the prokaryotes || bacterial enzymes

CHAPTER 34Bacterial Enzymes

Bacterial Enzymes

WIM J QUAX

Introduction

In the living world each chemical reaction iscatalyzed by its own enzyme Enzymes exhibit ahigh specificity as they are able to discriminatebetween slightly different substrate moleculesFurthermore they have the ability to operate atmoderate temperature pressure and pH whichmakes them attractive catalysts for industrial andhousehold conversion processes The first reportson the industrial use of enzyme products go backto the beginning of the last century It was theGerman scientist Roumlhm who introduced the useof bovine pancreas extracts for the removal ofstains in dirty clothing (Roumlhm 1915 Fig 1)Around the same time the Laboratoire Amyloin France experimented with the use of extractfrom Bacillus for conversion of starch into sugars(Fig 2) As a result the company Rapidase(Seclin France) which is now part of the lifescience division of DSM was formed With thedevelopment of microbial fermentations in thesecond half of the last century the number ofindustrial processes performed by enzymes andthe amount of enzymes produced have increasedsharply At present a renewed worldwide re-search effort has been directed to identifyingmore sustainable and environmentally friendlybiocatalytic processes The availability of highlyspecific and cheap enzymes resulting fromgenetic and protein engineering has been veryinstrumental in reviving interest in the industrialapplication of enzymes

Most classically used industrial enzymes arehydrolytic (proteolytic amylolytic or lipolytic)Hydrolytic enzymes hardly require any cofac-tors which allows their application in a greatvariety of conditions These enzymes are usuallyseparated from the cell broth after fermentationand formulated in more or less high concentra-tions Recently more specialized bioconversionshave been developed in which enzymatic activityis maintained only by special cofactors that mustbe regenerated or even worse by living cells Inthis chapter the emphasis will be on bacterialenzymes that can be used in isolated form

The organization of this chapter has focusedon application Owing to the versatility and sta-bility of hydrolytic enzymes the same enzymemay be used in totally different parts of indus-trial processes Table 1 summarizes the currentuse of enzymes in various industry and house-hold applications The data are compiled frominformation provided by enzyme producerscustomers and industry organizations and frominformation acquired as a result of my involve-ment with industrial enzyme production formany years As it relates to products of commer-cial importance access to the data is not alwayspossible and relating the biochemical andgenetic characteristics of production strainswhich are usually proprietary to better describedstrains in the literature is sometimes difficultNevertheless this manuscript should provide anoverview of the importance of bacterial enzymesfor sustainable and efficient conversion in indus-trial processes Of the $18 billion annual worldsales of industrial enzymes about 50 are salesof bacterial enzymes and most of the remaining50 are sales of fungal enzymes

Scientific Background the Source of Enzymes

Historically the selection of microorganisms thatproduce enzymes has been empirical startingwith samples from very diverse natural sourcesCultures enriched by growth on substrates wereused to inoculate fermentations In a later stagepure bacterial strains were selected Intellectualproperty protections associated with biopro-cesses have hampered the taxonomical charac-terization of industrially used strains For thesame reason it is not always possible to trace theorigin and history of currently used organismsOnce it became possible to protect man-madebacterial strains by a patent (Chakrabarty 1981)the taxonomy of the bacterial strains became akey element in the development of industrialenzymes Ever since the 16S rRNA sequence hasbeen routinely determined for every bacterial

Prokaryotes (2006) 1777ndash796DOI 1010070-387-30741-9_22

778 WJ Quax CHAPTER 34

Fig 1 A copy of the original patent by Roumlhm which describes for the first time the use of proteases as a cleaning aid

CHAPTER 34 Bacterial Enzymes 779

strain producing an enzyme with interestingproperties (Jones et al 1998b) Later the DNAsequence of the enzyme-encoding gene and itscorresponding amino acid sequence became thekey subjects for patent protection (Yamagataand Udaka 1994 Outtrup et al 1998 van Sol-ingen et al 2001) because genetic engineeringeliminated restrictions on enzyme production(ie the enzymes could be produced by both theoriginal host bacterium and specialized expres-sion hosts)

In general early important criteria for evalu-ating enzyme technology included the ease offermentation and recovery lack of adverse side-products yield and finally the properties of theenzyme (see Table 2) It is no surprise that thisemphasis on easily recovered enzymes hasresulted in industrial production organisms thatare predominantly secreting organisms Gram-positive species with only a single membrane arehighly represented among enzyme host cellsEspecially bacilli known for their high secretory

capability are often used Nevertheless in theabsence of good alternatives some interestingenzymes such as glucose isomerases expressed inStreptomyces (Jorgensen et al 1988) are recov-ered from the cytoplasm of bacteria Other prod-ucts are secreted from Gram-negative organismssuch as lipases from Pseudomonas (Gerritse etal 1998a)

Commercial Applications

Starch

Starch the primary storage polymer in higherplants consists of a mixture of amylose (15ndash30ww) and amylopectin (70ndash85 ww) Amyloseis composed of α-14-linked glucose units linkedin linear chains of molecular weight ca 60000ndash800000 Amylopectin is a branched polymercontaining α-16 branch points every 24ndash30 glu-cose units (Fig 3) its molecular weight may beas high as 100 million (Buleon et al 1998) Corn(maize) starch represents 75 of the worldstarch production Virtually all of the 20 million

Fig 2 The founders of the Rapidase Company the officialwebsite of the city of Seclin (France) (source httpwwwville-seclinfr where the history of industrial activityincluding the ldquoUsine Rapidaserdquo is described)

Table 1 Bacterial enzymes and their field of application

Abbreviations +++++ to + the importance of the enzyme class to the specific use is graded on the basis of the amount ofenzyme produced and its economic value and minus enzyme of no importance to this use

Starch Detergents Food Textile Fine chemicals Brewing and juices Paper and pulp Feed

Amylases +++++ ++ ++ ++ minus minus minus minusProteases minus +++++ ++ + minus + minus minusLipase minus ++ + minus ++++ minus minus minusEsterase minus minus minus +++ minus minus minusCellullase minus ++ + +++ minus minus + minusGlucanase minus minus + minus minus +++ minus minusXylanase minus minus + minus minus ++ ++ +Glucose isomerase ++++ minus minus minus minus minus minus minusβ-Lactam acylase minus minus minus minus ++++ minus minus minusPhytases minus minus minus minus minus minus minus ++

Table 2 Critical parameters for selecting an industrialenzyme

bull Enzyme activity Specific activity (unitsmg) Application dosage

bull Stability during storage application immobilization for multiple use

bull pH range for activity and stability broad range of pH broad range of process conditions

bull Safety (allergenicity) Non-toxic to men and environment Non allergenic

bull High yield production High yield expression in bacterial host Secretion for high yield and for easy purification


ton world production of corn starch (EconomicResearch Service personal communication) isconverted into glucose by a two-step enzyme cat-alyzed process involving 1) liquefaction of solidstarch with an endoamylase into dextrins consist-ing of 7ndash10 molecules of glucose and 2) saccha-rification of the resulting liquefied starch with anexoamylase (glucoamylase) into single glucosemonomers The industrially most importantendoamylases are α-amylases isolated frombacilli (Welker and Campbell 1967a Aiba et al1983 Yuuki et al 1985) The glucoamylase withthe best industrial specifications is producedfrom the fungus Aspergillus niger (Reilly 1979)The produced glucose is used in more or lessequal shares for the production of concentrateddextrose syrups fuel ethanol and high-fructosecorn syrup

Amylases α-Amylase (EC 3211) hydrolyzesstarch glycogen and related polysaccharides bycleaving internal α-14-glucosidic bonds at ran-dom The reports on the industrial use of bacte-rial amylase go back to the early 1920s with aproduct trade-named ldquoRapidaserdquo marketed by aEuropean company with the same name Thisenzyme introduced to replace the acid hydroly-sis process which suffered from large salt loadsand extreme yield losses has long been classifiedas a product of Bacillus subtilis Taxonomic dataof the 1970s have revealed however that theproduction organism is a related but distinct spe-cies nowadays known as Bacillus amyloliquefa-ciens (Welker and Campbell 1967b) Notablythe amylase (AmyE) from Bacillus subtilis 168has no liquefying activity at all and is in fact

unrelated In the classical process starch is firstheated in a jet cooking treatment that serves toopen up the starch granules for gelatinizationand after cooling the mixture to 60degC the α-amylase is added to the starch

In the early 1980s a major change was intro-duced in the industry Now the enzyme is addedduring the first step of the starch degradationprocess and gelatinization occurs at high tem-perature (up to 110degC) allowing the liquefactionduring the steam explosion step This hasspeeded up hydrolysis rates and decreased con-version costs significantly The introduction ofthe more thermostable α-amylase from Bacilluslicheniformis has been crucial for this improve-ment (Outtrup and Aunstrup 1975 Chandra etal 1980 Edman et al 1999) Next to the amy-lase from Bacillus licheniformis the enzymefrom Bacillus stearothermophilus has been intro-duced for industrial use This enzyme with sta-bility slightly higher than that of the Bacilluslicheniformis amylase however has never beenwidely used since it generates maltodextrins ina size distribution that is unfavorable for the sub-sequent glucoamylase treatment In an effort tocombine the best properties of these two amy-lases chimeric enzymes formed of the NH2-terminal portion of Bacillus stearothermophilusα-amylase and the COOH-terminal portion ofBacillus licheniformis α-amylase have beenmade (Gray et al 1986) The hybrid enzymemolecules however were shown to be less stablethan each of the parent wildtype α-amylasesFinally an enzyme mixture composed of theamylases from Bacillus licheniformis and Bacil-lus stearothermophilus was introduced with moresuccess Nowadays most commercial amylasesare produced from a small subgroup of Bacillusspecies such as Bacillus amyloliquefaciens Bacil-lus coagulans Bacillus licheniformis or Bacillusstearothermophilus These enzymes show a highdegree of homology and similarity (Yuuki et al1985 Nakajima et al 1986)

