005 Ukraine

Correspondence should be addressed to Oleh Smutok smutokcellbiollvivua

Received 25 April 2016 Accepted 7 June 2016


Copyright copy 2016 Maria Karkovska et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

D-lactate is a natural component of many fermented foods like yogurts sour milk cheeses and pickles vegetable productsD-lactate in high concentrations is toxic for children and people with short bowel syndrome and provokes encephalopathyThese facts convincingly demonstrate a need for effective tools for the D-lactate removal from some food products The mainidea of investigation is focused on application of recombinant thermotolerant methylotrophic yeast Hansenula polymorphaldquotr6rdquo overproducing D-lactate cytochrome c oxidoreductase (EC 1124 D-lactate cytochrome c oxidoreductase D-lactatedehydrogenase (cytochrome) DLDH) In addition to 6-fold overexpression of DLDH under a strong constitutive promoter(prAOX) the strain ofH polymorpha ldquotr6rdquo (gcr1 catXΔcyb2 prAOX DLDH) is characterized by impairment in glucose repressionof AOX promoter devoid of catalase and L-lactate-cytochrome 119888 oxidoreductase activities Overexpression of DLDH coupling withthe deletion of L-lactate-cytochrome 119888 oxidoreductase activity opens possibility for usage of the strain as a base for constructionof bioreactor for removing D-lactate from fermented products due to oxidation to nontoxic pyruvate A laboratory prototype ofcolumn-type bioreactor for removing a toxic D-lactate from model solution based on permeabilized cells of the H polymorphaldquotr6rdquo and alginate gel was constructed and efficiency of this process was tested

1 Introduction

D-lactate is a natural component of many fermented foodslike yogurts [1 2] sourmilk [3] cheeses [4] and pickles vege-table products [5]

D-lactate can be metabolized slowly in the human bodyin comparison with L-lactate isomer and can cause metabolicdisorders if ingested in excess Therefore the World HealthOrganization (WHO) recommends a limited daily consump-tion of D-lactate up to 100mgsdotkgminus1 bodyweight Moreoversome countries for example Germany have decided to min-imize the D-lactate content of fermented dairy products [6]The high concentration of D-lactate is toxic for children [7]and people with short bowel syndrome [8ndash10] Also D-lactatetoxicity depends on physiological state of renal excretionHingorani et al demonstrated that in healthy men continu-ously infusedwith aD-lactate solution excretion rates ranged

between 61 and 100 [11] In 1993 there was reported a case ofchronic D-lactate encephalopathy occurring in a patient withshort bowel syndrome and end-stage renal disease [12]

Originally it was believed that due to the lack of enzymeD-lactate dehydrogenase in humans they are not able tometabolize D-lactate to pyruvate However in the past twodecades there is an abundance of literature suggesting thepresence of a D-lactate metabolizing enzyme D-2-hydroxyacid dehydrogenase (D-2-HDH) that is mainly found inthe liver and kidney [10 13] This enzyme is inhibited bylow-pH states which assumes importance in the relativeoverproduction of D-lactate in certain clinical situations D-lactic acidosis or D-lactate encephalopathy is a rare conditionthat occurs primarily in individuals who have a history ofshort bowel syndrome (SBS)

Thepredominant organ systemaffected byD-lactic acido-sis is the central nervous system (CNS) Presenting symptoms



may include slurred speech ataxia altered mental statuspsychosis or even coma [8 9 14 15]

The aim of the current work is construction of aneffective laboratory prototype of bioreactor for removing D-lactate from fermented products based on the use of yeastcells overproducing D-lactate oxidoreductase The D-lactateoxidoreductase (cytochrome c-dependent dehydrogenaseDLDH EC1124) is a FAD- andZn2+-containingmembrane-associated protein found in yeast and bacteria The enzymecatalyzes D-lactate oxidation to pyruvate coupled with ferri-cytochrome c reduction to ferrocytochrome 119888 It is charac-terized as a mitochondrial protein with a molecular weightof 63 kDa for DLDH from Kluyveromyces lactis and 64 kDafor DLDH from Saccharomyces cerevisiae respectively [1617] DLDH is highly selective to D-lactate however DL-120572-hydroxybutyric acid can be used as an alternative electrondonor The enzyme is not selective with respect to electronacceptors and can reduce in addition to its native acceptor(cytochrome c) different lowmolecular artificial redoxmedi-ators (ferricyanide dichlorphenol indophenols etc)

We propose using recombinant DLDH-overproducingstrainH polymorpha ldquotr6rdquo (gcr1 catXΔcyb2 prAOX DLDH)as a base for construction of laboratory prototype of bioreac-tor for removing a toxic D-lactate from fermented productsThe column-type bioreactor includes alginate gel formationswith incorporated permeabilized yeast cells

2 Material and Methods

21 Materials Sodium D-lactate sodium alginate and phe-nylmethylsulfonyl fluoride (PMSF) were purchased fromSigma-Aldrich Corp (Deisenhofen Germany) and cetyltri-methylammonium bromide (CTAB) was purchased fromChemapol Sp (Bratislava Slovakia) D(+)-glucose monohy-dratewas purchased from J T Baker (DeventerNetherlands)(NH4

)2

SO4

Na2

HPO4

KX2

`4

MgSO4

and CaCl2

wereobtained from Merck (Darmstadt Germany) All chemicalsand reagents were of analytical grade and all solutions wereprepared using deionized water D-lactate standard solutionand appropriate dilutions were prepared in 100mM phos-phate buffer (PB) pH 78

22 Cultivation and Permeabilization of Yeast Cells Recom-binant DLDH-overproducing strain H polymorpha ldquotr6rdquo(gcr1 catXΔcyb2 prAOX DLDH) is characterized by 6-foldoverexpression of DLDH impaired in glucose repressiondevoid of catalase and specific L-lactate-cytochrome c oxi-doreductase activities which was constructed by us earlier[18]

Cultivation of the H polymorpha ldquotr6rdquo was performedin flasks on a shaker (200 rpm) at 28∘C until the middle ofthe stationary growth phase (sim52 h) in a medium containing(gsdotLminus1) (NH

4

)2

SO4

35 KH2

PO4

10 and MgSO4

times 7H2

O05 supplemented with 075 yeast extract A mixture ofglucose (10 gsdotLminus1) andD-lactate (2 gsdotLminus1) was used as a carbonand energy source and (1 gsdotLminus1) methanol as an inductor forAOX promoter After washing the cells were suspended in50mM PB pH 78 containing 1mM PMSF

Before experiments the freshly prepared yeast cells wereresuspended to 30mgsdotmLminus1 in 50mM PB pH 78 and storedin refrigerator

The procedure of the cells permeabilization was as fol-lows the same volume of permeabilizing reagent (085mMCTAB) was added to the cell suspension (30mgsdotmLminus1 in50mMPB pH 78)The resulting solution was treated at 30∘Cin a water bath for 15min under mixing every 3-4min Thepermeabilized cells were washed by centrifugation (6000timesg5min) in 10mM PB pH 78 The precipitated permeabilizedcells were resuspended to 30mgsdotmLminus1 in the same buffersolution and stored at +4∘C A half-life of the permeabilizedyeast cellswas about threeweeks of storage at such conditions

23 Assay of DLDH Activity in Permeabilized Cells One unit(1 U) of the DLDH activity is defined as that amount of theenzyme which forms 1 120583mol hexacyanoferrate(II) per minuteunder standard conditions of the assay (20∘C 30mMPB andpH 78) Activity was estimated by spectrophotometric moni-toring of hexacyanoferrate(III) reduction at 120582 = 420 nm [19]During this process optical density of the analyzed solutionbecomes lower Assaymixture consisted of 30mMPB aX 7816mM sodium D-lactate 083mM P

3

Fe(CN)6

and 20120583L ofdiluted permeabilized cell suspension (30mgsdotmLminus1)

The specific activity of DLDH was calculated by thefollowing formula

SA = Δ119864min sdot 119881 sdot 119899119864mM sdot 119862cells sdot 119881cells

(1)

where Δ119864min is change of optical density at 120582 = 420 nm permin 119881 is total volume of the assay solution mL 119899 is dilutionof the enzyme before assay 119864mM is millimolar extinction ofhexacyanoferrate(III) 104mMminus1sdotcmminus1 119881cells is volume ofthe added permeabilized cell suspension mL and 119862cells ispermeabilized cell concentration mgsdotmLminus1

Specific DLDH activity in the cells was calculated by thefollowing formula considering difference between specificDLDH activity (+D-lactate) and nonspecific ferrireductaseactivity (without D-lactate) 119860 = 119860

+D-lact minus 119860minusD-lact


The possibility of usage of DLDH-producer as a perspectivetool for D-lactate removing from fermented products wasinvestigated For analysis of D-lactate utilization ability of therecombinant thermotolerant methylotrophic yeastHansenulapolymorpha ldquotr6rdquo (gcr1 catXΔcyb2 prAOX DLDH) con-structed by us earlier overproducing DLDH was chosenThe H polymorpha DLDH-producer was constructed in twosteps First the gene CYB2 was deleted on the backgroundof the b-105 (gcr1 catX) strain of H polymorpha impairedin glucose repression and devoid of catalase activity to avoidspecific L-lactate-cytochrome c oxidoreductase activitySecond the homologous gene DLD1 coding for DLDH wasoverexpressed under the control of the strongH polymorphaalcohol oxidase promoter in the frame of a plasmid for multi-copy integration in the Δcyb2 strain The selected recombi-nant strain possesses 6-fold increased DLDH activity as


Table 1 The residual D-lactate concentration by Buchner methodin the samples selected using two types of the H polymorpha ldquotr6rdquocells analyzed

Time ofincubation min

D-lactate mV(permeabilized cells)

D-lactate mV(intact cells)

0 200 plusmn 000 200 plusmn 00010 193 plusmn 015 1987 plusmn 01420 155 plusmn 02 185 plusmn 01830 119 plusmn 023 1551 plusmn 02540 1198 plusmn 028 1448 plusmn 02950 125 plusmn 030 145 plusmn 022

0 10 20 30 40 500

5

10

15

20

25

30

35

40

45

50

Time of incubation (min)

o

xida

tion

of D

-lact

ate

(b)

(a)

Figure 1 The profile of D-lactate oxidation using recombinantH polymorpha ldquotr6rdquo cells (a) permeabilized and (b) living cellsConditions 10mMPB pH 78 30∘C and shaking (200 rpm) at 30∘Cthe initial concentration of D-lactate was 20mM

compared to the initial strain [18] Two types of yeast cells(intact and permeabilized) were tested The experiment wascarried out as follows to 50mLof 10mMPB pH78we addedD-lactate (with a final concentration 20mM) and 01 g of theintact yeast cells with specific enzymatic activity 126Usdotmgminus1An incubation of the same amount of permeabilized cells oftheH polymorpha ldquotr6rdquo (with DLDH activity of 117 Usdotmgminus1)in the same mixture was performed in parallel The amountof the added yeast cells of two types was calculated relatedto the specific DLDH activity The obtained cell mixture wasincubated about one hour at a shaker (200 revsdotminminus1) at 30∘CEvery 10 minutes 2mL of the mixture was taken centrifu-gated (6000timesg 119903 = 8 cm 2min) and the eluates werefrozen at minus20∘CThe residual D-lactate in the frozen sampleswas analyzed chemically by Buchner method [20] aftercompleting the experiment (Table 1)

The profile of D-lactate utilization using recombinant Hpolymorpha ldquotr6rdquo cells is represented in Figure 1

As shown in Figure 1 effectiveness of D-lactate oxidation(during 40-minute incubation) was calculated as 28 forintact yeast cells (activity ofDLDH 126Usdotmgminus1) whereas forthe permeabilized cells (with activity of DLDH 112 Usdotmgminus1)

Control column Bioreactor

Model solution of D-lactate

Figure 2 Principal scheme of bioreactor prototype based onpermeabilized recombinant yeast cells for removing D-lactate fromaqueous solution The control column contains alginate gel withoutthe cells

it was about 41The average productivity of the process (for30min incubation)was about 90mmolsdotLminus1sdothminus1 It was shownthat the permeabilized yeast cells are 15-fold more effectiveagent for removal of D-lactate in comparison with intactliving cells Probably this effect could be explained by higherpermeability of permeabilized cells to substrate Thus forconstruction of cell-based bioreactor for D-lactate oxidationpermeabilized yeast cells of H polymorpha ldquotr6rdquo are moreeffective

A column-type bioreactor based on permeabilized yeastcells of H polymorpha ldquotr6rdquo incorporated in alginate gel wasconstructed The immobilization of the yeast cells in calciumalginate gel was performed as follows 2mL suspension ofpermeabilized H polymorpha ldquotr6rdquo cells (60mgsdotmLminus1) wasmixed with 2mL of 4 (wv) sodium alginate The obtainedmixture was put to medical syringe and dropped througha needle into solution of CaCl

2

(40mgsdotmLminus1) The formedalginate beads (diameter around 3mm) with incorporatedpermeabilized yeast cells were placed to columns 1 times 10 cm(4mL gel) As a control ldquobarerdquo alginate gel without the cells(Figure 2) was used

The packed columns were washed by 3mL of PB pH78 prior to the experiment beginning As a model solution20mM of D-lactate in 50mM PB pH 78 containing 1mMCaCl2

was usedFor optimisation of flow rate for high efficiency of D-lac-

tate oxidation the model solution was simultaneously passedthrough both columns with different speed 50mLsdotminminus1and 10mLsdotminminus1 Due to difference in flow rate throughthe alginate gel the first column with a speed 50mLsdotminminus1was marked as a ldquobioreactor 1rdquo and the other one (flowrate 10mLsdotminminus1) was marked as ldquobioreactor 2rdquo respectivelyEvery 15min a small amount (02mL) of mixture flowingthrough the columnswas collected and frozen atminus20∘CAfterfinishing the experiment a residual D-lactate in the frozensamples was analyzed by Buhner chemical method of lactateassay (Figure 3)


000 015 030 045 060 075 090 105 12005

101520253035404550

9095

100

Enzy

mat

ic co

nver

sion

of D

-lact

ate (

)

Volume of eluate (mL)

(c)

(b)

(a)

Figure 3 Optimisation of flow rate for enzymatic D-lactateconversion using a column bioreactor (a) flow rate 50mLsdotminminus1and (b) flow rate 10mLsdotminminus1 and (c) control column flow rate10mLsdotminminus1 Conditions enzymatic activity of DLDH in perme-abilized cells was 80UsdotmLminus1 and the initial D-lactate concentrationwas 20mM in the presence of 1mM CaCl

2

Aswas shown in Figure 3 both columns are able to removeD-lactate from themodel solutionHowever the effectivenessof this processwas significantly different anddepended on theflow rate through the columns Thus in the case of ldquobiore-actor 1rdquo enzymatic conversion of 2mL 20mM D-lactate wasranged between 15 and 28 ldquoBioreactor 2rdquo showed a twofoldhigher efficiency of D-lactate oxidation at the same volume ofmodel solution (45ndash53) The control alginate column wasused to analyze the possibility of nonenzymatic conversionof D-lactate As was clearly shown in Figure 3 nonenzymaticconversion of D-lactate in control reactor did not occur andthe observed decrease inD-lactate concentration (about 14)was caused by a partial dilution of sample as a result ofwashing the column by buffer

The obtained results clearly confirmed the possibility ofusing permeabilized cells immobilized in alginate gels ofrecombinant DLDH-overproducing strain of H polymorphaldquotr6rdquo as a catalyst for bioreactor able to remove D-lactatefrom fermented food products It was shown that efficiencyof enzymatic conversion of D-lactate for bioreactor prototypetightly depends on the flow rate through the column

The usage of the developed cell-based prototype forremoving toxic D-lactate from fermented products is inprogress