Thermostability pattern of breakdown intodextrins ease of production and activity at lowpH (lt6) are important criteria used industriallyfor choosing amylases In recent years the amy-lases in commercial use have been optimized byprotein engineering and directed evolutionEnzyme properties such as heat stability sub-strate specificity or performance at different pHhave been altered (Quax et al 1991a see alsoFuture Prospects in this Chapter) The genera-tion of engineered variants and the availabilityof the corresponding cloned genes have inspiredthe development of host strains genetically engi-neered to optimize expression of amylases Tooptimize yields in fermentation processes classi-cal mutagenesis was used to develop industrialstrains for many decades and much effort was

Fig 3 Starch and actions of amylases on amylopectin The14 bonds (horizontal) are cleaved by α-amylases and the 16bonds (vertical) which are formed every 24ndash30 glucose unitscan be cleaved by pullulanase (debranching enzyme)

O

OH CH3OH

CH2OH CH2OHCH3

OH

OH OH

OH OHOH

OH

O

O O

O OOO

O


made to develop transformation protocols andgenetically stable multicopy systems for indus-trial host strains such as Bacillus licheniformis(Sanders et al 1985) For efficiency and regula-tory reasons host strains of the same species orgenus from which the α-amylase is derived arepreferred (Jorgensen and Jorgensen 1993) Par-ticularly for the production of mutant amylasesa Bacillus licheniformis strain without a wildtypeα-amylase gene and preferably a strain withoutother enzymatic activities such as proteases isused (Quax et al 1991b) The α-amylases areproduced throughout fermentation as a precur-sor with a signal sequence that is cleaved offduring secretion and secretion facilitates recov-ery As a matter of fact the secretion of amylaseis so efficient that a potent expressionsecretionsystem based on Bacillus licheniformis strain T9and the amylase expression signals has beendeveloped This host strain has been at the basisof the PlugBugreg concept that was introduced byGist-brocades in the late 1980s (Quax et al1993) This system has been used to produce highamounts of both mutant α-amylases and humaninterleukin-3 (Van Leen et al 1991)

Apart from the use of α-amylases for the pro-duction of sweeteners the enzyme has also beenapplied in fuel ethanol production from liquefiedstarch (Kosaric et al 1983) Though the demandfor fuel ethanol is fluctuating fuel ethanol hasthe potential to become a major product of cornstarch and concomitant growth of the amylasesupply will be required

Isomerases A major part of the glucose pro-duced from starch liquefaction and saccharifica-tion is processed further into high fructose cornsyrup (HFCS) Eight million tons are producedworldwide (Economic Research Service per-sonal communication) Glucose isomerases (EC5315) catalyze the reversible isomerization ofglucose to fructose Fructose is now commonlyused as a sugar substitute because it is sweeterthan sucrose or glucose Many microorganismsare known to produce glucose isomerase seefor example the review article by Wen-Pin Chen(1980) which lists a large number of micro-organisms capable of producing glucoseisomerase The best producers of industrial glu-cose isomerases are from the Actinomycesgroup including Streptomyces rubiginosis Acti-noplanes missouriensis and Ampullariella spp(Quax et al 1991b Wong et al 1991 Saari etal 1997)

Activity on glucose (these enzymes are in factxylose isomerases) no need for heavy metalcofactors (eg cobalt) amenability to immobili-zation thermal stability (process conditions areat 55degC) and ease of production are the mostimportant features of glucose isomerases Gen-

erally the naturally occurring glucose isomerasesalso show a high affinity for sugars other thanglucose such as D-xylose D-ribose and L-arabinose As a matter of fact the Km values forxylose are generally significantly lower and theVmax values usually higher than those for glucosewhich is reflected in the official name of theenzyme (D-xylose ketol isomerase EC 5315)The enzyme causes glucose isomerization tofructose until about a 11 equilibrium mixture(the ratio present in natural sucrose) is formedand the product has the same sweetness assucrose Because the enzyme is not secreted itscost of production is relatively high Thereforeglucose isomerase is immobilized in columnreactors allowing prolonged use of one batch ofenzyme Typically the reactors operate for 60ndash100 days of continuous conversion at 55ndash60degC

Glucose isomerase requires a bivalent cationsuch as Mg+2 Co+2 or Mn+2 for its catalytic activ-ity Determination of three-dimensional (3D)structures of different glucose isomerases hasrevealed the presence of two metal ions in themonomeric unit (Kreft et al 1983 Farber et al1987 Henrick et al 1987) Apart from a role inthe catalytic mechanism bivalent cations arealso reported to increase the thermostability ofsome glucose isomerases (Callens et al 1988)Although the pH optimum of glucose isomerasesis usually 70ndash90 use of glucose isomerase atlower pH is beneficial for the following reasons1) under alkaline conditions the formation ofcolored byproducts and a nonmetabolizablesugar (D-psicose) is a problem and 2) the pro-cess step preceding the isomerization is per-formed at pH 45 (Roels and Tilburg 1979)Despite an extensive screening of many microor-ganisms by industry researchers for a glucoseisomerase with a higher activity at lower pH(Van Straten et al 1997) no novel commercialglucose isomerase has been found

Protein engineering has been used with moresuccess to obtain glucose isomerases with a lowerpH optimum (Drummond et al 1989 Luiten etal 1990 Zhu et al 2000) The mutation oflysine253 into arginine253 of the isomerase fromActinoplanes missouriensis has almost doubledthe operation time of the immobilized productunder industrial conditions (Quax et al 1991bFig 4) In addition technical optimizations suchas an improved immobilization technique haveenhanced the performance of traditional glucoseisomerases such as that produced from Strepto-myces murinus (Jorgensen et al 1988) Themutants by definition are produced in geneticallymodified host strains Also the classical nonmod-ified versions of the enzymes are nowadays beingproduced efficiently in nonsecreted form ingenetically modified Streptomyces host cellsHowever the exact nature of the strains and the


genetic constructions used by industry for thesepurposes are poorly documented

Pullulanases and Cyclodextrin-Glucanotransferases The endoamylasescyclomaltodextrinase (CGTase EC 32154)maltogenic amylase (EC 3 21133) and neopul-lulanase (EC 321135) are minor enzymescapable of hydrolyzing two or three of the fol-lowing cyclomaltodextrins pullulan and starchThese enzymes hydrolyze cyclomaltodextrinsand starch to maltose and pullulan to panose bycleavage of α-14 glycosidic bonds (see Fig 3)whereas α-amylases are essentially inactive oncyclomaltodextrins and pullulan Uniquelypullulanases are also able to cleave the α-16bonds (see Fig 3) which makes them especiallyimportant for completely converting starch intoglucose monomers Pullulanases have beendescribed from many species but the enzymefrom Bacillus acidopullolyticus seems to be spe-cially suited for use in the starch processingindustry (Kelly et al 1994)

The cyclodextrins produced from glucose haveapplications ranging from the formulation ofpharmaceuticals to surfactants (solubility en-hancers Hesselink et al 1989 Albers andMuller 1995) The right endoamylase for cyclo-dextrin production should act quickly and yieldthe desired product spectrum at high tempera-tures and low pH The enzymes from Bacilluscoagulans and Bacillus circulans are well knownin the market (Kitahata et al 1983 Hofmann etal 1989) Recently variants of cyclodextrin-glucanotransferases (CGTases) with an alteredcyclodextrin product spectrum have been engi-neered by mutagenesis of specific residues (Nor-ris et al 1983 Wind et al 1998)

Detergents

Proteases Subtilisins (EC 342162) a largeclass of microbial serine proteases are responsi-ble for the breakthrough in industrial enzymedevelopment As early as 1959 the Swiss com-pany Gebraumlnder Schnyder AG marketed thefirst detergent powder with a protease producedfrom a Bacillus strain under the name Bio 40Schweizerische Ferment AG in Basel deliveredthe protease The name of the enzyme subtili-sin refers to the producing organism Bacillussubtilis In 1963 the Dutch company Kortmannand Schulte marketed the first bacterial-enzymecleaning product (Biotexreg with Alcalasereg) andit became a big success Alcalasereg the majorextracellular serine protease from Bacilluslicheniformis was manufactured by the Danishcompany Novo (now Novozymes) Between1965 and 1966 the big soap producers (Procterand Gamble Unilever Colgate and Henkel)realized the potential of the hydrolytic action ofbacterial protease in removing protein-basedstains and they began adding Alcalasereg and asimilar product Maxatasereg to their majordetergent brands This has led to the creation ofa worldwide industrial enzyme market based onBacillus licheniformis fermentation Proteaseshydrolyze the peptide bonds of proteins stain-ing fabric releasing smaller polypeptides andindividual amino acid units In 1969 a majordrawback (fatal allergic reactions of employeesexposed to dust set free during enzyme produc-tion) became apparent Thanks to improveddust-free formulations the enzyme industry wasable to recover To satisfy the desire to lowerthe temperature and concomitantly increase thealkalinity of laundry processes extreme alkalineproteases (Maxacalreg originating from Bacillusalcalophilus [Van Eekelen et al 1988 Van derLaan et al 1991] by Gist-brocades and Savi-nasereg from Bacillus lentus [Betzel et al 1988]by Novo-Nordisk) were introduced into themarket in the early 1980s Interestingly thegene sequences showed that these proteases dif-fered by only a single amino acid Recently thestrain producing Savinasereg has been reclassi-fied as Bacillus clausii (Christiansen et al2002) The gene for the Alcalasereg serine pro-tease also known as Carlsberg subtilisin wascloned in 1985 (Jacobs et al 1985) The avail-ability of the cloned genes and detailed 3Dstructures of various subtilisin molecules(Drenth et al 1972 McPhalen and James 1988Van der Laan et al 1992) has facilitated pro-tein-engineered improvements in enzymes andtheir adaptation to the detergent matrix Morestable variants and especially more bleach-sta-ble variants which were obtained by substitut-ing the methionine residue next to the active

Fig 4 The application test of protein engineered thermo-stable glucose isomerase The activity of immobilized enzymeis plotted as a function of time The stability at 70degC indicateshow the enzymes will behave under industrial conditionsThe variant Lys253Arg of Actinoplanes missouriensis glucoseisomerase has been shown to have (also under industrialconditions) a doubled half-life

Stability of Glucose Isomerase at 70 C

120

rela

tive

activ

ity (

)

100 80 60 40

1 2 3 4 5 6 7 8 9

Time (days)

10 11 121314

K253RWild Type

20 0


site serine are dominating the marketplace to-day (Estell et al 1985 Van Eekelen et al1989) For liquid detergent application themore neutral subtilisin BPN-P originating fromBacillus amyloliquefaciens has been the productof choice for many years In the United Statesabout 50 of liquid detergents and 25 ofpowder detergents contain proteases for im-proved cleaning In Europe where powder de-tergents are more popular virtually all brandshave protease additives

Lipases After the successful introduction of pro-teases for the removal of proteinaceous stains inlaundry detergents the next challenge was thedevelopment of lipases for the removal of greasystains The search for suitable lipases howeverturned out to be far more difficult than the intro-duction of proteases

Detergent lipases were selected according tothe following criteria a) broad activity on a vari-ety of fats and lipids b) stability in alkalinedetergent formulations c) sufficient solubility inwater to soak into fabrics d) compatibility withproteases present in detergent formulations ande) ease of production The first lipase introducedin detergent powder is a lipase of fungal originthat fits well with criteria c) and e) Howeverowing to the acidophilic nature of fungi thecompatibility of their lipases with the alkalineconditions in detergents is poor Therefore bac-terial lipases (EC 3113) originating fromPseudomonas species have received much atten-tion Especially the lipase from Pseudomonasalcaligenes has an excellent activity in the pHrange compatible with detergent conditionsCriterion e) is however far more problematicfor fungal lipase production The expression inheterologous host strains such as Bacillus orEscherichia coli turned out to be impossiblebecause a lipase-specific chaperone Lif (El-Khattabi et al 1999) or LipB was required Fur-thermore the lipase is secreted via the terminalbranch of the general secretion pathway (Xcp-machinery) which involves very specific interac-tions (for a review see Filloux et al 1998 Fig5) Apart from the expression yield also therecovery of Pseudomonas lipases from the fer-mentation broth requires special processesowing to the hydrophobic nature of lipases andthe presence of lipopolysaccharides Despitethese obstacles the lipase from Pseudomonasalcaligenes was introduced as a detergent addi-tive in 1995 by Gist-brocades under the tradename Lipomaxreg (Gerritse et al 1998b Cox etal 2001) As a result of a stepwise improvementof the production strain and fermentation pro-cess commercially viable yields of lipase wereobtained (Gerritse et al 1998a 1998b Cox etal 2001)

As most lipases do not meet all the above-mentioned criteria the first protein engineeringof lipases was based on amino acid sequenceinformation only (eg the study on lipase fromPseudomonas mendocina Gray et al 1995)When the first 3D-structures became availablein the late 1980s protein engineering effortsincreased dramatically A European-widefunded project focused on solving new lipasestructures and now more than 15 X-ray struc-tures of lipases are available in the proteindatabase (PDB) of which 12 are microbial and5 are of prokaryotic origin The X-ray structureof lipase containing a phosphonate inhibitorcovalently bound to its active site serinerevealed that a lid was displaced from the activesite by a hinge bending movement creating anincreased hydrophobic surface Many of thelipase structures are solved in both a closed andan open conformation ie with the lid or lidsdisplaced from the active site A list of selectedsolved bacterial lipase structures is given inTable 3 The overall structure of the triacylglyc-erol lipases has a central L-sheet with the activeserine placed in a loop termed the ldquocatalyticelbowrdquo Above the serine a hydrophobic cleft ispresent or formed after activation of the lipasesMolecular modeling of these structures has beenused to construct models of lipase homologues(eg the engineering of the Pseudomonas alcali-genes lipase Aehle et al 1995) The use oflipases for the generation of enzymatic peroxidebleach in detergents has been pioneered but notyet applied

Fig 5 The secretion machinery of Pseudomonas alcaligenesthe producer of Lipomaxreg an alkaline lipase for detergentcompositions The XcpQ protein forms a multimeric ring inthe outer membrane The Xcp T U V and W proteins arethought to be involved in the gating of the pore and proteinsP X Y and Z form a connection between the outer mem-brane pore and the inner membrane XcpA S and P play arole in the processing of other Xcp proteins Sec is innermem-brane translocase Ch is the periplasmic chaperone

Outer membrane

Periplasm TUVW

A S

R

Xcp machinery

Ch

Sec

QP

X

Y

Z


Cellulases Cellulases are enzymes capable ofhydrolyzing the 14 β-D-glucosidic linkages incellulose Cellulolytic enzymes have beentraditionally divided into three major classesendoglucanases exoglucanases (or cellobiohy-drolases) and β-glucosidases (Knowles et al1987) A large number of bacteria yeasts andfungi is known to produce this group of enzymesInitially cellulolytic enzymes have been devel-oped for application in converting wood and cel-lulose pulp into sugars for bio-ethanolproduction Later on it was discovered that cel-lulases can be used for the treatment of textilesFor example repeated washing of cotton-containing fabrics results in a grayish cast to thefabric which is believed to be due to fibrils dis-rupted and disordered by mechanical action Thisgrayish cast sometimes called ldquopillsrdquo is particu-larly noticeable on colored fabrics The ability ofcellulase to remove the disordered top layer ofthe fiber and thus improve the overall appear-ance of the fabric has been used to reconditionused fabrics to make their colors more vibrant

Despite the availability of fungal cellulaseshaving some of the above properties new cellu-lases that are more compatible with the alkalinedetergent formulations have been soughtAlkalophilic Bacillus species have been foundto express cellulases (EC 3214) with excellentproperties for detergent conditions and one ofthese cellulases is now expressed from Bacillussubtilis and marketed under the trade namePuradaxreg (Jones and Quax 1998a) Also cellu-lases from Thermomonospora fusca have beenfound to be of interest (Irwin et al 1998) totextile decorators Some of these cellulases canbe abundantly expressed in a Streptomyces liv-idans host cell (Jung et al 1993)

Amylases The thermostable α-amylase (EC3211) from Bacillus licheniformis is perfectlycompatible with detergent conditions and now-adays small amounts of this enzyme are widelyadded to detergent powder formulations for theremoval of starch stains A protein engineeredvariant Purastarreg Ox has been developed spe-cifically for inclusion in bleach-containing deter-gent formulations (Genencor 2001) This brings

the number of different enzyme systems addedto modern detergent powders up to four pro-teases lipases cellulases and amylases

Food Processing

Microorganisms play a major role in the process-ing of dairy products beer wine and many otherfood products Isolated enzymes are also beingused in specialized processes although in muchsmaller amounts than are used in the immensestarch processing industry which will be dis-cussed in a separate chapter

The baking of bread is one of the oldest bio-technological processes known to man Yeastenzymes and endogenous flour enzymes are theprimary modifiers and metabolizers of flour sug-ars and proteins However the levels of endoge-nous enzymes vary considerably depending onwheat growth harvest and storage conditionsCorrection and supplementation of the flourwith bacterial enzymes result in more tastefuland better quality bread Bacillus amyloliquefa-ciens α-amylase (EC 3211) is used to obtain animproved loaf volume and crumb structure (Linand Lineback 1990) In addition α-amylasecontributes to anti-staling by mildly hydrolyzingstarch polymers which prevents their crystalliza-tion and thereby hardening of bread The neutralprotease of the same bacterium is used forimproving the rheological properties of biscuitand cracker dough (Lyons 1982) This proteasefragments the gluten protein in wheat flourwhich gives the dough its elastic properties As aresult the dough requires a reduced fermenta-tion time and the resulting biscuits have a pro-longed freshness

Dairy products and beverages are processedunder mildly acidic conditions favoring the useof enzymes of fungal origin However in theprocessing of beer the enzymes from selectedBacillus strains play an essential role The α-amylase from Bacillus amyloliquefaciens is usedto improve the enzymatic liquefaction potentialof the malt A β-glucanase from the same bacte-rium (Hofemeister et al 1986) is used to reducethe viscosity of the wort which improves thefiltration of the beer

Table 3 Three-dimensional structures of prokaryotic lipases

aCode name for the corresponding file in the Protein Database Bank [wwwrcsborgpdb] (PDB code)

Species Molecular weight (kDa) Structurea Reference

Burkholderia glumae (Pseudomonas glumae Chromobacter viscosum)

23 PDB1QGE Noble et al 1994

Pseudomonas aeruginosa 30 PDB1EX9 Nardini et al 2000Bacillus subtilis 19 PDB1I6W van Pouderoyen et al 2001Burkholderia cepacia (Pseudomonas cepacia) 33 PDB2LIP Schrag et al 1997Bacillus stearothermophilus 43 PDB1KU0 Jeong et al 2002


Textiles

Amylases Woven fabrics from natural plant andanimal fibers represent the oldest forms of tex-tile The introduction of mechanical processes inthe nineteenth century prompted the introduc-tion of protective agents to prevent warp-endbreaks Starch added as a sizing agent strength-ens fibers and makes the yarn more resistant tohigh mechanical stress during the weaving pro-cess Traditionally malt extracts and animalderived preparations have been used to removestarch-based thickeners in the desizing opera-tion However as early as 1917 a high tem-perature stable bacterial enzyme preparationobtained by dedicated fermentation was intro-duced (Wallerstein 1939) Today we know thatthe bacterium used was Bacillus amyloliquefa-ciens (Welker and Campbell 1967a) At presentboth Bacillus amyloliquefaciens and Bacilluslicheniformis α-amylases are being used for thisprocess