4 Conclusions

A laboratory prototype of column-type bioreactor for D-lactate removing from model solution based on permeabi-lized yeast cells of the H polymorpha ldquotr6rdquo and alginategel has been constructed and efficiency of this processhas been demonstrated The optimal concentration of thepermeabilized cells (with DLDH activity of 112 Usdotmgminus1) wasestimated as 30mg per 1mL of 4 alginate It was shown

that efficiency of enzymatic oxidation of D-lactate by thebioreactor prototype tightly depends on flow rate through thecolumnThe laboratory cell-based bioreactor would be usefulin food technology for removing toxic D-lactate

Competing Interests


Acknowledgments

This research was supported in part by NAS of Ukraine in theframe of the Scientific-Technical Program ldquoSensor systemsfor medical ecological and industrial-technological needsmetrological assurance and research exploitationrdquo as well asby NATO Project SfP 984173

References

[1] M de Vrese B Koppenhoefer and C A Barth ldquoD-lactic acidmetabolism after an oral load of dl-lactaterdquo Clinical Nutritionvol 9 no 1 pp 23ndash28 1990

[2] C S K Mishra and P Champagne Eds Biotechnology Appli-cations I K International Publishing House New Delhi India2009

[3] C ScheeleThe Collected Papers of Carl WilhelmScheele G BellLondon UK 1931

[4] Megazyme ldquoD-lactate acid (D-lactate) and L-lactate acid (L-lactate) assay proceduresrdquo 2007 httpssecuremegazymecomD-Lactic-Acid-Assay-Kit

[5] D L Zhang Z W Jiang J Jiang B Cao and J S Li ldquoD-lacticacidosis secondary to short bowel syndromerdquo PostgraduateMedical Journal vol 79 no 928 pp 110ndash112 2003

[6] A Zourari J P Accolas and M J Desmazeaud ldquoMetabolismand biochemical characteristics of yogurt bacteria A reviewrdquoLe Lait vol 72 no 1 pp 1ndash34 1992

[7] L Stolberg R Rolfe N Gitlin et al ldquoD-Lactic acidosis due toabnormal gut flora Diagnosis and treatment of two casesrdquoTheNew England Journal of Medicine vol 306 no 22 pp 1344ndash1348 1982

[8] M Hudson R Pocknee and N A G Mowat ldquoD-Lacticacidosis in short bowel syndromemdashan examination of possiblemechanismsrdquoQuarterly Journal ofMedicine vol 74 no 274 pp157ndash163 1990

[9] A S Day and G D Abbott ldquoD-lactic acidosis in short bowelsyndromerdquoTheNew ZealandMedical Journal vol 112 no 1092pp 277ndash278 1999

[10] N Gurukripa Kowlgi and L Chhabra ldquoD-lactic acidosisan underrecognized complication of short bowel syndromerdquoGastroenterology Research and Practice vol 2015 Article ID476215 8 pages 2015

[11] A D Hingorani I C Macdougall M Browne R W HWalker and C R V Tomson ldquoSuccessful treatment of acute D-lactate encephalopathy by haemodialysisrdquo Nephrology DialysisTransplantation vol 8 no 11 pp 1283ndash1285 1993

[12] J R Thurn G L Pierpont C W Ludvigsen and J H EckfeldtldquoD-lactate encephalopathyrdquo The American Journal of Medicinevol 79 no 6 pp 717ndash721 1985


[13] H Hove and P B Mortensen ldquoColonic lactate metabolism andD-lactic acidosisrdquoDigestive Diseases and Sciences vol 40 no 2pp 320ndash330 1995

[14] B E Coronado S M Opal and D C Yoburn ldquoAntibiotic-induced D-lactic acidosisrdquoAnnals of Internal Medicine vol 122no 11 pp 839ndash842 1995

[15] S C Kadakia ldquoD-lactic acidosis in a patient with jejunoilealbypassrdquo Journal of Clinical Gastroenterology vol 20 no 2 pp154ndash156 1995

[16] T Lodi and I Ferrero ldquoIsolation of the DLD gene of Sac-charomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductaserdquo Molecular and Gen-eral Genetics vol 238 no 3 pp 315ndash324 1993

[17] T Lodi D OrsquoConnor P Goffrini and I Ferrero ldquoCarboncatabolite repression inKluyveromyces lactis isolation and char-acterization of the KINLD gene encoding the mitochondrialenzymeD-lactate ferricytochrome c oxidoreductaserdquoMolecularand General Genetics vol 244 no 6 pp 622ndash629 1994

[18] O V Smutok K V Dmytruk M I Karkovska W SchuhmannM V Gonchar and A A Sibirny ldquoD-lactate-selective ampero-metric biosensor based on the cell debris of the recombinantyeast Hansenula polymorphardquo Talanta vol 125 pp 227ndash2322014

[19] C A Appleby and R K Morton ldquoLactic dehydrogenase andcytochrome b

2

of bakerrsquos yeast purification and crystallizationrdquoThe Biochemical journal vol 71 no 3 pp 492ndash499 1959

[20] E Buchner and K Schottenhammer ldquoDie Einwirkung vonDiazo-essigester aufMesitylenrdquoBerichte der Deutschen Chemis-chen Gesellschaft (A and B Series) vol 53 no 6 pp 865ndash8731920

Research ArticleImproved Production of Aspergillus usamii endo-120573-14-Xylanasein Pichia pastoris via Combined Strategies

Jianrong Wang Yangyuan Li and Danni Liu

Guangdong VTR Bio-Tech Co Ltd Zhuhai Guangdong 519060 China

Correspondence should be addressed to Jianrong Wang believe1234126com

Received 11 November 2015 Accepted 24 January 2016


Copyright copy 2016 Jianrong Wang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A series of strategies were applied to improve expression level of recombinant endo-120573-14-xylanase from Aspergillus usamii (Ausamii) in Pichia pastoris (P pastoris) Firstly the endo-120573-14-xylanase (xynB) gene fromA usamiiwas optimized for P pastoris andexpressed in P pastoris The maximum xylanase activity of optimized (xynB-opt) gene was 33500UmL after methanol inductionfor 144 h in 50 L bioreactor which was 59 higher than that by wild-type (xynB) gene To further increase the expression of xynB-opt the Vitreoscilla hemoglobin (VHb) gene was transformed to the recombinant strain containing xynB-opt The results showedthat recombinant strain harboring the xynB-opt and VHb (named X33xynB-opt-VHb) displayed higher biomass cell viability andxylanase activity The maximum xylanase activity of X33xynB-opt-VHb in 50 L bioreactor was 45225UmL which was 35 and115 higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene Finally the induction temperature of X33xynB-opt-VHb was optimized in 50 L bioreactor The maximum xylanase activity of X33xynB-opt-VHb reached 58792UmL when theinduction temperature was 22∘C The results presented here will greatly contribute to improving the production of recombinantproteins in P pastoris

1 Introduction

Xylan the major hemicellulose component in plant cell walland the most abundant renewable hemicellulose is a hetero-geneous polysaccharide consisting of a 120573-14-linked D-xylosebackbone [1] Xylan occupies one-third of the overall plantcarbohydrate and is the second most prevalent biomass aftercellulose in nature [2] Xylanase (EC 3218) can hydrolyzexylan into xylooligosaccharides andD-xylose Xylanases havegenerated considerable research interest and are becominga major group of industrial enzymes Xylanases have widecommercial applications in industrial processes such as thepaper and pulp industry the foodstuff industry the feedindustry and the energy industry [3 4] In recent yearsincreasing numbers of xylanases have been identified andcharacterized from various microorganisms such as bacteriaand fungi [5] Among microbial sources the filamentousfungi Aspergillus are especially interesting as they secretethese enzymes into the medium and their xylanase activities

are higher than those produced by other microorganisms[6ndash8] In previous studies an endo-120573-14-xylanase with highspecific activity and good enzymatic properties was isolatedfrom A usamii Furthermore the gene encoding endo-120573-14-xylanase from A usamii was cloned and expressed in Ppastoris [9 10] However the low expression level does notallow the recombinant xylanase to be applied practically andeconomically in industry For commercial exploitation of therecombinant A usamii endo-120573-14-xylanase it is essential toachieve high yield of the protein

P pastoris is nowwidely used for heterologous productionof recombinant proteins As a single-celled microorganismP pastoris has been proved as a highly successful system forvariety of recombinant proteins There are many advantagesin this expression system such as high level expressionefficient extracellular protein secretion proper protein fold-ing posttranslational modifications and the potential tohigh cell density fermentation [11 12] Due to the wideuse of this system many strategies have been developed to



further improve expression level of heterologous protein inP pastoris including codon optimization intracellular coex-pression ofVitreoscilla hemoglobin (VHb) high heterologousgene copy number altering the secretion signal peptide inexpression vector high efficient transcriptional promotersand cultivation optimization [13ndash15] However there is littlereport about integrating these optimization methods as awhole to optimize gene expression

In order to improve production of A usamii endo-120573-14-xylanase in P pastoris the xynB from A usamii was firstlyexpressed in P pastoris To further improve the productionof recombinantA usamii endo-120573-14-xylanase we combinedcodon optimization intracellular coexpression of VHb andoptimization of induction temperature as a whole to optimizexynB expression in P pastoris To our knowledge this is thefirst report to improve A usamii endo-120573-14-xylanase pro-duction in P pastoris by integrating these three optimizationmethods The results presented here will greatly contributeto improving the production of recombinant proteins inP pastoris and offer a greater value in various industrialapplications


21 Strains Plasmids Reagents and Media The P pas-toris strain X33 and the expression vector (pPIC35K andpPICZ120572A) were purchased from Invitrogen (Carlsbad CAUSA) The E coli strain Top 10 is routinely conserved inour laboratory Restriction enzymes T4-DNA ligase andPfu DNA polymerase were purchased from Sangon Biotech(Shanghai China) All other chemicals used were analyticalgrade reagents unless otherwise stated Yeast extract peptonedextrose (YPD)medium buffered glycerol complex (BMGY)medium and buffered methanol complex (BMMY) mediumwere prepared according to the manual of Pichia Expres-sion Kit (Version F Invitrogen) Fermentation Basal SaltsMedium (BSM) and PTM1 Trace Salts used for fermentationwere prepared according to the Pichia Fermentation ProcessGuidelines (Invitrogen)

22 Codon Optimization and Screening ofHigh Xylanase Activity Clones

221 Codon Optimization and Synthesis of the Gene Thecodon usage of xynB gene (GenBank DQ302412) from Ausamii and VHb gene (GenBank AY278220) was analysedusing Graphical Codon Usage Analyser (httpgcuaschoedlde) and they were optimized by replacing the codonspredicted to be less frequently used in P pastoris withthe frequently used ones (httpswwwdna20com) Theoptimized genes (xynB-opt and VHb) were synthesized bySangon (Shanghai China)

222 Vector Construction Transformation and Screening ofHigh Xylanase Activity Clones The synthetic gene encodingthe mature region of xylanase without the predicted signalsequence was digested by EcoRI and NotI and then lig-ated into pPICZ120572A to form pPICZ120572A-xynB-opt The native

xylanase gene (xynB) was cloned from A usamii by reversetranscription PCR and then inserted into plasmid pMD20-Tto formpMD20-T-xynBThepMD20-T-xynBwas digested byEcoRI and NotI and then ligated into pPICZ120572A resulting inthe recombinant plasmid pPICZ120572A-xynBThe syntheticVHbgene was digested by EcoRI and NotI and then ligated intopPIC35K to formpPIC35K-VHbThe recombinant plasmidswere checked by DNA sequencing

P pastoris X33 was transformed with 10 120583g of PmeI-linearized pPICZ120572A-xynB-opt and pPICZ120572A-xynB vector byelectrotransformation according to Invitrogenrsquos recommen-dations Transformants were plated on YPDS plates (10 gLyeast extract 20 gL peptone 20 gL dextrose 20 gL agar and1M sorbitol) containing 100 120583gmL Zeocin to isolate resistantclonesThemethod for screening transformants was the sameas our previous described method except the microplates[16] In this study transformants were picked and culturedin 24-deep-well microplates containing 12mLwell BMGYmedium at 30∘C for 24 h After this the cells were harvestedby centrifugation resuspended and cultured in 1mLwellBMMY medium After 24 h plates were subjected to cen-trifugation again and supernatants were used in subsequentactivity assays The clones showing higher activities werechecked by shaking flask fermentation

223 Expression of xynB-opt and xynB in P pastoris Shake-Flask Cultures Thirty clones which had higher enzymeactivity were selected for shake-flask culturesThe seeds wereinoculated in 10mL of BMGY medium in a 100mL shakeflask and incubated at 30∘C until the culture reached OD

600

= 20ndash60 The cells were harvested by centrifugation andresuspended in 50mL of BMMY medium and incubated at30∘C The methanol induction temperature was set at 30∘Cand 07 (vv) methanol was fed at 24 h intervals for 5 daysThe activities of the xylanase were checked at 24 48 72 96and 120 h The colony with the highest activity was selectedas the host for the transformation of the pPIC35K-VHb andpPIC35K vectors

23 Intracellular Coexpression of VHb

231 Construction of Recombinant Strains Containing VHband xynB-opt A clone named X33xynB-opt (the recombi-nant strain containing xynB-opt) exhibiting the maximumxylanase activity under shake-flask cultures was chosenas the host for the transformation of the pPIC35K-VHband pPIC35K vectors The plasmids pPIC35K-VHb andpPIC35K were both linearized by SacI and transformed intoX33xynB-opt to form X33xynB-opt-VHb and X33xynB-opt-p The X33xynB-opt-p was used as control during theexperiments The transformants were plated on YPDG plates(10 gL yeast extract 20 gL peptone 20 gL dextrose 20 gLagar and 1M sorbitol) containing 4mgmL G418 to isolateresistant clones

232 Expression of Recombinant Strains in P pastoris Shake-Flask Cultures The G418-resistant clones were cultivatedin shaking flask The cultivation conditions and medium


composition were the same as described above The cloneexhibiting higher enzyme activity in shaking flask wasselected for further experiments

24 High Cell Density Fermentation The transformed strains(X33xynB X33xynB-opt X33xynB-opt-p and X33xynB-opt-VHb) showing the highest xylanase activity in shake-flask culture were cultivated in high cell density fermentorHigh cell density fermentation was carried out in 50 Lbioreactor (Baoxing Co Shanghai China) The cultivationconditions and medium composition were the same as theprevious described method [17] The enzyme activity of thesupernatant and dry cell weight (DCW) were monitoredthroughout the cultivation

25 Optimization of the Induction Temperature To investi-gate the effects of temperature on the production of xylanaseof recombinant strain X33xynB-opt-VHb the inductiontemperature was optimized in a 50 L bioreactor During themethanol fed-phase the temperature was designed in therange of 30∘C to 22∘C

26 Assay of Xylanase Activity Protein Determination CellViability Oxygen Uptake and Dry Cell Weight Xylanaseactivity was assayed according to the method described byprevious study and with some modification [18] All enzymeassays unless otherwise stated were carried out at 50∘C for30min in 100mM acetic acid-sodium acetic acid buffer (pH50) 2mL basic reaction mixture (containing 1mL of 10(wv) oat spelt xylan and 1mL of a suitably diluted enzymesolution) was incubated at 50∘C for 30min and reducingsugar (xylose) was measured by the dinitrosalicylic acid(DNS) according to the standardmethod and xylosewas usedas a standard One unit of xylanase activity was defined as theamount of enzyme that produced 1 120583M reducing sugar fromsubstrate solution perminute under the assay conditionsTheprotein content was determined according to the Bradfordmethod using BSA as standard The measurement of cellviability was performed by methylene blue dye exclusiontechnique as described by the previous study [19]The oxygenuptake (OUR) was determined according to the previousstudies [20 21] Cell density was expressed as grams of drycell weigh (DCW) per liter of broth and was obtained bycentrifuging 10mL samples in a preweighted centrifuge tubeat 8000 g for 10min and washing twice with deionized waterthen allowing the pellet to dry at 100∘C to constant weight


31 Improved Production of A usamii Xylanase in P pastorisby Codon Optimization As an easy and simple system Ppastoris is now widely used for heterologous production ofrecombinant proteins [22] Due to the discrepancy of codonusage between the host and their original strains researchershave used codon optimization to increase the expression ofheterologous gene Codon optimization by using frequentlyused codons in the host is an efficient strategy to improvethe expression level of heterologous gene Generally this is

accomplished by replacing all codons with preferred codonseliminating AT-rich stretches and adjusting the G+C content[8 23] Analysis of the DNA sequence of VHb and nativexynB using Graphical Codon Usage Analyser revealed thatsome amino acid residues were encoded by codons that arerarely used in P pastoris These codons TCG (Ser) CCG(Pro)GGC (Gly) AGC (Ser) andGCG(Ala) shared less than15 of usage percentage which may result in a much lowerexpression level of xynB in P pastoris (Tables 1 and 2) Tosolve this problem we took a strategy of rewriting the nativexynB andVHb according to P pastoris preferred codon usageG+C content affects the secondary structure of mRNA andthen affects the expression level of heterologous gene In thisstudy the G+C content of VHb was increased from 42 to 51and the native xynB was reduced from 576 to 551 which isin the appropriate range for Picha system Totally there were105 and 57 amino acids optimized in native xynB and VHbrespectively (Tables 1 and 2) As shown in Figures 1 and 2the optimized gene (xynB-opt and VHb-opt) shared 83 and86 of nucleotide sequence identity with that of the nativegene (xynB and VHb)

The recombinant plasmids pPICZ120572A-xynB-opt andpPICZ120572A-xynB were transformed into P pastoris X33 andhundreds of transformants were obtained on YPDZ platesThepositive cloneswere cultured in 24-deep-wellmicroplatesand further screened by xylanase activity assay Two clones(one carrying xynB-opt named X33xynB-opt and the othercarrying xynB named X33xynB) showing the higher activitywere chosen for shake-flask culturesThe recombinant strainsX33xynB-opt and X33xynBwere cultivated in shaking flaskAfter 120 h of cultivation under inducing conditions thexylanase activities of X33xynB-opt and X33xynB were920UmL and 520UmL respectively The total proteincontent of X33xynB-opt and X33xynB were 025mgmLand 015mgmL respectively The recombinant strainX33xynB-opt was chosen as the host for the transformationof the pPIC35K-VHb and pPIC35K vectors

32 Intracellular Coexpression of VHb Oxygen supply is oneof the most critical parameters for cell growth and heterolo-gous protein expression in recombinant P pastoris VHb isa suitable oxygen uptake improving protein for expressionin P pastoris due to its high oxygen trapping and releasingability enabling it to satisfy extremely high oxygen demandduring fermentations [24] In this study in order to enhanceoxygen uptake and improve the production of recombinantxylanase we attempted to coexpress the VHb with xynB-optin P pastoris The recombinant plasmids pPIC35K-VHb andpPIC35K were linearized and transformed into recombinantstrain X33xynB-opt to form recombinant strains VHb+(X33xynB-opt containing VHb) and VHbminus (X33xynB-opt containing pPIC35K) Transformants were plated onYPDG plates Then the G418-resistant clones were culturedin shaking flasks The VHb+ (named X33xynB-opt-VHb)showed higher cell density and xylanase activity than VHbminus(named X33xynB-opt-p) The cell density of X33xynB-opt-VHb was approximately 25 gL higher than X33xynB-opt-p Moreover the xylanase activity of X33xynB-opt-VHb was


ATGCTCACCAAGAACCTTCTCCTCTGCTTTGCCGCGGCTAAGGCTGCTCTGGCTGTTCCCCACGACTCTGTCGCCCAGCGTTCGGATGCCTTGCACATGC

ATGCTTACTAAGAACTTGCTTTTGTGTTTCGCCGCCGCCAAAGCTGCCTTGGCTGTTCCACACGATTCCGTCGCTCAGAGATCCGACGCCTTGCACATGT

TCTCTGAGCGCTCGACCCCGAGCTCGACCGGCGAGAACAACGGCTTCTACTACTCCTTCTGGACCGACGGCGGTGGCGACGTGACCTACACCAACGGAGA

TGTCCGAGAGATCTACCCCATCCTCCACCGGAGAGAACAACGGTTTCTACTACTCCTTCTGGACCGACGGTGGAGGTGACGTCACCTACACCAACGGTGA

TGCTGGTGCCTACACTGTTGAGTGGTCCAACGTGGGCAACTTTGTCGGTGGAAAGGGCTGGAACCCCGGAAGTGCGCAGGACATCACCTACAGCGGCACC

CGCTGGTGCCTACACCGTCGAGTGGTCCAACGTCGGAAACTTCGTCGGTGGTAAGGGATGGAACCCAGGTTCCGCTCAGGACATCACCTACTCCGGTACT

TTCACCCCTAGCGGCAACGGCTATCTCTCCGTCTATGGCTGGACCACTGACCCCCTGATCGAGTACTACATCGTCGAGTCCTACGGCGACTACAACCCCG

TTCACCCCATCCGGTAACGGTTACTTGTCCGTCTACGGTTGGACCACTGACCCATTGATCGAGTATTACATCGTCGAGTCTTACGGTGACTACAACCCAG

GCAGTGGAGGCACATACAAGGGCACCGTCACCTCGGACGGATCCGTTTACGATATCTACACGGCTACCCGTACCAATGCTGCTTCCATTCAGGGAACCGC

GTTCTGGAGGTACCTACAAGGGAACCGTCACCTCCGATGGATCCGTCTACGACATCTACACCGCCACCCGTACCAACGCCGCTTCCATCCAGGGTACCGC

TACCTTCACTCAGTACTGGTCCGTCCGCCAGAACAAGAGAGTTGGCGGAACTGTTACCACCTCCAACCACTTCAATGCTTGGGCTAAGCTGGGAATGAAC

CACCTTTACCCAGTACTGGTCTGTCCGTCAGAACAAGCGTGTCGGAGGTACCGTCACCACCTCCAACCACTTCAACGCTTGGGCCAAGTTGGGAATGAAC

CTGGGTACTCACAACTACCAGATCGTGGCTACCGAGGGTTACCAGAGCAGTGGATCTTCGTCCATCACTGTTCAGTAA

CTTGGAACCCACAACTACCAGATTGTCGCCACTGAGGGTTACCAGTCTTCTGGATCCTCCTCCATCACCGTCCAATAA

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

100100

200200

300300

400400

500500

600600

678

678

Figure 1 Sequence comparison between the original (xynB) and the optimized (xynB-opt) genes Identical residues are marked in blackbackground

100VHb100VHb-opt

ATGTTGGATCAACAGACTATCAACATCATCAAGGCTACTGTTCCAGTCTTGAAGGAACATGGTGTTACTATCACTACTACTTTCTACAAGAACTTGTTTG

ATGTTGGATCAACAAACCATTAACATCATCAAGGCCACCGTCCCAGTCTTGAAGGAGCACGGAGTCACCATTACCACCACCTTCTACAAAAACTTGTTCG

200VHb200VHb-opt

CTAAGCATCCAGAAGTTAGACCATTGTTTGATATGGGTAGACAAGAATCTTTGGAACAACCAAAGGCTTTGGCTATGACTGTCTTGGCTGCTGCTCAGAA

CTAAGCACCCAGAAGTCCGTCCATTGTTCGACATGGGTAGACAGGAGTCCTTGGAGCAGCCAAAGGCTTTGGCTATGACCGTTTTGGCTGCTGCCCAGAA

300VHb300VHb-opt

CATTGAGAACTTGCCAGCTATCTTGCCAGCTGTTAAGAAGATTGCTGTTAAGCATTGTCAAGCTGGTGTTGCTGCTGCTCATTACCCAATTGTTGGTCAA

CATCGAGAACTTGCCAGCCATCTTGCCAGCCGTTAAGAAGATCGCTGTCAAGCACTGTCAAGCTGGTGTTGCTGCTGCTCATTACCCTATCGTCGGTCAG

400VHb400VHb-opt

GAATTGTTGGGTGCTATCAAGGAAGTCTTGGGTGATGCTGCTACTGATGATATCTTGGATGCTTGGGGTAAGGCTTACGGTGTTATTGCTGATGTCTTCA

GAGTTGTTGGGTGCCATTAAGGAGGTCTTGGGTGACGCTGCTACTGACGACATTTTGGACGCTTGGGGTAAGGCTTACGGTGTCATTGCCGACGTCTTCA

440VHb440VHb-opt

TTCAAGTTGAAGCTGATTTGTACGCTCAAGCTGTTGAATA

TCCAGGTCGAGGCTGATTTGTACGCTCAGGCTGTTGAATA

Figure 2 Sequence comparison between the original (VHb) and the optimized (VHb-opt) genes Identical residues are marked in blackbackground

1300UmL which was 31 and 60 more than that ofX33xynB-opt-p and X33xynB

33 High Cell Density Fermentation In order to obtain a largeamount of active protein the recombinant strains X33xynB-opt X33xynB X33xynB-opt-p and X33xynB-opt-VHbwerecultivated in 50 L fermentor As shown in Figure 3 themaximum xylanase activity and total protein concentra-tion produced by X33xynB-opt reached 33500UmL and

38 gL respectively Compared with X33xynB the maxi-mum xylanase activity and total protein concentration wereincreased by 60 and 80 respectively In this study thefermentation conditions and DCW of X33xynB-opt andX33xynB were almost the same during the high cell densityfermentation (data not shown)These results showed that theimproved production of recombinant xylanase in P pastoriswas reached by codon optimization Codon optimizationis an effective method to improve the expression level of


Table 1 Comparison of the codon usage for native (xynB) andsynthetic (xynB-opt) targeted genes at Pichia pastoris for expression

AA Codon Host fraction xynB xynB-opt

Gly

GGG 010 0 0GGA 032 9 11GGT 044 5 18GGC 014 15 0

Glu GAG 043 6 6GAA 057 0 0

Asp GAT 058 3 2GAC 042 7 8

Val

GTG 019 3 0GTA 015 0 0GTT 042 6 1GTC 023 6 14

Ala

GCG 006 2 0GCA 023 0 0GCT 045 12 7GCC 026 4 11

Arg

AGG 016 0 0AGA 048 1 2CGG 005 0 0CGA 010 0 0CGT 016 2 3CGC 005 2 0

Lys AAG 053 6 5AAA 047 0 1

Ser

AGT 015 3 0AGC 009 4 0TCG 009 5 0TCA 019 0 0TCT 029 3 6TCC 020 9 18

Stop TAA 053 1 1

Asn AAT 049 2 0AAC 051 13 15

Met ATG 100 3 3

IleATA 019 0 0ATT 050 1 1ATC 030 6 6

Thr

ACG 011 1 0ACA 024 1 0ACT 040 4 4ACC 025 19 23

Trp TGG 100 6 6

Cys TGT 065 0 1TGC 035 1 0

Tyr TAT 046 2 1TAC 055 15 16

Leu

TTG 033 1 8TTA 016 0 0CTG 016 4 0CTA 011 0 0CTT 016 1 3CTC 008 5 0

Table 1 Continued


Phe TTT 054 2 1TTC 046 5 6

Gln CAG 039 8 7CAA 061 0 1

His CAT 057 0 0CAC 043 4 4

Pro

CCC 015 4 0CCG 009 1 0CCA 041 0 6CCT 035 1 0

0

5000

10000

15000

20000

25000

30000

35000

Activity of X33xynBActivity of X33xynB-optProtein content of X33xynBProtein content of X33xynB-opt

Induction time (h)

Activ

ity (U

mL)

00

05

10

15

20

25

30

35

40

45

50

Prot

ein

cont

ent (

mg

mL)

24 48 72 96 120 144 168

Figure 3 Xylanase activity and total protein content of X33xynB-opt and X33xynB during fed batch fermentation in 50 L bioreactor

heterologous gene in P pastoris In our previous study the120572-amylase gene from Bacillus licheniformis was codon opti-mization according to the codon usage of P pastoris and theoptimized gene was expressed at a significantly higher levelthan the wild-type gene [17] Through codon optimizationthe glucanase gene from Fibrobacter succinogenes resulted ina 234-fold increase of target protein production [25]

The growth and xylanase activity profile of X33xynB-opt-p and X33xynB-opt-VHb in 50 L bioreactor were shown inFigure 4 The DCW and xylanase activity of X33xynB-opt-VHb were higher than those of X33xynB-opt-p during themethanol induction phase The highest xylanase activity ofX33xynB-opt-VHb was 45225UmL which was about 135-fold higher than that of X33xynB-opt-p The higher xylanaseactivity and DCW of X33xynB-opt-VHb were probablycaused by coexpression of VHb The function of VHb isusually considered to be the enhancement of respirationcell viability and energy metabolism by facilitating oxygenuptake As shown in Figure 5(a) SOUR of both strains wasalmost the same before methanol induction After induc-tion SOUR of X33xynB-opt-VHb increased higher than


Table 2 Comparison of the codon usage for native (VHb) andsynthetic (VHb-opt) targeted genes at Pichia pastoris for expression

AA Codon Host fraction VHb VHb-opt

Gly

GGG 010 0 0GGA 032 1 0GGT 044 7 8GGC 014 0 0

Glu GAG 043 7 1GAA 057 2 8

Asp GAT 058 2 8GAC 042 6 0

Val

GTG 019 0 0GTA 015 0 0GTT 042 4 10GTC 023 10 4

Ala

GCG 006 0 0GCA 023 0 0GCT 045 17 23GCC 026 6 0

Arg


Lys AAG 053 9 10AAA 047 1 0

Ser


Stop TAA 053 1 1

Asn AAT 049 0 0AAC 051 4 4

Met ATG 100 3 3

IleATA 019 0 0ATT 050 5 5ATC 030 7 7

Thr

ACG 011 0 0ACA 024 0 0ACT 040 1 8ACC 025 7 0

Trp TGG 100 1 1

Cys TGT 065 1 1TGC 035 0 0

Tyr TAT 046 0 0TAC 055 4 4

Leu


Table 2 Continued


Phe TTT 054 0 2TTC 046 4 2

Gln CAG 039 6 2CAA 061 3 7

His CAT 057 1 4CAC 043 3 0

Pro

CCC 015 0 0CCG 009 0 0CCA 041 6 7CCT 035 1 0

0

10000

20000

30000

40000

50000

Induction time (h)

Activ

ity (U

mL)

50

100

150

200

250

300

Dry

cell

wei

ght (

gL)

Activity of X33xynB-opt-VHbActivity of X33xynB-opt-pDCW of X33xynB-opt-VHbDCW of X33xynB-opt-p

24 48 72 96 120 144 168

Figure 4 Xylanase activity and DCW of X33xynB-opt-p andX33xynB-opt-VHbduring fed batch fermentation in 50 L bioreactor

X33xynB-opt-p during the whole induction phase whichwas probably caused by VHb expression improving oxygenutilization and respiratory efficiency The VHb-expressingstrain with higher oxygen demand was similar to previousstudies [26] Furthermore we also compared the cell via-bilities of X33xynB-opt-p and X33xynB-opt-VHb The cellviabilities of X33xynB-opt-p and X33xynB-opt-VHb at theend of 144 h of cultivation were 75 and 60 respectively(Figure 5(b)) This indicates that VHb expression resultedin an increase in cell viability In this study coexpressionof VHb increased SOUR and then improved cell viabilityand DCW of X33xynB-opt-VHb Our results indicated thatcoexpression of VHb is also an effective method to improvethe production of heterologous protein in P pastoris

34 Optimization of the Induction Temperature Temperatureis a key factor for optimization of heterologous proteinsexpressed in P pastoris In order to evaluate the effects ofcultivation temperature on cell growth cell viability andxylanase production X33xynB-opt-VHb was grown in BSM(pH 50) at 22 25 28 and 30∘C (Figure 6) As shown in