Cellulases In various treatments of cottonfibers these enzymes have resulted in betterwash-down effects resistance to pilling soften-ing and better dye uptake Later it was discov-ered that the enzymatic treatment of textilescould result in decorative effects on clothing sim-ilar to the stone washing of denim (Gusakov etal 2000) This has resulted in a large market forcellulases in providing a worn look to jeans Theenzymatic production of stone-washed denimproducts (no need for pumice) has become a fastgrowing market with more than $40 million insales per year A variety of cellulase products(many of fungal origin) is marketed for this pur-pose Recently enzymes from the actinomyceteThermomonospora fusca have been developed(Spezio et al 1993) The cellulase (EC 3214)can be efficiently produced from a geneticallyengineered Streptomyces lividans (Jung et al1993) Care needs to be taken to prevent loss offiber strength from cellulase treatment that is toolengthy or intense

Proteases These enzymes (eg subtilisin [EC342162]) are used to treat protein fabrics suchas wool and silk By breaking down the fibrils onthe surface the look and feel of the fabric can besoftened

Fine Chemicals

In nature a huge repertoire of chemical trans-formations is catalyzed by many thousands ofenzymes Its precise 3D architecture allows eachenzyme to exhibit a remarkable specificity forthe conversion of a particular set of substratesThe introduction of these enzymes as biocata-

lysts in the industrial production of fine chemi-cals probably represents the uppermostinnovation in the enzyme field in recent yearsSince a company produces in-house many of thebiocatalysts used within industrial processes (ieproduction for captive use or captive consump-tion) the information on the actual scale andcommercial impact of many of these biocatalyticprocesses is often limited Nevertheless from thescarce publications on industrial use of biocata-lysts it can be concluded that numerous energyintensive chemical processes involving a highoutput of pollutants have now been replaced byenvironmentally friendly enzymatic processes(Schmid et al 2001)

Amidases

β-Lactam Acylases Penicillin G acylase (benzyl-penicillin amidohydrolase also named ldquopenicil-lin amidaserdquo EC 35111) is an enzyme usedcommercially to produce 6-aminopenicillanicacid (6-APA) the most important intermediatefor the industrial production of semisyntheticpenicillins This is achieved by the hydrolysis ofpenicillin G (for review see Bruggink et al 1998Fig 6 left column)

Numerous bacterial species have beendescribed in the literature as penicillin G acylase-producing strains but only certain strains of thespecies E coli Kluyvera citrophila and Alcali-genes faecalis were found to produce an enzymecompatible with the requirements of industrialdeacylation (Balasingham et al 1972 Barberoet al 1986 Verhaert et al 1997) Driven byenvironmental legislation in the past decade allchemical deacylation processes in industry havebeen replaced by the less polluting enzymaticcleavage process Recombinant DNA methodshave been applied not only to increase the yieldsof commercially used penicillin G acylases(Bruns et al 1985) but also to decipher the com-plex processing of these enzymes (Schumacheret al 1986) The penicillin G acylase of E coliATCC11105 was found to be produced as a largeprecursor protein which is secreted into the peri-plasm and further processed to the mature pro-tein constituting a small (α) and a large (β)subunit Cloning and sequencing has revealed aclose homology (90 identity) to the Kluyveracitrophila and a distant homology (50 identity)to the Alcaligenes faecalis acylase gene The het-erodimeric structure however is evolutionarilypreserved not only among penicillin acylases butalso within the much larger family of β-lactamacylases

Whereas the conversion of penicillin-Grequires an enzyme with a specificity for the aro-matic phenyl acetate side chain the processingof the second largest β-lactam fermentation


product cephalosporin-C requires the cleavageof aminoadipyl an aliphatic side chain from theβ-lactam nucleus Since a one-step enzymaticdeacylation (Aramori et al 1991a) was not fea-sible a combination of two enzyme-mediatedreactions has been introduced to produce 7-aminocephalosporanic acid (ACA) In this pro-cess D-amino acid oxidase and a glutaryl acylaseperform an enzymatic deacylation of cepha-losporin-C (see Fig 6 right column) This glu-taryl acylase (EC 351-) can be obtained fromseveral Pseudomonas species (Shibuya et al1981 Matsuda et al 1987 Aramori et al 1991aIshiye and Niwa 1992 Ishii et al 1994 Li et al1998) or from a Bacillus species (Aramori et al1991b) Research towards a one-step cepha-losporin-C deacylating enzyme so far has beenunsuccessful

A third important intermediate 7-aminode-sacetoxycephalosporanic acid (7-ADCA) isproduced from penicillin G by an expensivechemical ring expansion reaction Subsequentdeacylation of cephalosporin G can be achieved

enzymatically by a penicillin-G acylase such asthe enzyme from Alcaligenes faecalis Fig 7 leftcolumn) The latest development in the field isthe use of a genetically modified Penicilliumchrysogenum equipped with an expandase genefrom Streptomyces clavuligerus to produceadipyl-7-ADCA upon fermentation with adipatefeed (Crawford et al 1995 Fig 7 right column)Deacylation of adipyl-7-ADCA cannot be donewith penicillin acylases but requires an enzymewith affinity for the adipate side chain (Schroenet al 2000 Xie et al 2001) Some of the afore-mentioned glutaryl acylase enzymes have a lowactivity on this substrate Recently by directedevolution several mutants of Pseudomonas SY-77 acylase (EC 351) with a high activity onadipyl-7-ADCA have been isolated (Otten et al2002 Sio et al 2002 Fig 7)

Semisynthetic cephalosporins and penicillinsare industrially produced from intermediatesdepicted in Figs 6 and 7 As β-lactam acylasesare hydrolytic enzymes in theory the reactioncan be reversed under conditions of low water

Fig 6 The role of β-lactam acylases in the manufacturing of semisynthetic cephalosporins and penicillins In the left pathwaythe production of 6-amino penicillanic acid (6-APA) from the fermentation product penicillin-G is shown In the rightpathway the production of 7-aminocephalosporanic acid (7-ACA) from the fermentation product cephalosporin-C isdepicted

H2N H2N SH

NH

OO

O

N

N

H2N

S

S

CH3

CH3

CH3

CH3

CH3

NH2

NHHO

CH3

CH3

CH3

NH2

NH

OO

N

S

O

O

O

O

O

OO

O

OO N

N

S

S

COOH

CH3

H2NCOOH

COOHCOOH

COOH

COOH

COOH

HOOC

COOH

L-amino adipic acid

Penicilliumchrysogenum

Penicillin-G acylase

Clutaryl - acylase

Penicillin G

6-APA

7-ACA

COOH

+ L-cysteine + L-valine

Cephalosporiumacremonium

Cephalosporin C

D-amino acid oxidase

Glutaryl-7-ACA


concentration Precisely this property of β-lactam acylases is being used for the selectivecoupling of specific side-chains to form pharma-ceutically valuable β-lactams such as ampicillincephalexin (Boesten and Moody 1995) and lora-carbef (Koeller and Wong 2001) Directed evo-lution will undoubtedly result in the isolation ofvariants with novel synthetic properties (Alkemaet al 2000)

Other Amidases Aspartame is a dipeptide withan immense sweet taste The synthesis of thislow-calorie sweetener is performed with the neu-tral protease (EC 342427) from Bacillus ther-moproteolyticus also known as ldquothermolysinrdquoApplied as a reversal of the hydrolytic reactionthe enzyme shows a remarkable specificity in thecoupling of N-protected-L-aspartic acid and DLphenylalanine methyl ester Owing its extremethermostability the enzyme is very stable in thehigh solvent conditions used for the reactionAs an alternative to thermolysin a highly stable

variant of Bacillus stearothermophilus neutralprotease obtained by protein engineering is nowavailable (Mansfeld et al 1997 Van den Burget al 1998)

Amidases are also applied for the chiral reso-lution of racemic amino-acid amides to allow thebiocatalytic synthesis of non-natural L-aminoacids which are important building blocks forpharmaceuticals An amidase (EC 3514) fromPseudomonas putida has been developed for thekinetic resolution of a wide range of amino acidamides (Schmid et al 2001)

Lipases and Esterases Lipases from Pseu-domonas aeruginosa Pseudomonas cepacia andPseudomonas fluorescence (EC 3113) are beingused for a large number of different syntheticreactions in organic chemistry with specialemphasis on kinetic resolution of chiral com-pounds (Coffen 1997) As lipases are active inorganic solvents not only hydrolytic but alsotransesterification reactions can be performed

Fig 7 In the left panel the classical process for obtaining 7-ADCA is shown A novel biosynthetic pathway for adipyl-cephalosporin using Penicillium is depicted in the right column The final conversion towards 7-ADCA is done with an adipylcephalosporin acylase Using directed evolution the glutaryl acylase of Pseudomonas SY77 has been converted into an adipylacylase (Otten et al 2002 Sio et al 2002)

CH3

CH3

CH3

N

N

S

S

O

O

O

O

O

O

HO

Adipyl-penicillin

Adipyl-cephalosporin

Pseudomonasadipyl acylase

In vivo enzymaticring expansion

NH

New process

NHHO

COOH

CH3

CH3N

S

O

O

Penicillin G

Chemicalring expansion

Current process

NH

COOH

CH3

CH3

N

S

N

S

O

O

O

NH

H2N

COOH

COOH

penG acylase

7-ADCA

Cephalosporin G

E coli∆ G

COOH

CH3

N

S

O

N2H

COOH

7-ADCA

Semi-synthetic cephalosporins-Cephalexin-Cephalothin-Etc

Improved enzyme foundby directed evolution


For racemic alcohols this may be an enantiose-lective transformation with acyl donors such asvinyl esters anhydrides or diketenes (Koellerand Wong 2001 Schmid et al 2001) Using thisprocess enantiomerically pure alcohols are pro-duced on a several hundred ton scale byBadische Anilin und Soda Fabriken (BASF)Pseudomonas lipase can also react with aminesas nucleophiles allowing the resolution of opti-cally active amines such as S-methoxyisopropy-lamine an important building block for theherbicide ldquoFrontierX2rdquo

The enzymatic activity of lipases is very compa-rable to that of esterases with the main differencebeing the chain length and hydrophobicity of theacid moiety of the substrate Therefore in finechemical applications lipases and esterases arebeing used as alternatives for several conversionsFor instance for the kinetic resolution of 2-arylpropionic acids such as naproxen and ibuprofenboth a lipase and an esterase have been found thatcan perform a stereoselective hydrolysis yieldingthe pharmaceutically preferred enantiomer S-naproxen (Bertola et al 1992 Hedstrom et al1993) High activity and ease of production havemade the carboxylesterase from Bacillus subtilisThai I-8 the prime choice of industry (Quax andBroekhuizen 1994)

The markets for fine chemicals that can bemade from esterss are very important and di-verse Thus the ability to perform ester hy-drolysis or esterification reactions in a mannerthat ensures high specificity and high stereoselec-tivity is of great importance Therefore the useof genomics information to search for newesterases is of great interest (Robertson et al1999 Droge et al 2001)

Areas of Research

Feed

Animal feed is mainly composed of polymericstructures that have to be digested in the gutAny pretreatment of the agricultural stock maylead to an improved digestibility and hence yieldof feedstuffs It is therefore no surprise that mostof the hydrolytic enzymes including the pancre-atic extracts used as the first enzyme preparationin the 1920s have been tested in one way oranother for the processing of animal feed Onlyafter an increased understanding of the digestivephysiology did realistic applications come withinreach The examples described below are theresult of expert advice and evaluation of feedindustry experiences

Phytases Phosphorus is an important compo-nent of feed as it is crucial for bone and skeleton

formation About 70 of phosphorus in vegeta-ble feed ingredients is present in the form ofphytate an inositol-bound organic form of phos-phorus that has a low bioavailability in monogas-tric animals For this reason the diet formonogastric animals like pigs and chickens issupplemented with significant amounts of inor-ganic phosphate that causes eutrophication inregions of the world with a dense monogastricanimal population such as the Netherlands Theaddition of microbial phytases (EC 31326) hasresulted in a doubling of the bioavailability ofphytate obviating the need for addition of inor-ganic phosphate (Simons et al 1990) This hasled to lowering phosphate in manure to unprec-edented levels in the Netherlands and to phos-phate pollution reductions that are moresignificant than the reductions from the deter-gent phosphate ban in the mid-1970s

Phytase from fungi has been shown to beextremely compatible with the low pH condi-tions of the animal gastric tract (Jongbloed et al1992) but also phytases from bacteria such asBacillus subtilis are being developed for use asa feed additive (Kerovuo et al 2000b Park etal 1999 Kerovuo and Tynkkynen 2000a)

Xylanases Pentosans present in wheat and ryediets are often poorly metabolized Especiallyarabinoxylans negatively influence the digestionand absorption of nutrients in the foregut of ani-mals When a xylanase (EC 3218) treated ara-binoxylan fraction was used the nutritionalparameters were similar to those when an arabi-nose and xylan monomeric mixture was usedindicating that xylanases are a valuable feedadditive Especially sought are enzymes withendo-14-β-xylanase activity that are stable in thedigestive tract of poultry (Mondou et al 1986)

Paper and Pulp

In the pulp and paper manufacturing processelemental chlorine is applied for the bleaching ofthe pulp As a byproduct of this process toxicchlorinated phenols as well as polychlorinatedbiphenyls are formed Next to alternativebleaching chemicals such as ozone the use ofenzymes has gained more interest Especially theremoval of residual lignin results in a lowerrequired amount of bleaching chemicals allow-ing the replacement of elementary chlorine bythe less polluting chlorine dioxide The removalof lignin can be facilitated by a pretreatment ofthe pulp with xylanases or by laccases This xyla-nase pretreatment cleaves the hemicellulosefraction that links the lignin to the cellulose Thelaccase treatment results in a direct oxidativedegradation of the lignin The search for suffi-ciently active laccase systems is still in its infancy


but xylanases have been developed for commer-cial use

The pulping process in a paper mill is per-formed at temperatures of 65ndash80degC at pH 9ndash12Xylanases (EC 32132 endo-13-β-xylanase)from some thermophilic bacilli were found to becompliant with these conditions (Gat et al1994) and the xylanase from Bacillus stearother-mophilus T6 was developed and tested on a largescale (Lundgren et al 1994) This enzyme showsactivity at high temperature (60ndash70degC) and highpH (7ndash9) The enzyme can be expressed andpurified in high yields from Bacillus subtilis(Lapidot et al 1996) The search for even morethermostable and more alkaline-stable xylanaseshas been targeted towards extremophiles (Saulet al 1995 Outtrup et al 1998)

General Expression Hosts

Bacteria are attractive for large scale manufac-turing of commercially relevant proteins owingto their fast growth rate and their high proteinsynthesis capacity Enhanced levels of geneexpression however often result in the intracel-lular accumulation of inactive protein aggregatesalso known as inclusion bodies For most enzymemanufacturing processes the recovery of activeprotein from these aggregates is uneconomicalThe only enzyme process that has been in use formany years has been the manufacturing ofbovine chymosin (rennin) with the Gram-negative bacterium E coli as a host (Nishimoriet al 1981 Emtage et al 1983)

Export of overexpressed heterologousenzymes from the cytoplasm has been exploredas a solution to prevent inclusion body formationand to produce functional proteins in an easilyrecoverable form With the identification ofsome periplasmic chaperone and foldase func-tions in Gram-negative bacteria the concept ofusing the periplasm as a ldquoconstruction compart-mentrdquo in which chaperones aid the folding andfunctional assembly of proteins has come withinreach The ultimate goal from the viewpoint ofindustrial scale recoverymdashaccumulation of pro-teins on a gram per liter scale in the extracellularmediummdashrequires however the passage throughtwo membranes Recently described have beensome nonpathogenic species such as Pseudomo-nas alcaligenes that have the capacity to secretecommercially important enzymes (lipases pro-teases cellulases and phospholipases) in signifi-cant amounts into the extracellular medium(Gerritse et al 1998a) The outer membranesecretion machinery is crucial for the export ofproteins from the periplasm At high expressionlevels the outer membrane can become a barrieras exemplified by the effect on Pseudomonasalcaligenes lipase overexpression of selecting the

Xcp gene cluster using the phenotype enhance-ment method (Gerritse et al 1998b) The xcpgene cluster encodes the type II secretion path-way in Gram-negative bacteria also referred toas the main terminal branch (MTB) of the gen-eral secretion pathway (GSP) Proteins secretedvia the GSP pass the cell envelope in two sepa-rate steps First they are translocated across theinner membrane into the periplasm a processmediated by the Sec machinery Subsequentlythe periplasmic intermediates are translocatedacross the outer membrane as fully folded pro-teins (Fig 5) Several nonspecific chaperonesfunction in the periplasm of E coli The peptidyl-prolyl-cis-trans-isomerases (PPI) catalyze thecis-trans isomerization of X-proline peptidebonds which was found to be rate limiting uponhigh level production of functional single chainFv (scFV) fragments in the periplasm of E coli(Jager and Pluckthun 1997) A second class ofnonspecific chaperones the thiol-disulfide oxido-reductases (Dsb) that catalyze the formationof disulfide bonds has been shown to play acrucial role in the formation of disulfide bondsin heterologous proteins expressed in E coli(Joly and Swartz 1997 Joly et al 1998)Recently homologues of dsb genes have beenfound in Pseudomonas aeruginosa to be involvedlipase folding (Reetz and Jaeger 1998)

In addition to nonspecific chaperones thefolding of a variety of extracellular proteinsrequires the action of specific chaperones Forexample the correct folding of lipases is medi-ated by the lipase-specific foldases (Lif) It hasbeen shown that folding of the lipase ofPseudomonas aeruginosa when expressed in Ecoli is dependent on the coexpression of thePseudomonas aeruginosa lif gene (El-Khattabiet al 1999) Interestingly it was found that theamount of Lif can become limiting in an indus-trial Pseudomonas alcaligenes strain upon over-expression of the endogenous lipase gene(Gerritse et al 1998a)

Bacillus species have always been the para-digm hosts for the production of bacterialenzymes and around 50 of the total worldwideenzyme production is by bacilli Neverthelessthe protein secretion machinery of Bacillus hascertain limitations and in a systematic analysismembers of the European Bacillus SecretionGroup (EBSG) over the past years have identi-fied bottlenecks in the secretion pathway ofBacillus subtilis that relate to different stages inthe secretion process Different proteins can runinto different limiting factors (Bolhuis et al1999) During transport over the membrane sig-nal peptidases can become limiting factors inpre-protein processing For example overpro-duction of signal peptidase was shown to bebeneficial for the secretion of heterologous β-


lactamase from Bacillus subtilis (Van Dijl et al1992) Alternatively signal peptidases can inter-fere with efficient pre-protein processing underconditions of high-level overproduction of secre-tory proteins This is illustrated by the observa-tion that the disruption of the sipS geneencoding one of the five signal peptidases ofBacillus subtilis resulted in highly increasedrates of processing of an α-amylase precursor(Tjalsma et al 1997)