00

5

10

15

20

25

30

35

40

Induction time (h)

X33xynB-opt-VHbX33xynB-opt-p

SOU

R (120583

mol

O2m

ing

DCW

)

24 48 72 96 120 144 168 192 216

(a)

50

60

70

80

90

100

Cel

l via

bilit

y (

)

Induction time (h)


24 48 72 96 120 144

(b)

Figure 5 Comparison of specific oxygen uptake (a) and cell viability (b) profile between X33xynB-opt-p and X33xynB-opt-VHb during fedbatch fermentation in 50 L bioreactor

0

10000

20000

30000

40000

50000

60000

30282522

Xyla

nase

activ

ity (U

mL)

0

50

100

150

200

250

DCW

(gL

)

Induction temperature (∘C)

Xylanase activityDCW

(a)

60

70

80

90

100

Cel

l via

bilit

y (

)

Induction time (h)24 48 72 96 120 144

30∘C28∘C

25∘C22∘C

(b)

Figure 6 Xylanase activity and DCW of X33xynB-opt-VHb during fed batch fermentation in 50 L bioreactor under different inductiontemperature (a) Cell viability profile of X33xynB-opt-VHb during fed batch fermentation in 50 L bioreactor under different inductiontemperature (b)

Figure 6(a) xylanase activities at lower induction temper-ature were higher than that at higher induction tempera-ture The maximum xylanase activity of 58792UmL witha cell density of 213 g DCW was obtained after 144 h ofculture at 22∘C which was 129-fold and 117-fold higherthan that at 30∘C Until now several Aspergillus endo-120573-14-xylanases have also been successfully expressed in P pastorisThe Aspergillus sulphureus and Aspergillus niger endo-120573-14-xylanases were functionally expressed and secreted in theP pastoris the enzyme activity of which reached 105 and

20424UmL [8 27] which were lower than the expressionlevel ofA usamii endo-120573-14-xylanase in this study Howeverthese values are not fully comparable since different cultiva-tion conditions and activity assays with different substratesand conditions have been usedMeanwhile the cell viabilitiesof X33xynB-opt-VHb under different temperature were alsodetermined The cell viability remained below 80 underthe temperature at 30∘C and 28∘C and above 90 in thecultivation with a temperature of 22∘C (Figure 6(b)) Theseresults indicated that lower induction temperature could


facilitate production of recombinant xylanase According tothe findings by other researchers lowering induction tem-perature can reduce cell death and increase cell viability andthen improved the production of heterologous proteins in Ppastoris [28] Furthermore lowering induction temperaturecan enlarge VHb effect on cell performance of P pastorisand then obtain a higher final cell density and viability incomparison with higher temperature [24]

4 Conclusions

In this study we combined codon optimization intracel-lular coexpression of VHb and optimization of inductiontemperature as a whole to improve the expression level ofrecombinant A usamii endo-120573-14-xylanase in P pastorisTo our knowledge this is the first report to combine thesemethods as a whole to improve the production of A usamiiendo-120573-14-xylanase in P pastoris Our results indicated thatcombined codon optimization intracellular coexpression ofVHb and optimization of induction temperature are aneffective method to improve the production of heterologousprotein in P pastoris Furthermore our results presentedhere will greatly contribute to improving the production ofrecombinant proteins in P pastoris and offer a greater valuein various industrial applications

Competing Interests


Authorsrsquo Contributions

Danni Liu made substantial contributions to the design ofthe experiments and the draft the paper Jianrong Wangand Yangyuan Li carried out this research interpreted thedata and drafted the paper Jianrong Wang and Yangyuan Licontributed equally to this work

Acknowledgments

This work was supported by the National High-TechnologyProject of China (no 2014AA093514) and theKey Science andTechnology Program of Zhuhai (no 2012D0201990042)

References

[1] Q K Beg M Kapoor L Mahajan and G S HoondalldquoMicrobial xylanases and their industrial applications a reviewrdquoApplied Microbiology and Biotechnology vol 56 no 3-4 pp326ndash338 2001

[2] M L T M Polizeli A C S Rizzatti R Monti H F Terenzi JA Jorge and D S Amorim ldquoXylanases from fungi propertiesand industrial applicationsrdquo Applied Microbiology and Biotech-nology vol 67 no 5 pp 577ndash591 2005

[3] A C E Gregory A P OrsquoConnell and G P Bolwell ldquoXylansrdquoBiotechnology and Genetic Engineering Reviews vol 15 no 1 pp439ndash456 1998

[4] R A Prade ldquoXylanases from biology to biotechnologyrdquoBiotechnology and Genetic Engineering Reviews vol 13 pp 101ndash131 1996

[5] S Subramaniyan and P Prema ldquoBiotechnology of microbialxylanases enzymology molecular biology and applicationrdquoCritical Reviews in Biotechnology vol 22 no 1 pp 33ndash64 2002

[6] Y Takahashi H Kawabata and S Murakami ldquoAnalysis offunctional xylanases in xylan degradation by Aspergillus nigerE-1 and characterization of the GH family 10 xylanase XynVIIrdquoSpringerPlus vol 2 article 447 2013

[7] A Hmida-Sayari S Taktek F Elgharbi and S Bejar ldquoBio-chemical characterization cloning and molecular modelingof a detergent and organic solvent-stable family 11 xylanasefrom the newly isolated Aspergillus niger US368 strainrdquo ProcessBiochemistry vol 47 no 12 pp 1839ndash1847 2012

[8] Y Li B Zhang X Chen Y Chen and Y Cao ldquoImprovement ofAspergillus sulphureus endo-120573-14-xylanase expression in Pichiapastoris by codon optimization and analysis of the enzymiccharacterizationrdquo Applied Biochemistry and Biotechnology vol160 no 5 pp 1321ndash1331 2010

[9] C Zhou J Bai S Deng et al ldquoCloning of a xylanase genefrom Aspergillus usamii and its expression in Escherichia colirdquoBioresource Technology vol 99 no 4 pp 831ndash838 2008

[10] H-M Zhang J-QWang M-CWu S-J Gao J-F Li and Y-JYang ldquoOptimized expression purification and characterizationof a family 11 xylanase (AuXyn11A) fromAspergillus usamiiE001in Pichia pastorisrdquo Journal of the Science of Food andAgriculturevol 94 no 4 pp 699ndash706 2014

[11] J R Wang Y Y Li S D Xu P Li J S Liu and D N LiuldquoHigh-level expression of pro-form lipase fromRhizopus oryzaein Pichia pastoris and its purification and characterizationrdquoInternational Journal of Molecular Sciences vol 15 no 1 pp203ndash217 2014

[12] D Teng Y Fan Y-L Yang Z-G Tian J Luo and J-HWang ldquoCodon optimization of Bacillus licheniformis 120573-13-14-glucanase gene and its expression in Pichia pastorisrdquo AppliedMicrobiology and Biotechnology vol 74 no 5 pp 1074ndash10832007

[13] R Ramon P Ferrer and F Valero ldquoSorbitol co-feeding reducesmetabolic burden caused by the overexpression of a Rhizopusoryzae lipase in Pichia pastorisrdquo Journal of Biotechnology vol130 no 1 pp 39ndash46 2007

[14] X Li Z Liu G Wang D Pan L Jiao and Y Yan ldquoOverexpres-sion of Candida rugosa lipase Lip1 via combined strategies inPichia pastorisrdquo Enzyme and Microbial Technology vol 82 pp115ndash124 2016

[15] M R Yu S Wen and T W Tan ldquoEnhancing production ofYarrowia lipolytica lipase Lip2 in Pichia pastorisrdquo Engineeringin Life Sciences vol 10 no 5 pp 458ndash464 2010

[16] J Wang Y Li and D Liu ldquoGene cloning high-level expressionand characterization of an alkaline and thermostable lipasefrom Trichosporon coremiiforme V3rdquo Journal of Microbiologyand Biotechnology vol 25 no 6 pp 845ndash855 2015

[17] J R Wang Y Y Li D N Liu et al ldquoCodon optimizationsignificantly improves the expression level of 120572-amylase genefrom Bacillus licheniformis in Pichia pastorisrdquo BioMed ResearchInternational vol 2015 Article ID 248680 9 pages 2015

[18] H Y Cai P J Shi Y G Bai et al ldquoA novel thermoacidophilicfamily 10 xylanase from Penicillium pinophilum C1rdquo ProcessBiochemistry vol 46 no 12 pp 2341ndash2346 2011

[19] X Wang Y Sun X Shen et al ldquoIntracellular expressionof Vitreoscilla hemoglobin improves production of Yarrowia


lipolytica lipase LIP2 in a recombinant Pichia pastorisrdquo Enzymeand Microbial Technology vol 50 no 1 pp 22ndash28 2012

[20] M Urgun-Demirtas K R Pagilla and B Stark ldquoEnhancedkinetics of genetically engineered Burkholderia cepacia role ofvgb in the hypoxic cometabolism of 2-CBArdquo Biotechnology andBioengineering vol 87 no 1 pp 110ndash118 2004

[21] J-M Wu and W-C Fu ldquoIntracellular co-expression ofVitreoscilla hemoglobin enhances cell performance and 120573-galactosidase production in Pichia pastorisrdquo Journal of Bio-science and Bioengineering vol 113 no 3 pp 332ndash337 2012

[22] A-S Xiong Q-H Yao R-H Peng et al ldquoHigh level expres-sion of a synthetic gene encoding Peniophora lycii phytase inmethylotrophic yeast Pichia pastorisrdquo Applied Microbiology andBiotechnology vol 72 no 5 pp 1039ndash1047 2006

[23] J K Yang and L Y Liu ldquoCodon optimization through a two-step gene synthesis leads to a high-level expression ofAspergillusniger lip2 gene in Pichia pastorisrdquo Journal of Molecular CatalysisB Enzymatic vol 63 no 3-4 pp 164ndash169 2010

[24] J-M Wu S-Y Wang and W-C Fu ldquoLower temperature cul-tures enlarge the effects ofVitreoscilla hemoglobin expression onrecombinant pichia pastorisrdquo International Journal of MolecularSciences vol 13 no 10 pp 13212ndash13226 2012

[25] H Huang P Yang H Luo et al ldquoHigh-level expression of atruncated 13-14-120573-D-glucanase from Fibrobacter succinogenesin Pichia pastoris by optimization of codons and fermentationrdquoApplied Microbiology and Biotechnology vol 78 no 1 pp 95ndash103 2008

[26] R Mora-Lugo M Madrigal V Yelemane and M Fernandez-Lahore ldquoImproved biomass and protein production in solid-state cultures of an Aspergillus sojae strain harboring the Vit-reoscilla hemoglobinrdquo Applied Microbiology and Biotechnologyvol 99 no 22 pp 9699ndash9708 2015

[27] F Li S Yang L Zhao Q Li and J Pei ldquoSynonymous condonusage bias and overexpression of a synthetic xynB gene fromAspergillus Niger NL-1 in Pichia pastorisrdquo BioResources vol 7no 2 pp 2330ndash2343 2012

[28] Y Wang Z Wang Q Xu et al ldquoLowering induction temper-ature for enhanced production of polygalacturonate lyase inrecombinant Pichia pastorisrdquo Process Biochemistry vol 44 no9 pp 949ndash954 2009







Contents





























References



















1 Introduction









2 Experimental










119878minus 119860119877)(119860119878+ 119860119877)


119877is peak areas









N435

F

COOHCOOH

COO OO O

O

OHOH

HOHO

HO HOHO

F

F

H3C

H2O

CH3


+

+ +

H3C

-Flurbiprofen(S R)

(a)

F

FO

F

O


O O

OHOH

HOHO

HOHO

HO

COO

CH3


CH3OH


++ +

N435

-Flurbiprofen(S R)

(b)








3032343638404244464850

Con

vers

ion

()

170 190 210 230 250 270150Shaken speed (rpm)






100150200250300350400450500550600

Con

vers

ion

()

10 20 30 40 50 60 70 800Enzyme (mg)



200

250

300

350

400

450

500

Con

vers

ion

()







0

10

20

30

40

50

60

70

Con

vers

ion

()






Esterification

Transesterification

0 5 10 15 20 25 3002468

1012141618

Lmiddothmiddotm

molminus

1)

1V

(

Lmiddotmolminus1

)1C (





4 Conclusion




Competing Interests


Acknowledgments


References

























1 Introduction







(a) (b)



3 Isolation Methods



4 Microbial Amylase



Digestion

Glucose

Digestion

Starch

Glucose

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

H

O

H

H

O

H OH

OH

H

O

H

H

O

H OH

OH

H

O

H

HO

O

H OH

OH

H

O

H

H

O

Starch

H


HH


H


CH2

CH2OH

CH2OH CH2OH

CH2OHCH2OHCH2OH








Region 1


Domain B

Domain C

N-term

C-term

1205731 1205732 1205733

12057341205735120573612057371205738






Promotersequence











Select amylase gene














A versicolor

(a)

(b)









Nor

mal

enzy

mat

ic p

roce

ssEn

zym

e enc

apsu

lated

bea

ds

StarchStarch

Glucose



Competing Interests


Acknowledgments


References




















































1 Introduction











2HPO4 004 CaCl

2 001

MgSO4times 7H

2O 003 MnSO

4 001 NaCl 03) Bac-


590)



6(BioRad USA)








1

1

1

61

61

61

bpu

bpu

bli

bli

bam

bam

121

121

121

bpublibam

SP

MP

PP

lowast

lowast




∙∙∙

∙∙

∙ ∙ ∙ ∙∙

∙∙

∙∙∙∙

∙∙




Sec pathway

∙∙

∙ ∙ ∙ ∙ ∙ ∙∙∙∙

∙∙∙∙

∙∙

∙∙

∙∙∙∙

∙∙∙ ∙ ∙ ∙∙

∙∙

∙∙

∙

(a)

yvdB

yvdA


yvcNBLi03719yrdF

ydfFydfE

yvcTyvdA

yvcL

yvcL

yvcK

yvcK

COG391

COG1660COG1481

COG-FruBCOG-NhoA

BPUM_3110

BPUM_3109BPUM_3107

COG_LdhA

COG-CynTCOG-SUL1

yvcJ

crh

crh




(b)

1

1

1

42

60

120

119

179

159

239

127

102

59

bpu

bpubli

blibam

bam

bpu

bpu

bpuSD

bli

bli

bli

bam

bam

bam


minus35 minus10




------------------------------------------------------------


(c)






N

R86

E72 R82

C

D53H101 K26

(a)

N

R86E72 R82

C

D53H101

K26

(b)

N

R86E72

R82

C

D53

H101

K26

(c)

92

61

41

56

81

9042

9981

100

96

02

98100

100

61

95









(d)


















280was




























18

16

14

12

10

8

6

4

2

0

RNas

e act

ivity

(U

mL)

302826242220181614121086420

Time (h)

Gro

wth

(OD590

) sp

ecifi

cra

te o

f bac

teria

l gro

wth

(sca

le18

1)

spec

ific r

ate o

f RN

ase

secr

etio

n (s

cale18

1)


7000

6000

5000

4000

3000

2000

1000

0

(a)

100

075

050

025

000

minus025

000 1000 2000 3000 4000

Time (min)


1000

00

minus1000

(AU

)

(mSmiddot

cmminus1)

(b)



4 Conclusions







280





10

15

25

35

40

55

70

100

130

180

M 1 2 3 4 5(kDa)

(a)

25

15

10

35

M 1 2 3(kDa)

(b)

10

15

25

35

40

55

70

100

130

180

M 1 2(kDa)

(c)

25

15

10

35

M 1 2(kDa)

(d)





1 binase 2 balifase



Competing Interests


Acknowledgments


References











































119898of 511mM


1 Introduction


2O2toH2OandO

2[1] H2O2
















2HPO4









2PO4


2PO4 300mM










2O2(diluted with


2O2permin


2O2


2O2





2O2





M 1

(bp)

100

250

500

7501000

2000






















100

100

100

85

51

97

91

99

99

100

98

8665

005












96P26901100O9366296P3356997AJO270321

Consensus

M

G

E

KM

MNMA

SSTD

SSQT

NKGK

KVPKl

LLLLt

TTTTt

TTTT

SGES

WFAWg

GGGG

AIAAp

PPPPv

VVVV

GGAGd

DDDD

NDNNq

QQQQn

NNNNs

SSSS

MLEIt

TTTTa

AAAAg

GGGG

SNVN

RRGPg

GGGGp

PPPP

TVVTl

LLLL

IMVIq

QQQQd

DDDD

VVQV

HHLHl

LLLL

LLLI

EDEEk

KKKKl

LLLL

ASAAh

HHHHf

FFFF

NDNN

RHRRe

EEEEr

RRRR

VIIVp

PPPPe

EEEEr

RRRRv

VVVVv

VVVVh

HHHHa

AAAA

KKRKg

GGGGa

AAAAg

GGGGa

AAAA

HGYHg

GGGG

YYTYf

FFFF

EETE

VVLVt

TTTT

NARNd

DDDD

VVVM

TTSS

KKRK

YYWYt

TTTT

KKRKa

AAAA

AKAK

FFFV

LLLF

SSSN

EEEG

VIVVg

GGGGk

KKKKr

RRRRt

TTTT

PEEP

LVTVf

FFFF

IVLVr

RRRRf

FFFFs

SSSSt

TTTTv

VVVV

AGAA

196P26901200O93662196P33569197AJO270321

Consensus g

GGGG

EESE

LKLLg

GGGG

SSASa

AAAAd

DDDD

TSAT

VAVVr

RRRRd

DDDDp

PPPPr

RRRRg

GGGG

FFWFa

AAAA

VVLVk

KKKKf

FFFFy

YYYYt

TTTTe

EEEE

EDEEg

GGGGn

NNNNy

YYYYd

DDDD

ILLIv

VVVVg

GGGGn

NNNNn

NNNNt

TTTTp

PPPP

VVVIf

FFFFf

FFFFi

IIII

RRKRd

DDDD

APAA

ILIIk

KKKKf

FFFFp

PPPPd

DDDDf

FFFFi

IIIIh

HHHHt

TTTTq

QQQQk

KKKKr

RRRR

DNDDp

PPPP

KAYRt

TTTT

HNGH

LCSL

KKQK

NDEN

PPAP

TDDT

AMNA

VFVMw

WWWWd

DDDDf

FFFF

WLWW

SSGSl

LLLL

STSSp

PPPPe

EEEEs

SSSS

LITLh

HHHHq

QQQQv

VVVVt

TTTT

IIWYl

LLLL

MFFF

SSGGd

DDDDr

RRRRg

GGGG

ITIIp

PPPP

AAAL

TTST

LYYYr

RRRR

HNHHm

MMMM

HNNNg

GGGG

FYYY

GSGGs

SSSS

296P26901300O93662296P33569297AJO270321

Consensus h

HHHHt

TTTT

FYYF

KKQKw

WWWW

TYNVn

NNNN

AEEE

EKAKg

GGGGe

EEEE

GYVA

VFFVw

WWWW

IVVV

KQKKy

YYYYh

HHHHf

FFFFk

KKKKt

TTTT

EDDNq

QQQQg

GGGG

VIIVk

KKKKn

NNNN

LLLM

DTTD

VLQP

NEDE

TEELa

AAAA

AENV

KKRK

IILI

AGAAg

GGGG

ESEE

NDDNp

PPPPd

DDDD

YHSY

HAHH

TTQT

ERREd

DDDDl

LLLL

FYRY

NEENa

AAAAi

IIII

EKEE

NKRKg

GGGGd

DDDD

YYFYp

PPPP

ASTSw

WWWW

KTTT

LLVL

YEQY

VMVVq

QQQQi

IIIIm

MMMM

PTPP

LPAL

EEAE

DQDDa

AAAA

NEAK

TDGTy

YYYYr

RRRRf

FFFF

DDNN

PIPP

FRFFd

DDDD

VILVt

TTTTk

KKKKv

VVVVw

WWWW

SPPS

QHHH

KGEKd

DDDD

YFYYp

PPPP

LTPL

IMVI

EKEE

VIIVg

GGGG

RKTR

MLLM

VVEV

395P26901400O93662395P33569396AJO270321

Consensus l

LLLL

DNNNr

RRRRn

NNNNp

PPPP

ETEEn

NNNN

YYIYf

FFFFa

AAAAe

EEEEv

VVVVe

EEEEq

QQQQ

AASA

TAITf

FFFFs

SSSSp

PPPP

GAAG

TNHN

LLFLv

VVVVp

PPPPg

GGGG

IIIV

DGGE

VIPPs

SSSSp

PPPPd

DDDDk

KKKKm

MMMMl

LLLLq

QQQQ

GGGAr

RRRR

LVLLf

FFFF

ASAAy

YYYY

HHGAd

DDDD

ATAAh

HHHH

RIRR

YHYYr

RRRR

VLVVg

GGGG

APIVn

NNNN

HYAH

QNDN

ALHL

LILLp

PPPP

IVVIn

NNNN

RARR

APPP

RKHR

NYAV

KSTE

VPEV

NEAN

NNRN

ST

YYHY

QQSQr

RRRRd

DDDDg

GGGG

QFFF

MMLM

RRR

FVYFd

DDDD

DAGN

NNRN

GGHG

GGKG

GSGG

SGAS

VPKV

YNNNy

YYYY

EWEEp

PPPPn

NNNNs

SSSSf

FFFFg

GGGGg

GGGGp

PPPP

KSVT

EPQE

SDTV

PSDS

EVRE

483P26901497O93662483P33569488AJO270321

Consensus

DYPH

KLLK

QEWT

APQT

APPP

YFTF

PGPPv

VVVV

QSTSg

GGGG

ILVM

AATA

G

DADE

SRHS

VTAV

SLAP

YYPY

DTSD

HHHD

PA

E

YNDDd

DDDD

HDDH

YFFY

TVTTq

QQQQa

AAAAg

GGGG

DNDDl

LLLLy

YYYYr

RRRR

D

LVLLm

MMMM

STSS

EDEE

DYDE

EDEE

RRKK

TEGA

RNRRl

LLLL

VVIV

EGDKn

NNNN

IILI

VVSV

NSGE

AHFS

MLIL

KSAK

PAKQ

VAVV

EQST

KKRK

ERDE

EDEi

IIII

KQAK

LLELr

RRRR

QQAQ

ITII

EAGR

HLNHf

FFFF

YFRY

KKRKa

AAAAd

DDDD

PREP

EDDD

YYFYg

GGGG

KSKRr

RRRR

VVLV

AAEA

EKAE

GGAG

LLVL

GEQG

LLAL

PDLQ

IIRV

KKGP

KED

DVD

SEV

RI

LT

AN

NA

M

T

N

E

483P26901504O93662483P33569488AJO270321

Consensus

E

R

A

R

A

T

E



RBS

f1Ori

Amp

pBR322 ori

LacI

T7Kat





M 1 2 3 4

25

70

200

85120

5060

40

30

20

15

(kDa)



0 10 20 30

Time (h)

40000

30000

20000

10000

0

Cata

lase

activ

ity (U

mL)


100806040200

Temperature (∘C)

70

80

90

100

110

120

Rela

tive a

ctiv

ity (

)

(a)

150

100

50

010050

Time (h)

Resid

ual a

ctiv

ity (

)

40∘C50∘C60∘C

70∘C80∘C90∘C

(b)









4 Conclusions



Competing Interests



lativ

e act

ivity

()

150

100

50

0

4 5 6 7 8 9 10

pH11


minus005

0

005

01

015

02

0 01 02 03 04 05minus01

1s (mmolL)minus1

R2 = 09972y = 03393x + 00066

1v

(min

120583m

ol)middot1

03


Acknowledgments


References


















































1 Introduction













)2

SO4

Na2

HPO4

KX2

`4

MgSO4

and CaCl2




4

)2

SO4

35 KH2

PO4

10 and MgSO4

times 7H2





3

Fe(CN)6




(1)












0 10 20 30 40 500

5

10

15

20

25

30

35

40

45

50


o

xida

tion

of D

-lact

ate

(b)

(a)










2






000 015 030 045 060 075 090 105 12005

101520253035404550

9095

100

Enzy

mat

ic co

nver

sion

of D

-lact

ate (

)


(c)

(b)

(a)


2




4 Conclusions



Competing Interests


Acknowledgments


References





















2











1 Introduction
















600






























xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

100100

200200

300300

400400

500500

600600

678

678


100VHb100VHb-opt



200VHb200VHb-opt



300VHb300VHb-opt



400VHb400VHb-opt



440VHb440VHb-opt










Gly

GGG 010 0 0GGA 032 9 11GGT 044 5 18GGC 014 15 0

Glu GAG 043 6 6GAA 057 0 0

Asp GAT 058 3 2GAC 042 7 8

Val

GTG 019 3 0GTA 015 0 0GTT 042 6 1GTC 023 6 14

Ala

GCG 006 2 0GCA 023 0 0GCT 045 12 7GCC 026 4 11

Arg


Lys AAG 053 6 5AAA 047 0 1

Ser


Stop TAA 053 1 1

Asn AAT 049 2 0AAC 051 13 15

Met ATG 100 3 3

IleATA 019 0 0ATT 050 1 1ATC 030 6 6

Thr

ACG 011 1 0ACA 024 1 0ACT 040 4 4ACC 025 19 23

Trp TGG 100 6 6

Cys TGT 065 0 1TGC 035 1 0

Tyr TAT 046 2 1TAC 055 15 16

Leu


Table 1 Continued


Phe TTT 054 2 1TTC 046 5 6

Gln CAG 039 8 7CAA 061 0 1

His CAT 057 0 0CAC 043 4 4

Pro

CCC 015 4 0CCG 009 1 0CCA 041 0 6CCT 035 1 0

0

5000

10000

15000

20000

25000

30000

35000


Induction time (h)

Activ

ity (U

mL)

00

05

10

15

20

25

30

35

40

45

50

Prot

ein

cont

ent (

mg

mL)

24 48 72 96 120 144 168







Gly

GGG 010 0 0GGA 032 1 0GGT 044 7 8GGC 014 0 0

Glu GAG 043 7 1GAA 057 2 8

Asp GAT 058 2 8GAC 042 6 0

Val

GTG 019 0 0GTA 015 0 0GTT 042 4 10GTC 023 10 4

Ala

GCG 006 0 0GCA 023 0 0GCT 045 17 23GCC 026 6 0

Arg


Lys AAG 053 9 10AAA 047 1 0

Ser


Stop TAA 053 1 1

Asn AAT 049 0 0AAC 051 4 4

Met ATG 100 3 3

IleATA 019 0 0ATT 050 5 5ATC 030 7 7

Thr

ACG 011 0 0ACA 024 0 0ACT 040 1 8ACC 025 7 0

Trp TGG 100 1 1

Cys TGT 065 1 1TGC 035 0 0

Tyr TAT 046 0 0TAC 055 4 4

Leu


Table 2 Continued


Phe TTT 054 0 2TTC 046 4 2

Gln CAG 039 6 2CAA 061 3 7

His CAT 057 1 4CAC 043 3 0

Pro

CCC 015 0 0CCG 009 0 0CCA 041 6 7CCT 035 1 0

0

10000

20000

30000

40000

50000

Induction time (h)

Activ

ity (U

mL)

50

100

150

200

250

300

Dry

cell

wei

ght (

gL)


24 48 72 96 120 144 168





00

5

10

15

20

25

30

35

40

Induction time (h)


SOU

R (120583

mol

O2m

ing

DCW

)

24 48 72 96 120 144 168 192 216

(a)

50

60

70

80

90

100

Cel

l via

bilit

y (

)

Induction time (h)


24 48 72 96 120 144

(b)


0

10000

20000

30000

40000

50000

60000

30282522

Xyla

nase

activ

ity (U

mL)

0

50

100

150

200

250

DCW

(gL

)



(a)

60

70

80

90

100

Cel

l via

bilit

y (

)

Induction time (h)24 48 72 96 120 144

30∘C28∘C

25∘C22∘C

(b)






4 Conclusions


Competing Interests




Acknowledgments


References




















































References



















1 Introduction









2 Experimental










119878minus 119860119877)(119860119878+ 119860119877)


119877is peak areas









N435

F

COOHCOOH

COO OO O

O

OHOH

HOHO

HO HOHO

F

F

H3C

H2O

CH3


+

+ +

H3C

-Flurbiprofen(S R)

(a)

F

FO

F

O


O O

OHOH

HOHO

HOHO

HO

COO

CH3


CH3OH


++ +

N435

-Flurbiprofen(S R)

(b)








3032343638404244464850

Con

vers

ion

()

170 190 210 230 250 270150Shaken speed (rpm)






100150200250300350400450500550600

Con

vers

ion

()

10 20 30 40 50 60 70 800Enzyme (mg)



200

250

300

350

400

450

500

Con

vers

ion

()







0

10

20

30

40

50

60

70

Con

vers

ion

()






Esterification

Transesterification

0 5 10 15 20 25 3002468

1012141618

Lmiddothmiddotm

molminus

1)

1V

(

Lmiddotmolminus1

)1C (





4 Conclusion




Competing Interests


Acknowledgments


References

























1 Introduction







(a) (b)



3 Isolation Methods



4 Microbial Amylase



Digestion

Glucose

Digestion

Starch

Glucose

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

H

O

H

H

O

H OH

OH

H

O

H

H

O

H OH

OH

H

O

H

HO

O

H OH

OH

H

O

H

H

O

Starch

H


HH


H


CH2

CH2OH

CH2OH CH2OH

CH2OHCH2OHCH2OH








Region 1


Domain B

Domain C

N-term

C-term

1205731 1205732 1205733

12057341205735120573612057371205738






Promotersequence











Select amylase gene














A versicolor

(a)

(b)









Nor

mal

enzy

mat

ic p

roce

ssEn

zym

e enc

apsu

lated

bea

ds

StarchStarch

Glucose



Competing Interests


Acknowledgments


References




















































1 Introduction











2HPO4 004 CaCl

2 001

MgSO4times 7H

2O 003 MnSO

4 001 NaCl 03) Bac-


590)



6(BioRad USA)








1

1

1

61

61

61

bpu

bpu

bli

bli

bam

bam

121

121

121

bpublibam

SP

MP

PP

lowast

lowast




∙∙∙

∙∙

∙ ∙ ∙ ∙∙

∙∙

∙∙∙∙

∙∙




Sec pathway

∙∙

∙ ∙ ∙ ∙ ∙ ∙∙∙∙

∙∙∙∙

∙∙

∙∙

∙∙∙∙

∙∙∙ ∙ ∙ ∙∙

∙∙

∙∙

∙

(a)

yvdB

yvdA


yvcNBLi03719yrdF

ydfFydfE

yvcTyvdA

yvcL

yvcL

yvcK

yvcK

COG391

COG1660COG1481

COG-FruBCOG-NhoA

BPUM_3110

BPUM_3109BPUM_3107

COG_LdhA

COG-CynTCOG-SUL1

yvcJ

crh

crh




(b)

1

1

1

42

60

120

119

179

159

239

127

102

59

bpu

bpubli

blibam

bam

bpu

bpu

bpuSD

bli

bli

bli

bam

bam

bam


minus35 minus10




------------------------------------------------------------


(c)






N

R86

E72 R82

C

D53H101 K26

(a)

N

R86E72 R82

C

D53H101

K26

(b)

N

R86E72

R82

C

D53

H101

K26

(c)