Finally late stages in the secretion processincluding the folding of mature proteins and cellwall passage can become secretion bottlenecksIt was found that the lipoprotein PrsA becomeslimiting under conditions of high-level secretionof α-amylases as it is required for the foldinginto a protease-resistant conformation upontranslocation (Kontinen and Sarvas 1993) Inanother experiment it was found that the cellwall which is relatively thick (10ndash50 nm) andcontains a high concentration of immobilizednegative charge (eg teichoic or teichuronicacids) can act as a barrier in translocation (Saun-ders and Guyer 1986 Stephenson et al 1998b)Thus proteins with a net positive charge mightbe retained in the wall Furthermore it wasshown that the wall-bound serine proteaseCWBP52 encoded by the wprA gene candegrade slowly folding enzymes at the site of pre-protein translocation Hence CWBP52 deple-tion has resulted in an increased yield of secretedα-amylase (Stephenson and Harwood 1998a)

More successful approaches to remove bottle-necks in the production of proteins from Bacillusinvolve the elimination of detrimental factorssuch as extracellular proteases In a stepwiseapproach strains with an increasing number ofprotease gene deletions have been constructedresulting in a sevenfold protease negative strainthat shows significant higher yields of susceptiblebacterial enzymes (Ye et al 1999)

Patents and Regulatory Systems

Regulations and Enzymes

Bacterial enzymes for food applications mustcomply with the regulations put forward by theUnited States Food and Drug Administration(FDA) or comparable bodies in other countriesMost enzymes are considered as food processingaids and usually do not end up in the final con-sumer end product Nevertheless all productsundergo a strict testing program including toxic-ity and efficacy testing Finally the industrial pro-duction process has to comply with theregulations stipulated by the EnvironmentalProtection Agency (EPA) These documents maybe accessed through at the Office of Pollution

Prevention and Toxicsrsquo Biotechnology Pro-gram homepage (httpwwwepagovopptintrbiotech) Alternatively the documents areavailable from the EPA homepage (httpwwwepagovfedrgstr) at the EnvironmentalSub Set entry for this document underldquoRegulationsrdquo

The industrial and household enzyme productsnot used for food applications must comply withthe regulations of the EPA and general productsafety regulations Especially with respect to pre-venting allergenicity there are strict specifica-tions for formulating enzymes and preventingdust formation The production host strains mustbe nontoxic and preferably with a record of safeuse Most of the enzyme products have GenerallyRecognized as Safe (GRAS) status

Patents and Taxonomy

Purified enzyme products can be covered by abroad substance patent claim as long as the dis-closure complies with the three elements of apatent application the substance should benovel the disclosure should involve an inventivestep and the substance should have a use Theaspect of novelty can be readily checked sincethe amino acid sequence of a newly describedenzyme can be easily compared to a protein orDNA database As the number of describedamino acid and DNA sequences has exploded inthe past years and since patent examiners tendto use the criterion of 70 amino acid sequenceidentity to specify homologous enzymes it isclear that broad substance patent claims will bedifficult to obtain in future Rather patent pro-tection will be sought more for specific methodsand applications of certain enzymes Enzymesthat have been obtained by protein engineeringor directed evolution represent a special groupof patent claims As the sequence identity toexisting enzymes will generally be very high(gt99) the variant will need to have a propertythat distinguishes it from wildtype enzymes tobecome patentable Patent claims in those caseshave mostly been restricted to the specific exam-ples shown in the description

With the granting of patent claims on livingorganisms (Chakrabarty 1981) a new dimensionwas added to the intellectual property protectionPatent claims on the bacteria themselves wereinitially rejected because living things were notconsidered patentable Finally the United StatesSupreme Court reversed the initial decisionmaking the argument that a genetically engi-neered microorganism is not a product of naturebut rather a product of a personrsquos work and isthus patentable under the United States law Thisdecision has added a new element to the patent-ing of bacterial enzymes and the host cells pro-


ducing them Now also the bacterial strains asisolated from natural sources could be patented(Collins et al 1998a Collins et al 1998b Out-trup et al 1998) A detailed description in theform of a correct taxonomic determination of thestrain is now essential to obtain good patent pro-tection This has led to the development of mod-ern tools for the description of claimed speciessuch as the 16S RNA identification

Prospects

Extremophiles

Enzymes isolated from microorganisms livingunder harsh conditions are adapted to thoseextreme conditions For example an amylase anda protease that are fully stable and active at 95degChave been isolated from Pyrococcus furiosus ahyperthermophile living in a 90degC hotspring(Brown et al 1990 Eggen et al 1990) Espe-cially the progress in research on archaea and theability to culture these strains in the laboratoryhave generated a lot of enthusiasm for house-hold and industrial uses of enzymes from extre-mophiles As the growth conditions for theseextremophiles are difficult to create on an indus-trial scale the goal is to express the genetic mate-rial encoding these enzymes in mesophilic hostsNumerous novel genes encoding thermostable(Koch et al 1990 Hakamada et al 2000) alkalistable (Shendye and Rao 1993 Kobayashi et al1995 Saeki et al 2000) and acid stable (Tamuriet al 1997) enzymes have been characterized inrecent years This can result in not only enzymesbetter suited to existing applications (such asdetergents [alkaline] and starch [high tempera-ture]) but also completely new applications suchas the enzymatic bleaching of pulp a processrequiring both high temperature and very alka-line conditions The yields in production ofenzymes from extremophiles however are gen-erally low because compatibility of these pro-teins with the folding and secretion machinery ofmesophilic hosts is low The impact of thesenovel enzymes on the household and industrialenzyme market therefore remains to be seen andldquoexpressibilityrdquo must be considered when select-ing extremophilic enzymes with desired proper-ties (Van Solingen et al 2001) The best resultshave been obtained with enzymes from extremo-philic eubacterial origin such as the thermo-stable xylanase (produced on a large scale forenzymatic pulp treatment) from Bacillus stearo-thermophilus (Lundgren et al 1994) In researchand diagnostic laboratories the thermostableDNA polymerases (such as the Taq polymerasefrom Thermus aquaticus and Pfu polymerasefrom Pyrococcus furiosis) have shown their tre-

mendous value already (Peterson 1988 Picardet al 1994) The diagnostic enzymes includingthe huge diversity of restriction enzymes andpolymerases are however beyond the scope ofthis chapter

Directed Evolution

In the past two decades the technique of proteinengineering has allowed investigators to createnew enzymes and proteins Interestingly someof the most striking commercial successes havenot been the result of rational design based ona 3D structure but merely the payoff of smartcombinations of random mutagenesis andscreening The power of this combination residesin the fact that many variants with subtle differ-ences can be probed quickly In practice how-ever major weaknesses are still encountered asmost screening assays for enzymatic activity arerather limited in throughput A major improve-ment can be made if a selection instead of ascreening can be introduced This combination ofgene pool diversification and selection for func-tion (collectively termed ldquodirected evolutionrdquo) isnow considered as one of the most successfulprotein engineering strategies Two processesplay a key role in evolution mutation andselection Gene mutation methods have beenexpanded enormously with the advent of thepolymerase chain reaction (PCR) techniques(error prone PCR and PCR with spiked oligonu-cleotide primers and staggered extension pro-cess) and DNA shuffling (Crameri et al 1997Zhao et al 1998 Matsumura et al 1999) How-ever the selection for function is less obvious asthe majority of industrial enzymes are secretedinto the extracellular medium which interfereswith growth selection Most of the newlydescribed directed evolution studies have beenon intracellular enzymes with in vivo selectablefunctions such as β-lactamase which can beselected for by increasing the antibiotic concen-trations (Stemmer 1994) Attempts have beenmade to use display techniques involving cou-pling of the phenotype of an extracellularenzyme with the genotype As demonstratedwith the industrially important enzyme α-amylase from Bacillus licheniformis it is possibleto use phage display for the selection of enzymeswith improved substrate binding properties(Verhaert et al 2002) Binding to substrate tran-sition state analogues has been used to selectfor enzymes with altered catalytic propertiesAlthough binding of phages to transition stateanalogues is feasible the use of this technique toselect for industrially relevant catalytic proper-ties remains to be established (Legendre et al2000) More success has been obtained with thecompartmentalization of bacteria that are secret-


ing mutant enzymes By fixing the mutant bacte-rial cells in a solid matrix the diffusion of thesecreted mutant protease was delayed This pro-vides a way of coupling the phenotype to thegenotype inasmuch as the converted growthsubstrate remains in the same compartment asthe bacterial cell (Tawfik and Griffiths 1998Griffiths and Tawfik 2003) Finally a noveldimension has been given to evolution tech-niques by the use of genes isolated directly fromsoil samples (without culturing the donor organ-ism) In a large experiment genes encodingamylases were cloned directly from soil andidentified by expression on starch plates and theresulting genes have been ldquoevolvedrdquo using DNAshuffling This has resulted in a very thermo-stable α-amylase (Richardson et al 2002) Thisshows that isolating enzyme encoding genesfrom extremophiles combined with directed evo-lution in the laboratory can be a path forward forenzyme engineering

Acknowledgments This chapter is a compilationof numerous collaborations that have allowedthe author to develop insights and ideas in thebacterial enzyme field Special thanks to all thecolleagues and students who have contributedmaterial and illustrations presented in this chap-ter The sponsoring of EU under contractsBIO2-CT950119 BIO4-9-98-0249 QLK3-CT-1999-00413 QLTR-2001-00519 and of NWOSTW under contract GBI4707 is highlyacknowledged

Literature Cited

Aehle W G Gerritse and H B Lenting 1995 Lipases withImproved Surfactant Resistance Patent WO 9530744

Aiba S K Kitai and T Imanaka 1983 Cloning and expres-sion of thermostable alpha-amylase gene from Bacillusstearothermophilus in Bacillus stearothermophilus andBacillus subtilis Appl Environ Microbiol 461059ndash1065

Albers E and B W Muller 1995 Cyclodextrin derivativesPharmaceut Crit Rev Ther Drug Carrier Syst 12311ndash337