92

61

41

56

81

9042

9981

100

96

02

98100

100

61

95









(d)


















280was




























18

16

14

12

10

8

6

4

2

0

RNas

e act

ivity

(U

mL)

302826242220181614121086420

Time (h)

Gro

wth

(OD590

) sp

ecifi

cra

te o

f bac

teria

l gro

wth

(sca

le18

1)

spec

ific r

ate o

f RN

ase

secr

etio

n (s

cale18

1)


7000

6000

5000

4000

3000

2000

1000

0

(a)

100

075

050

025

000

minus025

000 1000 2000 3000 4000

Time (min)


1000

00

minus1000

(AU

)

(mSmiddot

cmminus1)

(b)



4 Conclusions







280





10

15

25

35

40

55

70

100

130

180

M 1 2 3 4 5(kDa)

(a)

25

15

10

35

M 1 2 3(kDa)

(b)

10

15

25

35

40

55

70

100

130

180

M 1 2(kDa)

(c)

25

15

10

35

M 1 2(kDa)

(d)





1 binase 2 balifase



Competing Interests


Acknowledgments


References











































119898of 511mM


1 Introduction


2O2toH2OandO

2[1] H2O2
















2HPO4









2PO4


2PO4 300mM










2O2(diluted with


2O2permin


2O2


2O2





2O2





M 1

(bp)

100

250

500

7501000

2000






















100

100

100

85

51

97

91

99

99

100

98

8665

005












96P26901100O9366296P3356997AJO270321

Consensus

M

G

E

KM

MNMA

SSTD

SSQT

NKGK

KVPKl

LLLLt

TTTTt

TTTT

SGES

WFAWg

GGGG

AIAAp

PPPPv

VVVV

GGAGd

DDDD

NDNNq

QQQQn

NNNNs

SSSS

MLEIt

TTTTa

AAAAg

GGGG

SNVN

RRGPg

GGGGp

PPPP

TVVTl

LLLL

IMVIq

QQQQd

DDDD

VVQV

HHLHl

LLLL

LLLI

EDEEk

KKKKl

LLLL

ASAAh

HHHHf

FFFF

NDNN

RHRRe

EEEEr

RRRR

VIIVp

PPPPe

EEEEr

RRRRv

VVVVv

VVVVh

HHHHa

AAAA

KKRKg

GGGGa

AAAAg

GGGGa

AAAA

HGYHg

GGGG

YYTYf

FFFF

EETE

VVLVt

TTTT

NARNd

DDDD

VVVM

TTSS

KKRK

YYWYt

TTTT

KKRKa

AAAA

AKAK

FFFV

LLLF

SSSN

EEEG

VIVVg

GGGGk

KKKKr

RRRRt

TTTT

PEEP

LVTVf

FFFF

IVLVr

RRRRf

FFFFs

SSSSt

TTTTv

VVVV

AGAA

196P26901200O93662196P33569197AJO270321

Consensus g

GGGG

EESE

LKLLg

GGGG

SSASa

AAAAd

DDDD

TSAT

VAVVr

RRRRd

DDDDp

PPPPr

RRRRg

GGGG

FFWFa

AAAA

VVLVk

KKKKf

FFFFy

YYYYt

TTTTe

EEEE

EDEEg

GGGGn

NNNNy

YYYYd

DDDD

ILLIv

VVVVg

GGGGn

NNNNn

NNNNt

TTTTp

PPPP

VVVIf

FFFFf

FFFFi

IIII

RRKRd

DDDD

APAA

ILIIk

KKKKf

FFFFp

PPPPd

DDDDf

FFFFi

IIIIh

HHHHt

TTTTq

QQQQk

KKKKr

RRRR

DNDDp

PPPP

KAYRt

TTTT

HNGH

LCSL

KKQK

NDEN

PPAP

TDDT

AMNA

VFVMw

WWWWd

DDDDf

FFFF

WLWW

SSGSl

LLLL

STSSp

PPPPe

EEEEs

SSSS

LITLh

HHHHq

QQQQv

VVVVt

TTTT

IIWYl

LLLL

MFFF

SSGGd

DDDDr

RRRRg

GGGG

ITIIp

PPPP

AAAL

TTST

LYYYr

RRRR

HNHHm

MMMM

HNNNg

GGGG

FYYY

GSGGs

SSSS

296P26901300O93662296P33569297AJO270321

Consensus h

HHHHt

TTTT

FYYF

KKQKw

WWWW

TYNVn

NNNN

AEEE

EKAKg

GGGGe

EEEE

GYVA

VFFVw

WWWW

IVVV

KQKKy

YYYYh

HHHHf

FFFFk

KKKKt

TTTT

EDDNq

QQQQg

GGGG

VIIVk

KKKKn

NNNN

LLLM

DTTD

VLQP

NEDE

TEELa

AAAA

AENV

KKRK

IILI

AGAAg

GGGG

ESEE

NDDNp

PPPPd

DDDD

YHSY

HAHH

TTQT

ERREd

DDDDl

LLLL

FYRY

NEENa

AAAAi

IIII

EKEE

NKRKg

GGGGd

DDDD

YYFYp

PPPP

ASTSw

WWWW

KTTT

LLVL

YEQY

VMVVq

QQQQi

IIIIm

MMMM

PTPP

LPAL

EEAE

DQDDa

AAAA

NEAK

TDGTy

YYYYr

RRRRf

FFFF

DDNN

PIPP

FRFFd

DDDD

VILVt

TTTTk

KKKKv

VVVVw

WWWW

SPPS

QHHH

KGEKd

DDDD

YFYYp

PPPP

LTPL

IMVI

EKEE

VIIVg

GGGG

RKTR

MLLM

VVEV

395P26901400O93662395P33569396AJO270321

Consensus l

LLLL

DNNNr

RRRRn

NNNNp

PPPP

ETEEn

NNNN

YYIYf

FFFFa

AAAAe

EEEEv

VVVVe

EEEEq

QQQQ

AASA

TAITf

FFFFs

SSSSp

PPPP

GAAG

TNHN

LLFLv

VVVVp

PPPPg

GGGG

IIIV

DGGE

VIPPs

SSSSp

PPPPd

DDDDk

KKKKm

MMMMl

LLLLq

QQQQ

GGGAr

RRRR

LVLLf

FFFF

ASAAy

YYYY

HHGAd

DDDD

ATAAh

HHHH

RIRR

YHYYr

RRRR

VLVVg

GGGG

APIVn

NNNN

HYAH

QNDN

ALHL

LILLp

PPPP

IVVIn

NNNN

RARR

APPP

RKHR

NYAV

KSTE

VPEV

NEAN

NNRN

ST

YYHY

QQSQr

RRRRd

DDDDg

GGGG

QFFF

MMLM

RRR

FVYFd

DDDD

DAGN

NNRN

GGHG

GGKG

GSGG

SGAS

VPKV

YNNNy

YYYY

EWEEp

PPPPn

NNNNs

SSSSf

FFFFg

GGGGg

GGGGp

PPPP

KSVT

EPQE

SDTV

PSDS

EVRE

483P26901497O93662483P33569488AJO270321

Consensus

DYPH

KLLK

QEWT

APQT

APPP

YFTF

PGPPv

VVVV

QSTSg

GGGG

ILVM

AATA

G

DADE

SRHS

VTAV

SLAP

YYPY

DTSD

HHHD

PA

E

YNDDd

DDDD

HDDH

YFFY

TVTTq

QQQQa

AAAAg

GGGG

DNDDl

LLLLy

YYYYr

RRRR

D

LVLLm

MMMM

STSS

EDEE

DYDE

EDEE

RRKK

TEGA

RNRRl

LLLL

VVIV

EGDKn

NNNN

IILI

VVSV

NSGE

AHFS

MLIL

KSAK

PAKQ

VAVV

EQST

KKRK

ERDE

EDEi

IIII

KQAK

LLELr

RRRR

QQAQ

ITII

EAGR

HLNHf

FFFF

YFRY

KKRKa

AAAAd

DDDD

PREP

EDDD

YYFYg

GGGG

KSKRr

RRRR

VVLV

AAEA

EKAE

GGAG

LLVL

GEQG

LLAL

PDLQ

IIRV

KKGP

KED

DVD

SEV

RI

LT

AN

NA

M

T

N

E

483P26901504O93662483P33569488AJO270321

Consensus

E

R

A

R

A

T

E



RBS

f1Ori

Amp

pBR322 ori

LacI

T7Kat





M 1 2 3 4

25

70

200

85120

5060

40

30

20

15

(kDa)



0 10 20 30

Time (h)

40000

30000

20000

10000

0

Cata

lase

activ

ity (U

mL)


100806040200

Temperature (∘C)

70

80

90

100

110

120

Rela

tive a

ctiv

ity (

)

(a)

150

100

50

010050

Time (h)

Resid

ual a

ctiv

ity (

)

40∘C50∘C60∘C

70∘C80∘C90∘C

(b)









4 Conclusions



Competing Interests



lativ

e act

ivity

()

150

100

50

0

4 5 6 7 8 9 10

pH11


minus005

0

005

01

015

02

0 01 02 03 04 05minus01

1s (mmolL)minus1

R2 = 09972y = 03393x + 00066

1v

(min

120583m

ol)middot1

03


Acknowledgments


References


















































1 Introduction













)2

SO4

Na2

HPO4

KX2

`4

MgSO4

and CaCl2




4

)2

SO4

35 KH2

PO4

10 and MgSO4

times 7H2





3

Fe(CN)6




(1)












0 10 20 30 40 500

5

10

15

20

25

30

35

40

45

50


o

xida

tion

of D

-lact

ate

(b)

(a)










2






000 015 030 045 060 075 090 105 12005

101520253035404550

9095

100

Enzy

mat

ic co

nver

sion

of D

-lact

ate (

)


(c)

(b)

(a)


2




4 Conclusions



Competing Interests


Acknowledgments


References





















2











1 Introduction
















600






























xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

100100

200200

300300

400400

500500

600600

678

678


100VHb100VHb-opt



200VHb200VHb-opt



300VHb300VHb-opt



400VHb400VHb-opt



440VHb440VHb-opt










Gly

GGG 010 0 0GGA 032 9 11GGT 044 5 18GGC 014 15 0

Glu GAG 043 6 6GAA 057 0 0

Asp GAT 058 3 2GAC 042 7 8

Val

GTG 019 3 0GTA 015 0 0GTT 042 6 1GTC 023 6 14

Ala

GCG 006 2 0GCA 023 0 0GCT 045 12 7GCC 026 4 11

Arg


Lys AAG 053 6 5AAA 047 0 1

Ser


Stop TAA 053 1 1

Asn AAT 049 2 0AAC 051 13 15

Met ATG 100 3 3

IleATA 019 0 0ATT 050 1 1ATC 030 6 6

Thr

ACG 011 1 0ACA 024 1 0ACT 040 4 4ACC 025 19 23

Trp TGG 100 6 6

Cys TGT 065 0 1TGC 035 1 0

Tyr TAT 046 2 1TAC 055 15 16

Leu


Table 1 Continued


Phe TTT 054 2 1TTC 046 5 6

Gln CAG 039 8 7CAA 061 0 1

His CAT 057 0 0CAC 043 4 4

Pro

CCC 015 4 0CCG 009 1 0CCA 041 0 6CCT 035 1 0

0

5000

10000

15000

20000

25000

30000

35000


Induction time (h)

Activ

ity (U

mL)

00

05

10

15

20

25

30

35

40

45

50

Prot

ein

cont

ent (

mg

mL)

24 48 72 96 120 144 168







Gly

GGG 010 0 0GGA 032 1 0GGT 044 7 8GGC 014 0 0

Glu GAG 043 7 1GAA 057 2 8

Asp GAT 058 2 8GAC 042 6 0

Val

GTG 019 0 0GTA 015 0 0GTT 042 4 10GTC 023 10 4

Ala

GCG 006 0 0GCA 023 0 0GCT 045 17 23GCC 026 6 0

Arg


Lys AAG 053 9 10AAA 047 1 0

Ser


Stop TAA 053 1 1

Asn AAT 049 0 0AAC 051 4 4

Met ATG 100 3 3

IleATA 019 0 0ATT 050 5 5ATC 030 7 7

Thr

ACG 011 0 0ACA 024 0 0ACT 040 1 8ACC 025 7 0

Trp TGG 100 1 1

Cys TGT 065 1 1TGC 035 0 0

Tyr TAT 046 0 0TAC 055 4 4

Leu


Table 2 Continued


Phe TTT 054 0 2TTC 046 4 2

Gln CAG 039 6 2CAA 061 3 7

His CAT 057 1 4CAC 043 3 0

Pro

CCC 015 0 0CCG 009 0 0CCA 041 6 7CCT 035 1 0

0

10000

20000

30000

40000

50000

Induction time (h)

Activ

ity (U

mL)

50

100

150

200

250

300

Dry

cell

wei

ght (

gL)


24 48 72 96 120 144 168





00

5

10

15

20

25

30

35

40

Induction time (h)


SOU

R (120583

mol

O2m

ing

DCW

)

24 48 72 96 120 144 168 192 216

(a)

50

60

70

80

90

100

Cel

l via

bilit

y (

)

Induction time (h)


24 48 72 96 120 144

(b)


0

10000

20000

30000

40000

50000

60000

30282522

Xyla

nase

activ

ity (U

mL)

0

50

100

150

200

250

DCW

(gL

)



(a)

60

70

80

90

100

Cel

l via

bilit

y (

)

Induction time (h)24 48 72 96 120 144

30∘C28∘C

25∘C22∘C

(b)






4 Conclusions


Competing Interests




Acknowledgments


References









































1 Introduction









2 Experimental










119878minus 119860119877)(119860119878+ 119860119877)


119877is peak areas









N435

F

COOHCOOH

COO OO O

O

OHOH

HOHO

HO HOHO

F

F

H3C

H2O

CH3


+

+ +

H3C

-Flurbiprofen(S R)

(a)

F

FO

F

O


O O

OHOH

HOHO

HOHO

HO

COO

CH3


CH3OH


++ +

N435

-Flurbiprofen(S R)

(b)








3032343638404244464850

Con

vers

ion

()

170 190 210 230 250 270150Shaken speed (rpm)






100150200250300350400450500550600

Con

vers

ion

()

10 20 30 40 50 60 70 800Enzyme (mg)



200

250

300

350

400

450

500

Con

vers

ion

()







0

10

20

30

40

50

60

70

Con

vers

ion

()






Esterification

Transesterification

0 5 10 15 20 25 3002468

1012141618

Lmiddothmiddotm

molminus

1)

1V

(

Lmiddotmolminus1

)1C (





4 Conclusion




Competing Interests


Acknowledgments


References

























1 Introduction







(a) (b)



3 Isolation Methods



4 Microbial Amylase



Digestion

Glucose

Digestion

Starch

Glucose

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

H

O

H

H

O

H OH

OH

H

O

H

H

O

H OH

OH

H

O

H

HO

O

H OH

OH

H

O

H

H

O

Starch

H


HH


H


CH2

CH2OH

CH2OH CH2OH

CH2OHCH2OHCH2OH








Region 1


Domain B

Domain C

N-term

C-term

1205731 1205732 1205733

12057341205735120573612057371205738






Promotersequence











Select amylase gene














A versicolor

(a)

(b)









Nor

mal

enzy

mat

ic p

roce

ssEn

zym

e enc

apsu

lated

bea

ds

StarchStarch

Glucose



Competing Interests


Acknowledgments


References




















































1 Introduction











2HPO4 004 CaCl

2 001

MgSO4times 7H

2O 003 MnSO

4 001 NaCl 03) Bac-


590)



6(BioRad USA)








1

1

1

61

61

61

bpu

bpu

bli

bli

bam

bam

121

121

121

bpublibam

SP

MP

PP

lowast

lowast




∙∙∙

∙∙

∙ ∙ ∙ ∙∙

∙∙

∙∙∙∙

∙∙




Sec pathway

∙∙

∙ ∙ ∙ ∙ ∙ ∙∙∙∙

∙∙∙∙

∙∙

∙∙

∙∙∙∙

∙∙∙ ∙ ∙ ∙∙

∙∙

∙∙

∙

(a)

yvdB

yvdA


yvcNBLi03719yrdF

ydfFydfE

yvcTyvdA

yvcL

yvcL

yvcK

yvcK

COG391

COG1660COG1481

COG-FruBCOG-NhoA

BPUM_3110

BPUM_3109BPUM_3107

COG_LdhA

COG-CynTCOG-SUL1

yvcJ

crh

crh




(b)

1

1

1

42

60

120

119

179

159

239

127

102

59

bpu

bpubli

blibam

bam

bpu

bpu

bpuSD

bli

bli

bli

bam

bam

bam


minus35 minus10




------------------------------------------------------------


(c)






N

R86

E72 R82

C

D53H101 K26

(a)

N

R86E72 R82

C

D53H101

K26

(b)

N

R86E72

R82

C

D53

H101

K26

(c)

92

61

41

56

81

9042

9981

100

96

02

98100

100

61

95









(d)


















280was




























18

16

14

12

10

8

6

4

2

0

RNas

e act

ivity

(U

mL)

302826242220181614121086420

Time (h)

Gro

wth

(OD590

) sp

ecifi

cra

te o

f bac

teria

l gro

wth

(sca

le18

1)

spec

ific r

ate o

f RN

ase

secr

etio

n (s

cale18

1)


7000

6000

5000

4000

3000

2000

1000

0

(a)

100

075

050

025

000

minus025

000 1000 2000 3000 4000

Time (min)


1000

00

minus1000

(AU

)

(mSmiddot

cmminus1)

(b)



4 Conclusions







280





10

15

25

35

40

55

70

100

130

180

M 1 2 3 4 5(kDa)

(a)

25

15

10

35

M 1 2 3(kDa)

(b)

10

15

25

35

40

55

70

100

130

180

M 1 2(kDa)

(c)

25

15

10

35

M 1 2(kDa)

(d)





1 binase 2 balifase



Competing Interests


Acknowledgments


References











































119898of 511mM


1 Introduction


2O2toH2OandO

2[1] H2O2
















2HPO4









2PO4


2PO4 300mM










2O2(diluted with


2O2permin


2O2


2O2





2O2





M 1

(bp)

100

250

500

7501000

2000






















100

100

100

85

51

97

91

99

99

100

98

8665

005












96P26901100O9366296P3356997AJO270321

Consensus

M

G

E

KM

MNMA

SSTD

SSQT

NKGK

KVPKl

LLLLt

TTTTt

TTTT

SGES

WFAWg

GGGG

AIAAp

PPPPv

VVVV

GGAGd

DDDD

NDNNq

QQQQn

NNNNs

SSSS

MLEIt

TTTTa

AAAAg

GGGG

SNVN

RRGPg

GGGGp

PPPP

TVVTl

LLLL

IMVIq

QQQQd

DDDD

VVQV

HHLHl

LLLL

LLLI

EDEEk

KKKKl

LLLL

ASAAh

HHHHf

FFFF

NDNN

RHRRe

EEEEr

RRRR

VIIVp

PPPPe

EEEEr

RRRRv

VVVVv

VVVVh

HHHHa

AAAA

KKRKg

GGGGa

AAAAg

GGGGa

AAAA

HGYHg

GGGG

YYTYf

FFFF

EETE

VVLVt

TTTT

NARNd

DDDD

VVVM

TTSS

KKRK

YYWYt

TTTT

KKRKa

AAAA

AKAK

FFFV

LLLF

SSSN

EEEG

VIVVg

GGGGk

KKKKr

RRRRt

TTTT

PEEP

LVTVf

FFFF

IVLVr

RRRRf

FFFFs

SSSSt

TTTTv

VVVV

AGAA

196P26901200O93662196P33569197AJO270321

Consensus g

GGGG

EESE

LKLLg

GGGG

SSASa

AAAAd

DDDD

TSAT

VAVVr

RRRRd

DDDDp

PPPPr

RRRRg

GGGG

FFWFa

AAAA

VVLVk

KKKKf

FFFFy

YYYYt

TTTTe

EEEE

EDEEg

GGGGn

NNNNy

YYYYd

DDDD

ILLIv

VVVVg

GGGGn

NNNNn

NNNNt

TTTTp

PPPP

VVVIf

FFFFf

FFFFi

IIII

RRKRd

DDDD

APAA

ILIIk

KKKKf

FFFFp

PPPPd

DDDDf

FFFFi

IIIIh

HHHHt

TTTTq

QQQQk

KKKKr

RRRR

DNDDp

PPPP

KAYRt

TTTT

HNGH

LCSL

KKQK

NDEN

PPAP

TDDT

AMNA

VFVMw

WWWWd

DDDDf

FFFF

WLWW

SSGSl

LLLL

STSSp

PPPPe

EEEEs

SSSS

LITLh

HHHHq

QQQQv

VVVVt

TTTT

IIWYl

LLLL

MFFF

SSGGd

DDDDr

RRRRg

GGGG

ITIIp

PPPP

AAAL

TTST

LYYYr

RRRR

HNHHm

MMMM

HNNNg

GGGG

FYYY

GSGGs

SSSS

296P26901300O93662296P33569297AJO270321

Consensus h

HHHHt

TTTT

FYYF

KKQKw

WWWW

TYNVn

NNNN

AEEE

EKAKg

GGGGe

EEEE

GYVA

VFFVw

WWWW

IVVV

KQKKy

YYYYh

HHHHf

FFFFk

KKKKt

TTTT

EDDNq

QQQQg

GGGG

VIIVk

KKKKn

NNNN

LLLM

DTTD

VLQP

NEDE

TEELa

AAAA

AENV

KKRK

IILI

AGAAg

GGGG

ESEE

NDDNp

PPPPd

DDDD

YHSY

HAHH

TTQT

ERREd

DDDDl

LLLL

FYRY

NEENa

AAAAi

IIII

EKEE

NKRKg

GGGGd

DDDD

YYFYp

PPPP

ASTSw

WWWW

KTTT

LLVL

YEQY

VMVVq

QQQQi

IIIIm

MMMM

PTPP

LPAL

EEAE

DQDDa

AAAA

NEAK

TDGTy

YYYYr

RRRRf

FFFF

DDNN

PIPP

FRFFd

DDDD

VILVt

TTTTk

KKKKv

VVVVw

WWWW

SPPS

QHHH

KGEKd

DDDD

YFYYp

PPPP

LTPL

IMVI

EKEE

VIIVg

GGGG

RKTR

MLLM

VVEV

395P26901400O93662395P33569396AJO270321

Consensus l

LLLL

DNNNr

RRRRn

NNNNp

PPPP

ETEEn

NNNN

YYIYf

FFFFa

AAAAe

EEEEv

VVVVe

EEEEq

QQQQ

AASA

TAITf

FFFFs

SSSSp

PPPP

GAAG

TNHN

LLFLv

VVVVp

PPPPg

GGGG

IIIV

DGGE

VIPPs

SSSSp

PPPPd

DDDDk

KKKKm

MMMMl

LLLLq

QQQQ

GGGAr

RRRR

LVLLf

FFFF

ASAAy

YYYY

HHGAd

DDDD

ATAAh

HHHH

RIRR

YHYYr

RRRR

VLVVg

GGGG

APIVn

NNNN

HYAH

QNDN

ALHL

LILLp

PPPP

IVVIn

NNNN

RARR

APPP

RKHR

NYAV

KSTE

VPEV

NEAN

NNRN

ST

YYHY

QQSQr

RRRRd

DDDDg

GGGG

QFFF

MMLM

RRR

FVYFd

DDDD

DAGN

NNRN

GGHG

GGKG

GSGG

SGAS

VPKV

YNNNy

YYYY

EWEEp

PPPPn

NNNNs

SSSSf

FFFFg

GGGGg

GGGGp

PPPP

KSVT

EPQE

SDTV

PSDS

EVRE

483P26901497O93662483P33569488AJO270321

Consensus

DYPH

KLLK

QEWT

APQT

APPP

YFTF

PGPPv

VVVV

QSTSg

GGGG

ILVM

AATA

G

DADE

SRHS

VTAV

SLAP

YYPY

DTSD

HHHD

PA

E

YNDDd

DDDD

HDDH

YFFY

TVTTq

QQQQa

AAAAg

GGGG

DNDDl

LLLLy

YYYYr

RRRR

D

LVLLm

MMMM

STSS

EDEE

DYDE

EDEE

RRKK

TEGA

RNRRl

LLLL

VVIV

EGDKn

NNNN

IILI

VVSV

NSGE

AHFS

MLIL

KSAK

PAKQ

VAVV

EQST

KKRK

ERDE

EDEi

IIII

KQAK

LLELr

RRRR

QQAQ

ITII

EAGR

HLNHf

FFFF

YFRY

KKRKa

AAAAd

DDDD

PREP

EDDD

YYFYg

GGGG

KSKRr

RRRR

VVLV

AAEA

EKAE

GGAG

LLVL

GEQG

LLAL

PDLQ

IIRV

KKGP

KED

DVD

SEV

RI

LT

AN

NA

M

T

N

E

483P26901504O93662483P33569488AJO270321

Consensus

E

R

A

R

A

T

E



RBS

f1Ori

Amp

pBR322 ori

LacI

T7Kat





M 1 2 3 4

25

70

200

85120

5060

40

30

20

15

(kDa)



0 10 20 30

Time (h)

40000

30000

20000

10000

0

Cata

lase

activ

ity (U

mL)


100806040200

Temperature (∘C)

70

80

90

100

110

120

Rela

tive a

ctiv

ity (

)

(a)

150

100

50

010050

Time (h)

Resid

ual a

ctiv

ity (

)

40∘C50∘C60∘C

70∘C80∘C90∘C

(b)









4 Conclusions



Competing Interests



lativ

e act

ivity

()

150

100

50

0

4 5 6 7 8 9 10

pH11


minus005

0

005

01

015

02

0 01 02 03 04 05minus01

1s (mmolL)minus1

R2 = 09972y = 03393x + 00066

1v

(min

120583m

ol)middot1

03


Acknowledgments


References


















































1 Introduction













)2

SO4

Na2

HPO4

KX2

`4

MgSO4

and CaCl2




4

)2

SO4

35 KH2

PO4

10 and MgSO4

times 7H2





3

Fe(CN)6




(1)












0 10 20 30 40 500

5

10

15

20

25

30

35

40

45

50


o

xida

tion

of D

-lact

ate

(b)

(a)










2






000 015 030 045 060 075 090 105 12005

101520253035404550

9095

100

Enzy

mat

ic co

nver

sion

of D

-lact

ate (

)


(c)

(b)

(a)


2




4 Conclusions



Competing Interests


Acknowledgments


References





















2











1 Introduction
















600






























xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

100100

200200

300300

400400

500500

600600

678

678


100VHb100VHb-opt



200VHb200VHb-opt



300VHb300VHb-opt



400VHb400VHb-opt



440VHb440VHb-opt










Gly

GGG 010 0 0GGA 032 9 11GGT 044 5 18GGC 014 15 0

Glu GAG 043 6 6GAA 057 0 0

Asp GAT 058 3 2GAC 042 7 8

Val

GTG 019 3 0GTA 015 0 0GTT 042 6 1GTC 023 6 14

Ala

GCG 006 2 0GCA 023 0 0GCT 045 12 7GCC 026 4 11

Arg


Lys AAG 053 6 5AAA 047 0 1

Ser


Stop TAA 053 1 1

Asn AAT 049 2 0AAC 051 13 15

Met ATG 100 3 3

IleATA 019 0 0ATT 050 1 1ATC 030 6 6

Thr

ACG 011 1 0ACA 024 1 0ACT 040 4 4ACC 025 19 23

Trp TGG 100 6 6

Cys TGT 065 0 1TGC 035 1 0

Tyr TAT 046 2 1TAC 055 15 16

Leu


Table 1 Continued


Phe TTT 054 2 1TTC 046 5 6

Gln CAG 039 8 7CAA 061 0 1

His CAT 057 0 0CAC 043 4 4

Pro

CCC 015 4 0CCG 009 1 0CCA 041 0 6CCT 035 1 0

0

5000

10000

15000

20000

25000

30000

35000


Induction time (h)

Activ

ity (U

mL)

00

05

10

15

20

25

30

35

40

45

50

Prot

ein

cont

ent (

mg

mL)

24 48 72 96 120 144 168







Gly

GGG 010 0 0GGA 032 1 0GGT 044 7 8GGC 014 0 0

Glu GAG 043 7 1GAA 057 2 8

Asp GAT 058 2 8GAC 042 6 0

Val

GTG 019 0 0GTA 015 0 0GTT 042 4 10GTC 023 10 4

Ala

GCG 006 0 0GCA 023 0 0GCT 045 17 23GCC 026 6 0

Arg


Lys AAG 053 9 10AAA 047 1 0

Ser


Stop TAA 053 1 1

Asn AAT 049 0 0AAC 051 4 4

Met ATG 100 3 3

IleATA 019 0 0ATT 050 5 5ATC 030 7 7

Thr

ACG 011 0 0ACA 024 0 0ACT 040 1 8ACC 025 7 0

Trp TGG 100 1 1

Cys TGT 065 1 1TGC 035 0 0

Tyr TAT 046 0 0TAC 055 4 4

Leu


Table 2 Continued


Phe TTT 054 0 2TTC 046 4 2

Gln CAG 039 6 2CAA 061 3 7

His CAT 057 1 4CAC 043 3 0

Pro

CCC 015 0 0CCG 009 0 0CCA 041 6 7CCT 035 1 0

0

10000

20000

30000

40000

50000

Induction time (h)

Activ

ity (U

mL)

50

100

150

200

250

300

Dry

cell

wei

ght (

gL)


24 48 72 96 120 144 168





00

5

10

15

20

25

30

35

40

Induction time (h)


SOU

R (120583

mol

O2m

ing

DCW

)

24 48 72 96 120 144 168 192 216

(a)

50

60

70

80

90

100

Cel

l via

bilit

y (

)

Induction time (h)


24 48 72 96 120 144

(b)


0

10000

20000

30000

40000

50000

60000

30282522

Xyla

nase

activ

ity (U

mL)

0

50

100

150

200

250

DCW

(gL

)



(a)

60

70

80

90

100

Cel

l via

bilit

y (

)

Induction time (h)24 48 72 96 120 144

30∘C28∘C

25∘C22∘C

(b)






4 Conclusions


Competing Interests




Acknowledgments


References


































2 Experimental










119878minus 119860119877)(119860119878+ 119860119877)


119877is peak areas









N435

F

COOHCOOH

COO OO O

O

OHOH

HOHO

HO HOHO

F

F

H3C

H2O

CH3


+

+ +

H3C

-Flurbiprofen(S R)

(a)

F

FO

F

O


O O

OHOH

HOHO

HOHO

HO

COO

CH3


CH3OH


++ +

N435

-Flurbiprofen(S R)

(b)








3032343638404244464850

Con

vers

ion

()

170 190 210 230 250 270150Shaken speed (rpm)






100150200250300350400450500550600

Con

vers

ion

()

10 20 30 40 50 60 70 800Enzyme (mg)



200

250

300

350

400

450

500

Con

vers

ion

()







0

10

20

30

40

50

60

70

Con

vers

ion

()






Esterification

Transesterification

0 5 10 15 20 25 3002468

1012141618

Lmiddothmiddotm

molminus

1)

1V

(

Lmiddotmolminus1

)1C (





4 Conclusion




Competing Interests


Acknowledgments


References

























1 Introduction







(a) (b)