Alkema W B C M Hensgens E H Kroezinga E DeVries R Floris J M Van der Laan B W Dijkstra andD B Janssen 2000 Characterization of the beta-lactambinding site of penicillin acylase of Escherichia coli bystructural and site-directed mutagenesis studies ProteinEngin 13857ndash863

Genencor Cleaning Enzymes Product List 2001 GenencorCleaning Enzymes Product List Genencor Interna-tional Rochester NY

Aramori I M Fukagawa M Tsumura M Iwami T IsogaiH Ono Y Ishitani H Kojo M Kohsaka Y Ueda andH Imanaka 1991a Cloning and nucleotide sequencingof new glutaryl 7-aca and cephalosporin c acylase genesfrom pseudomonas strains J Ferment Bioengin72(4)232ndash243

Aramori I M Fukagawa M Tsumura M Iwami H OnoH Kojo M Kohsaka Y Ueda and H Imanaka 1991bCloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase geneof Bacillus laterosporus and its expression in Escheri-chia coli and Bacillus subtilis J Bacteriol 1737848ndash7855

Balasingham K D Warburton P Dunnill and M D Lilly1972 The isolation and kinetics of penicillin amidasefrom Escherichia coli Biochim Biophys Acta 276250ndash256

Barbero J L J M Buesa G Gonzalez De Buitrago EMendez A Perez-Aranda and J L Garcia 1986 Com-plete nucleotide sequence of penicillin acylase genefrom Kluyvera citrophila Gene 4969ndash80

Bertola M A W J Quax B W Robertson A F Marx andC J van der Laken 1992 Microbial Esterases and Pro-cess for the Preparation of 2-arylpropionic Acids PatentEP 233656-B

Betzel C K S Wilson and S Branner 1988 Crystallizationand preliminary X-ray diffraction studies of an alkalineprotease from Bacillus lentus J Molec Biol 204803ndash804

Boesten W H J and H M Moody 1995 Process for theEnzymatic Preparation of a Beta-lactam DerivativePatent WO 9503420

Bolhuis A H Tjalsma H E Smith A De Jong R MeimaG Venema S Bron and J M van Dijl 1999 Evaluationof bottlenecks in the late stages of protein secretionin Bacillus subtilis Appl Environ Microbiol 652934ndash2941

Brown S H H R Costantino and R M Kelly 1990 Char-acterization of amylolytic enzyme activities associatedwith the hyperthermophilic archaebacterium Pyro-coccus furiosus Appl Environ Microbiol 561985ndash1991

Bruggink A E C Roos and E Devroom 1998 Penicillinacylase in the industrial production of beta-lactam anti-biotics Organ Proc Res Devel 2128ndash133

Bruns W Hoppe J Tsai H Bruning H J Maywald FCollins J Mayer H 1985 Structure of the penicillinacylase gene from Escherichia coli a periplasmicenzyme that undergoes multiple proteolytic processingJ Mol Appl Genet 3(1)36ndash44

Buleon A P Colonna V Planchot and S Ball 1998 Starchgranules Structure and biosynthesis Int J Biol Macro-mol 2385ndash112

Callens M H Kersters-Hilderson W Vangrysperre andC K De Bruyne 1988 D-xylose isomerase from Strep-tomyces violaceoruber Structural and catalytic roles ofbivalent metal ions Enzyme Microb Technol 10695ndash700

Chakrabarty A M 1981 Microorganisms Having MultipleCompatible Degradative Energy-generating Plasmidsand Preparation Thereof US Patent 4259444

Chandra A K S Medda and A K Bhadra 1980 Produc-tion Of extracellular thermostable alpha-amylase byBacillus licheniformis J Ferment Technol 581ndash10

Chen W-P 1980 Glucose isomerase [review] Proc Bio-chem 1536ndash41

Christiansen T B Christensen and J Nielsen 2002 Meta-bolic network analysis of Bacillus clausii on minimal andSemirich medium using (13)C-labeled glucose MetabEngin 4159ndash169

Coffen D L 1997 Enzyme-catalyzed reactions In S EAhuja (Ed) Chiral Separations Applications and Tech-


nology American Chemical Society Washington DC59ndash91

Collins N C W D Grant and B E Jones 1998a Gram-negative Alkaliphilic Microorganisms US Patent5733767

Collins N C W D Grant and B E Jones 1998b Gram-positive Alkaliphilic Microorganisms US Patent5707851

Cox M G Gerritse L Dankmeyer and W J Quax 2001Characterization of the promoter and upstream activat-ing sequence from the Pseudomonas alcaligenes lipasegene J Biotechnol 869ndash17

Crameri A G Dawes E Rodriguez Jr S Silver and W PStemmer 1997 Molecular evolution of an arsenatedetoxification pathway by DNA shuffling Nature Bio-technol 15436ndash438

Crawford L A M Stepan P C Mcada J A RambosekM J ConderV A Vinci and C D Reeves 1995 Pro-duction of cephalosporin intermediates by feeding adi-pic acid to recombinant Penicillium chrysogenum strainsexpressing ring expansion activity Biotechnology NY1358ndash62

Drenth J W G J Hol J N Jansonius and R Koekoek1972 Subtilisin novo The three-dimensional structureand its comparison with subtilisin Bpn Eur J Biochem26177ndash181

Droge M J R Bos and W J Quax 2001 Paralogous geneanalysis reveals a highly enantioselective 12-o-isopropy-lideneglycerol caprylate esterase of Bacillus subtilisEur J Biochem 2683332ndash3338

Drummond R J W Bloch B W Matthews P L Toy andH H Nicholson 1989 Procaryotic xylose isomerasemuteins and method to increase protein stability PatentWO 8901520

Edman M T Jarhede M Sjostrom and A Wieslander1999 Different sequence patterns in signal peptidesfrom mycoplasmas other Gram-positive bacteria andEscherichia coli A multivariate data analysis ProtStruct Funct Genet 35195ndash205

Eggen R A Geerling J Watts and W M Devos 1990Characterization of pyrolysin a hyperthermoactiveserine protease from the archaebacterium Pyrococcusfuriosus FEMS Microbiol Lett 7117ndash20

El-Khattabi M C Ockhuijsen W Bitter K E Jaeger andJ Tommassen 1999 Specificity of the lipase-specific fol-dases of Gram-negative bacteria and the role of themembrane anchor Molec Gen Genet 261770ndash776

Emtage J S S Angal M T Doel T J R Harris B LoweP A Jenkins and G Lilley 1983 Synthesis of calf pro-chymosin (prorennin) in Escherichia coli (synthetic oli-gonucleotidegene expressionindustrial enzyme) ProcNatl Acad Sci USA 803671ndash3675

Estell D A T P Graycar and J A Wells 1985 Engineeringan enzyme by site-directed mutagenesis to be resistantto chemical oxidation J Biol Chem 2606518ndash6521

Farber G K G A Petsko and D Ringe 1987 The 30 Acrystal structure of xylose isomerase from Streptomycesolivochromogenes Protein Engin 1459ndash466

Filloux A G Michel and M Bally 1998 GSP-dependentprotein secretion in Gram-negative bacteria The Xcpsystem of Pseudomonas aeruginosa FEMS MicrobiolRev 22177ndash198

Gat O A Lapidot I Alchanati C Regueros and Y Sho-ham 1994 Cloning and dna sequence of the gene codingfor Bacillus stearothermophilus T-6 xylanase ApplEnviron Microbiol 601889ndash1896

Gerritse G R W Hommes and W J Quax 1998a Devel-opment of a lipase fermentation process that uses arecombinant Pseudomonas alcaligenes strain ApplEnviron Microbiol 642644ndash2651

Gerritse G R Ure F Bizoullier and W J Quax 1998b Thephenotype enhancement method identifies the Xcpouter membrane secretion machinery from Pseudomo-nas alcaligenes as a bottleneck for lipase production JBiotechnol 6423ndash38

Gray G L S E Mainzer M W Rey M H Lamsa K LKindle C Carmona and C Requadt 1986 Structuralgenes encoding the thermophilic alpha-amylases ofBacillus stearothermophilus and Bacillus licheniformisJ Bacteriol 166635ndash643

Gray G L S D Power and A J Poulouse 1995 Lipasefrom Pseudomonas Mendocina Having Cutinase Activ-ity US Patent 5389536

Griffiths A D and D S Tawfik 2003 Directed evolution ofan extremely fast phosphotriesterase by in vitro com-partmentalization EMBO J 2224ndash35

Gusakov A V A P Sinitsyn A G Berlin A V Markovand N V Ankudimova 2000 Surface hydrophobicamino acid residues in cellulase molecules as a structuralfactor responsible for their high denim-washing perfor-mance Enz Microb Technol 27664ndash671

Hakamada Y Y Hatada K Koike T Yoshimatsu SKawai T Kobayashi and S Ito 2000 Deduced aminoacid sequence and possible catalytic residues of athermostable alkaline cellulase from an alkaliphilicbacillus strain Biosci Biotechnol Biochem 642281ndash2289

Hedstrom G M Backlund and J P Slotte 1993 Enantiose-lective synthesis of ibuprofen esters in Aot isooctanemicroemulsions by Candida cylindracea lipase Biotech-nol Bioengin 42618ndash624

Henrick K D M Blow H L Carrell and J P Glusker1987 Comparison of backbone structures of glucoseisomerase from streptomyces and arthrobacter ProteinEngin 1467ndash469

Hesselink P G M S van Vliet H De Vries and B Witholt1989 Optimization of steroid side chain cleavage byMycobacterium sp in the presence of cyclodextrinsEnz Microb Technol 11398ndash404

Hofemeister J A Kurtz R Borriss and J Knowles 1986The beta-glucanase gene from Bacillus amyloliquefa-ciens shows extensive homology with that of Bacillussubtilis Gene 49177ndash187