3 Isolation Methods



4 Microbial Amylase



Digestion

Glucose

Digestion

Starch

Glucose

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

OH

HO

H

HH

O

H OH

OH

H

O

H

H

O

H OH

OH

H

O

H

H

O

H OH

OH

H

O

H

HO

O

H OH

OH

H

O

H

H

O

Starch

H


HH


H


CH2

CH2OH

CH2OH CH2OH

CH2OHCH2OHCH2OH








Region 1


Domain B

Domain C

N-term

C-term

1205731 1205732 1205733

12057341205735120573612057371205738






Promotersequence











Select amylase gene














A versicolor

(a)

(b)









Nor

mal

enzy

mat

ic p

roce

ssEn

zym

e enc

apsu

lated

bea

ds

StarchStarch

Glucose



Competing Interests


Acknowledgments


References




















































1 Introduction











2HPO4 004 CaCl

2 001

MgSO4times 7H

2O 003 MnSO

4 001 NaCl 03) Bac-


590)



6(BioRad USA)








1

1

1

61

61

61

bpu

bpu

bli

bli

bam

bam

121

121

121

bpublibam

SP

MP

PP

lowast

lowast




∙∙∙

∙∙

∙ ∙ ∙ ∙∙

∙∙

∙∙∙∙

∙∙




Sec pathway

∙∙

∙ ∙ ∙ ∙ ∙ ∙∙∙∙

∙∙∙∙

∙∙

∙∙

∙∙∙∙

∙∙∙ ∙ ∙ ∙∙

∙∙

∙∙

∙

(a)

yvdB

yvdA


yvcNBLi03719yrdF

ydfFydfE

yvcTyvdA

yvcL

yvcL

yvcK

yvcK

COG391

COG1660COG1481

COG-FruBCOG-NhoA

BPUM_3110

BPUM_3109BPUM_3107

COG_LdhA

COG-CynTCOG-SUL1

yvcJ

crh

crh




(b)

1

1

1

42

60

120

119

179

159

239

127

102

59

bpu

bpubli

blibam

bam

bpu

bpu

bpuSD

bli

bli

bli

bam

bam

bam


minus35 minus10




------------------------------------------------------------


(c)






N

R86

E72 R82

C

D53H101 K26

(a)

N

R86E72 R82

C

D53H101

K26

(b)

N

R86E72

R82

C

D53

H101

K26

(c)

92

61

41

56

81

9042

9981

100

96

02

98100

100

61

95









(d)


















280was




























18

16

14

12

10

8

6

4

2

0

RNas

e act

ivity

(U

mL)

302826242220181614121086420

Time (h)

Gro

wth

(OD590

) sp

ecifi

cra

te o

f bac

teria

l gro

wth

(sca

le18

1)

spec

ific r

ate o

f RN

ase

secr

etio

n (s

cale18

1)


7000

6000

5000

4000

3000

2000

1000

0

(a)

100

075

050

025

000

minus025

000 1000 2000 3000 4000

Time (min)


1000

00

minus1000

(AU

)

(mSmiddot

cmminus1)

(b)



4 Conclusions







280





10

15

25

35

40

55

70

100

130

180

M 1 2 3 4 5(kDa)

(a)

25

15

10

35

M 1 2 3(kDa)

(b)

10

15

25

35

40

55

70

100

130

180

M 1 2(kDa)

(c)

25

15

10

35

M 1 2(kDa)

(d)





1 binase 2 balifase



Competing Interests


Acknowledgments


References











































119898of 511mM


1 Introduction


2O2toH2OandO

2[1] H2O2
















2HPO4









2PO4


2PO4 300mM










2O2(diluted with


2O2permin


2O2


2O2





2O2





M 1

(bp)

100

250

500

7501000

2000






















100

100

100

85

51

97

91

99

99

100

98

8665

005












96P26901100O9366296P3356997AJO270321

Consensus

M

G

E

KM

MNMA

SSTD

SSQT

NKGK

KVPKl

LLLLt

TTTTt

TTTT

SGES

WFAWg

GGGG

AIAAp

PPPPv

VVVV

GGAGd

DDDD

NDNNq

QQQQn

NNNNs

SSSS

MLEIt

TTTTa

AAAAg

GGGG

SNVN

RRGPg

GGGGp

PPPP

TVVTl

LLLL

IMVIq

QQQQd

DDDD

VVQV

HHLHl

LLLL

LLLI

EDEEk

KKKKl

LLLL

ASAAh

HHHHf

FFFF

NDNN

RHRRe

EEEEr

RRRR

VIIVp

PPPPe

EEEEr

RRRRv

VVVVv

VVVVh

HHHHa

AAAA

KKRKg

GGGGa

AAAAg

GGGGa

AAAA

HGYHg

GGGG

YYTYf

FFFF

EETE

VVLVt

TTTT

NARNd

DDDD

VVVM

TTSS

KKRK

YYWYt

TTTT

KKRKa

AAAA

AKAK

FFFV

LLLF

SSSN

EEEG

VIVVg

GGGGk

KKKKr

RRRRt

TTTT

PEEP

LVTVf

FFFF

IVLVr

RRRRf

FFFFs

SSSSt

TTTTv

VVVV

AGAA

196P26901200O93662196P33569197AJO270321

Consensus g

GGGG

EESE

LKLLg

GGGG

SSASa

AAAAd

DDDD

TSAT

VAVVr

RRRRd

DDDDp

PPPPr

RRRRg

GGGG

FFWFa

AAAA

VVLVk

KKKKf

FFFFy

YYYYt

TTTTe

EEEE

EDEEg

GGGGn

NNNNy

YYYYd

DDDD

ILLIv

VVVVg

GGGGn

NNNNn

NNNNt

TTTTp

PPPP

VVVIf

FFFFf

FFFFi

IIII

RRKRd

DDDD

APAA

ILIIk

KKKKf

FFFFp

PPPPd

DDDDf

FFFFi

IIIIh

HHHHt

TTTTq

QQQQk

KKKKr

RRRR

DNDDp

PPPP

KAYRt

TTTT

HNGH

LCSL

KKQK

NDEN

PPAP

TDDT

AMNA

VFVMw

WWWWd

DDDDf

FFFF

WLWW

SSGSl

LLLL

STSSp

PPPPe

EEEEs

SSSS

LITLh

HHHHq

QQQQv

VVVVt

TTTT

IIWYl

LLLL

MFFF

SSGGd

DDDDr

RRRRg

GGGG

ITIIp

PPPP

AAAL

TTST

LYYYr

RRRR

HNHHm

MMMM

HNNNg

GGGG

FYYY

GSGGs

SSSS

296P26901300O93662296P33569297AJO270321

Consensus h

HHHHt

TTTT

FYYF

KKQKw

WWWW

TYNVn

NNNN

AEEE

EKAKg

GGGGe

EEEE

GYVA

VFFVw

WWWW

IVVV

KQKKy

YYYYh

HHHHf

FFFFk

KKKKt

TTTT

EDDNq

QQQQg

GGGG

VIIVk

KKKKn

NNNN

LLLM

DTTD

VLQP

NEDE

TEELa

AAAA

AENV

KKRK

IILI

AGAAg

GGGG

ESEE

NDDNp

PPPPd

DDDD

YHSY

HAHH

TTQT

ERREd

DDDDl

LLLL

FYRY

NEENa

AAAAi

IIII

EKEE

NKRKg

GGGGd

DDDD

YYFYp

PPPP

ASTSw

WWWW

KTTT

LLVL

YEQY

VMVVq

QQQQi

IIIIm

MMMM

PTPP

LPAL

EEAE

DQDDa

AAAA

NEAK

TDGTy

YYYYr

RRRRf

FFFF

DDNN

PIPP

FRFFd

DDDD

VILVt

TTTTk

KKKKv

VVVVw

WWWW

SPPS

QHHH

KGEKd

DDDD

YFYYp

PPPP

LTPL

IMVI

EKEE

VIIVg

GGGG

RKTR

MLLM

VVEV

395P26901400O93662395P33569396AJO270321

Consensus l

LLLL

DNNNr

RRRRn

NNNNp

PPPP

ETEEn

NNNN

YYIYf

FFFFa

AAAAe

EEEEv

VVVVe

EEEEq

QQQQ

AASA

TAITf

FFFFs

SSSSp

PPPP

GAAG

TNHN

LLFLv

VVVVp

PPPPg

GGGG

IIIV

DGGE

VIPPs

SSSSp

PPPPd

DDDDk

KKKKm

MMMMl

LLLLq

QQQQ

GGGAr

RRRR

LVLLf

FFFF

ASAAy

YYYY

HHGAd

DDDD

ATAAh

HHHH

RIRR

YHYYr

RRRR

VLVVg

GGGG

APIVn

NNNN

HYAH

QNDN

ALHL

LILLp

PPPP

IVVIn

NNNN

RARR

APPP

RKHR

NYAV

KSTE

VPEV

NEAN

NNRN

ST

YYHY

QQSQr

RRRRd

DDDDg

GGGG

QFFF

MMLM

RRR

FVYFd

DDDD

DAGN

NNRN

GGHG

GGKG

GSGG

SGAS

VPKV

YNNNy

YYYY

EWEEp

PPPPn

NNNNs

SSSSf

FFFFg

GGGGg

GGGGp

PPPP

KSVT

EPQE

SDTV

PSDS

EVRE

483P26901497O93662483P33569488AJO270321

Consensus

DYPH

KLLK

QEWT

APQT

APPP

YFTF

PGPPv

VVVV

QSTSg

GGGG

ILVM

AATA

G

DADE

SRHS

VTAV

SLAP

YYPY

DTSD

HHHD

PA

E

YNDDd

DDDD

HDDH

YFFY

TVTTq

QQQQa

AAAAg

GGGG

DNDDl

LLLLy

YYYYr

RRRR

D

LVLLm

MMMM

STSS

EDEE

DYDE

EDEE

RRKK

TEGA

RNRRl

LLLL

VVIV

EGDKn

NNNN

IILI

VVSV

NSGE

AHFS

MLIL

KSAK

PAKQ

VAVV

EQST

KKRK

ERDE

EDEi

IIII

KQAK

LLELr

RRRR

QQAQ

ITII

EAGR

HLNHf

FFFF

YFRY

KKRKa

AAAAd

DDDD

PREP

EDDD

YYFYg

GGGG

KSKRr

RRRR

VVLV

AAEA

EKAE

GGAG

LLVL

GEQG

LLAL

PDLQ

IIRV

KKGP

KED

DVD

SEV

RI

LT

AN

NA

M

T

N

E

483P26901504O93662483P33569488AJO270321

Consensus

E

R

A

R

A

T

E



RBS

f1Ori

Amp

pBR322 ori

LacI

T7Kat





M 1 2 3 4

25

70

200

85120

5060

40

30

20

15

(kDa)



0 10 20 30

Time (h)

40000

30000

20000

10000

0

Cata

lase

activ

ity (U

mL)


100806040200

Temperature (∘C)

70

80

90

100

110

120

Rela

tive a

ctiv

ity (

)

(a)

150

100

50

010050

Time (h)

Resid

ual a

ctiv

ity (

)

40∘C50∘C60∘C

70∘C80∘C90∘C

(b)









4 Conclusions



Competing Interests



lativ

e act

ivity

()

150

100

50

0

4 5 6 7 8 9 10

pH11


minus005

0

005

01

015

02

0 01 02 03 04 05minus01

1s (mmolL)minus1

R2 = 09972y = 03393x + 00066

1v

(min

120583m

ol)middot1

03


Acknowledgments


References


















































1 Introduction













)2

SO4

Na2

HPO4

KX2

`4

MgSO4

and CaCl2




4

)2

SO4

35 KH2

PO4

10 and MgSO4

times 7H2





3

Fe(CN)6




(1)












0 10 20 30 40 500

5

10

15

20

25

30

35

40

45

50


o

xida

tion

of D

-lact

ate

(b)

(a)










2






000 015 030 045 060 075 090 105 12005

101520253035404550

9095

100

Enzy

mat

ic co

nver

sion

of D

-lact

ate (

)


(c)

(b)

(a)


2




4 Conclusions



Competing Interests


Acknowledgments


References





















2











1 Introduction
















600






























xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

xynBxynB-opt

100100

200200

300300

400400

500500

600600

678

678


100VHb100VHb-opt



200VHb200VHb-opt



300VHb300VHb-opt



400VHb400VHb-opt



440VHb440VHb-opt










Gly

GGG 010 0 0GGA 032 9 11GGT 044 5 18GGC 014 15 0

Glu GAG 043 6 6GAA 057 0 0

Asp GAT 058 3 2GAC 042 7 8

Val

GTG 019 3 0GTA 015 0 0GTT 042 6 1GTC 023 6 14

Ala

GCG 006 2 0GCA 023 0 0GCT 045 12 7GCC 026 4 11

Arg


Lys AAG 053 6 5AAA 047 0 1

Ser


Stop TAA 053 1 1

Asn AAT 049 2 0AAC 051 13 15

Met ATG 100 3 3

IleATA 019 0 0ATT 050 1 1ATC 030 6 6

Thr

ACG 011 1 0ACA 024 1 0ACT 040 4 4ACC 025 19 23

Trp TGG 100 6 6

Cys TGT 065 0 1TGC 035 1 0

Tyr TAT 046 2 1TAC 055 15 16

Leu


Table 1 Continued


Phe TTT 054 2 1TTC 046 5 6

Gln CAG 039 8 7CAA 061 0 1

His CAT 057 0 0CAC 043 4 4

Pro

CCC 015 4 0CCG 009 1 0CCA 041 0 6CCT 035 1 0

0

5000

10000

15000

20000

25000

30000

35000


Induction time (h)

Activ

ity (U

mL)

00

05

10

15

20

25

30

35

40

45

50

Prot

ein

cont

ent (

mg

mL)

24 48 72 96 120 144 168







Gly

GGG 010 0 0GGA 032 1 0GGT 044 7 8GGC 014 0 0

Glu GAG 043 7 1GAA 057 2 8

Asp GAT 058 2 8GAC 042 6 0

Val

GTG 019 0 0GTA 015 0 0GTT 042 4 10GTC 023 10 4

Ala

GCG 006 0 0GCA 023 0 0GCT 045 17 23GCC 026 6 0

Arg


Lys AAG 053 9 10AAA 047 1 0

Ser


Stop TAA 053 1 1

Asn AAT 049 0 0AAC 051 4 4

Met ATG 100 3 3

IleATA 019 0 0ATT 050 5 5ATC 030 7 7

Thr

ACG 011 0 0ACA 024 0 0ACT 040 1 8ACC 025 7 0

Trp TGG 100 1 1

Cys TGT 065 1 1TGC 035 0 0

Tyr TAT 046 0 0TAC 055 4 4

Leu


Table 2 Continued


Phe TTT 054 0 2TTC 046 4 2

Gln CAG 039 6 2CAA 061 3 7

His CAT 057 1 4CAC 043 3 0

Pro

CCC 015 0 0CCG 009 0 0CCA 041 6 7CCT 035 1 0

0

10000

20000

30000

40000

50000

Induction time (h)

Activ

ity (U

mL)

50

100

150

200

250

300

Dry

cell

wei

ght (

gL)


24 48 72 96 120 144 168





00

5

10

15

20

25

30

35

40

Induction time (h)


SOU

R (120583

mol

O2m

ing

DCW

)

24 48 72 96 120 144 168 192 216

(a)

50

60

70

80

90

100

Cel

l via

bilit

y (

)

Induction time (h)


24 48 72 96 120 144

(b)


0

10000

20000

30000

40000

50000

60000

30282522

Xyla

nase

activ

ity (U

mL)

0

50

100

150

200

250

DCW

(gL

)



(a)

60

70

80

90

100

Cel

l via

bilit

y (

)

Induction time (h)24 48 72 96 120 144

30∘C28∘C

25∘C22∘C

(b)






4 Conclusions


Competing Interests




Acknowledgments


References































microbial enzymes and their applications in industries and

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