Hofmann B E H Bender and G E Schulz 1989 Three-dimensional structure of cyclodextrin glycosyltrans-ferase from Bacillus circulans at 34 A resolution JMolec Biol 209793ndash800

Irwin D D H Shin S Zhang B K Barr J Sakon P AKarplus and D B Wilson 1998 Roles of the catalyticdomain and two cellulose binding domains of Ther-momonospora fusca E4 in cellulose hydrolysis J Bacte-riol 1801709ndash1714

Ishii Y Y Saito T Fujimura T Isogai H Kojo MYamashita M Niwa and M Kohsaka 1994 A novel 7-beta-(4-carboxybutanamido)-cephalosporanic acid acy-lase isolated from Pseudomonas strain C427 and itshigh-level production in Escherichia coli J FermentBioengin 77591ndash597

Ishiye M and M Niwa 1992 Nucleotide sequence andexpression in Escherichia coli of the cephalosporin acy-lase gene of a Pseudomonas strain Biochim BiophysActa 1132233ndash239


Jacobs M M Eliasson M Uhlen and J I Flock 1985Cloning sequencing and expression of subtilisin Carls-berg from Bacillus lichenformis Nucleic Acids Res138913ndash8927

Jager M and A Pluckthun 1997 The rate-limiting steps forthe folding of an antibody Scfv fragment FEBS Lett418106ndash110

Jeong S T H K Kim S J Kim S W Chi J G Pan T KOh and S E Ryu 2002 Novel zinc-binding center anda temperature switch in the Bacillus stearothermophilusL1 lipase J Biol Chem 27717041ndash17047

Joly J C and J R Swartz 1997 In vitro and in vivo redoxstates of the Escherichia coli periplasmic oxidoreduc-tases Dsba and Dsbc Biochemistry 3610067ndash10072

Joly J C W S Leung and J R Swartz 1998 Overexpres-sion of Escherichia coli oxidoreductases increasesrecombinant insulin-like growth factor-I accumulationProc Natl Acad Sci USA 952773ndash2777

Jones B and W Quax 1998a Alzheimer tau test and deter-gent cellulase made by genetic engineering [no 9 in aseries of articles to promote a better understanding ofthe use of genetic engineering] J Biotechnol 66229ndash233

Jones B E W D Grant A W Duckworth and G G Owen-son 1998b Microbial diversity of soda lakes Extremo-philes 2191ndash200

Jongbloed A W Z Mroz and P A Kemme 1992 The effectof supplementary Aspergillus niger phytase in diets forpigs on concentration and apparent digestability of drymatter total phosphorus and phytic acid in differentsections of the animentary tract J Anim Sci 701168

Jorgensen O B L G Karlsen N B Nielsen S Pedersenand S Rugh 1988 A new immobolized glucoseisomerase with high productivity produced by a strain ofStreptomyces murinus StarchStarke 40307ndash313

Jorgensen S T and P L Jorgensen 1993 A Process forExpressing Genes in Bacillus Licheniformis Patent WO9310248

Jung E D G Lao D Irwin B K Barr A Benjamin andD B Wilson 1993 DNA sequences and expression inStreptomyces lividans of an exoglucanase gene and anendoglucanase gene from Thermomonospora fuscaAppl Environ Microbiol 593032ndash3043

Kelly A P B Diderichsen S Jorgensen and D JMcConnell 1994 Molecular genetic analysis of the pul-lulanase b gene of Bacillus acidopullulyticus FEMSMicrobiol Lett 11597ndash105

Kerovuo J and S Tynkkynen 2000a Expression of Bacillussubtilis phytase in Lactobacillus plantarum 755 LettAppl Microbiol 30325ndash329

Kerovuo J J Rouvinen and F Hatzack 2000b Analysis ofmyo-inositol hexakisphosphate hydrolysis by bacillusphytase Indication of a novel reaction mechanism Bio-chem J 352623ndash628

Kitahata S M Taniguchi S D Beltran T Sugimoto and SOkada 1983 Purification and some properties of cyclo-dextrinase from Bacillus coagulans Agric Biol Chem471441ndash1447

Knowles J P Lehtovaara M Penttila T Teeri A Harkkiand I Salovuori 1987 The cellulase genes of Tricho-derma Ant v Leeuwenhoek 53335ndash341

Kobayashi T Y Hakamada S Adachi J Hitomi T Yoshi-matsu K Koike S Kawai and S Ito 1995 Purificationand properties of an alkaline protease from alkalophilicBacillus sp KSM-K16 Appl Microbiol Biotechnol43473ndash481

Koch R P Zablowski A Spreinat and G Antranikian1990 Extremely thermostable amylolytic enzyme fromthe archaebacterium Pyrococcus furiosus FEMS Micro-biol Lett 7121ndash26

Koeller K M and C H Wong 2001 Enzymes for chemicalsynthesis Nature 409232ndash240

Kontinen V P and M Sarvas 1993 The PrsA lipoprotein isessential for protein secretion in Bacillus subtilis andsets a limit for high-level secretion Molec Microbiol8727ndash737

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Kreft J H Berger M Haertlein B Mueller G Goebel andW Weidinger 1983 Cloning and expression in E coliand Bacillus subtilis of the hemolysin determinant fromBacillus cereus J Bacteriol 155681ndash689

Lapidot A A Mechaly and Y Shoham 1996 Overexpres-sion and single-step purification of a thermostablexylanase from Bacillus stearothermophilus T-6 J Bio-technol 51259ndash264

Legendre D N Laraki T Graslund M E Bjornvad MBouchet P A Nygren T V Borchert and J Fastrez 2000Display of active subtilisin 309 on phage Analysis ofparameters influencing the selection of subtilisin variantswith changed substrate specificity from libraries usingphosphonylating inhibitors J Molec Biol 29687ndash102

Li Y W Jiang Y Yang G Zhao and E Wang 1998 Over-production and purification of glutaryl 7-amino cepha-losporanic acid acylase Protein Expr Purif 12233ndash238

Lin W and D R Lineback 1990 Changes in carbohydratefractions in enzyme-supplemented bread and the poten-tial relationship to staling Starch 42385ndash394

Luiten R G M W J Quax P W Schuurhuizen and NMrabet 1990 Novel Glucose Isomerase Enzymes andTheir Use Patent EP 0351029-A

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Lyons T P 1982 Proteinase enzymes relevant to the bakingindustry Biochem Soc Trans 10287ndash290

Mansfeld J G Vriend B W Dijkstra O R Veltman B Bvan Den G Venema R Ulbrich-Hofmann and V GEijsink 1997 Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond J BiolChem 27211152ndash11156

Matsuda A K Matsuyama K Yamamoto S Ichikawa andK Komatsu 1987 Cloning and characterization of thegenes for two distinct cephalosporin acylases from aPseudomonas strain J Bacteriol 1695815ndash5820

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Mondou F F Shareck R Morosoli and D Kluepfel 1986Cloning of the xylanase gene of Streptomyces lividansGene 49323ndash329

Nakajima R T Imanaka and S Aiba 1986 Comparisonof animo acid sequences of eleven different alpha-amylases Appl Microbiol Biotechnol 23355ndash360


Nardini M D A Lang K Liebeton K E Jaeger andB W Dijkstra 2000 Crystal structure of Pseudomonasaeruginosa lipase in the open conformation The proto-type for Family I1 of bacterial lipases J Biol Chem27531219ndash31225

Nishimori K Y Kawaguchi M Hidaka T Uozumi and TBeppu 1981 Communication Cloning in Escherichiacoli of the structural gene of prorennin the precursor ofcalf milk-clotting enzyme rennin J Biochem 90901ndash904

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Norris L F Norris L Christiansen and N Fiil 1983 Effi-cient site-directed mutagenesis by simultaneous use oftwo primers Nucleic Acids Res 115103ndash5112

Saari G C Kumar A A Kawasaki G H Insley M YOrsquoHara PJ 1987 Sequence of the Ampullariella spstrain 3876 gene coding for xylose isomerase J Bacte-riol 169(2)612ndash618

Otten L G C F Sio J Vrielink R H Cool and W J Quax2002 Altering the substrate specificity of cephalosporinacylase by directed evolution of the beta-subunit J BiolChem 27742121ndash42127

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Park S C Y W Choi and T K Oh 1999 Comparativeenzymatic hydrolysis of phytate in various animal feed-stuff with two different phytases J Vet Med Sci611257ndash1259

Peterson M G 1988 DNA sequencing using Taq poly-merase Nucleic Acids Res 1610915

Picard V E Ersdalbadju A Q Lu and S C Bock 1994 Arapid and efficient one-tube PCR-based mutagenesistechnique using PFU DNA polymerase Nucleic AcidsRes 222587ndash2591

Quax W J Y Laroche A W H Vollebregt P Stanssensand M Lauwereys 1991a Mutant Microbial Alpha-amylases with Increased Thermal Acid andor AlkalineStability Patent WO 9100353

Quax W J N T Mrabet R G Luiten P W SchuurhuizenP Stanssens and I Lasters 1991b Enhancing the ther-mostability of glucose isomerase by protein engineeringBiotechnology NY 9738ndash742

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Quax W J and C P Broekhuizen 1994 Development of anew bacillus carboxyl esterase for use in the resolutionof chiral drugs Appl Microbiol Biotechnol 41425ndash431

Reetz M T and K E Jaeger 1998 Overexpressionimmobilization and biotechnological application ofPseudomonas lipases Chem Phys Lipids 933ndash14

Reilly P J 1979 Starch hydrolysis with soluble and immobi-lized glucoamylase Appl Biochem Bioengin 2185ndash207

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Robertson D E D Murphy J Reid M M Antony S LinkR V Swanson P V Warren and A Kosmotka 1999Esterases US Patent 5942430

Roels J A and R van Tilburg 1979 Temperaturedependence of the stability and the activity of immo-bilized glucose isomerase ACS Symp Series 106147ndash172

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Saeki K M Okuda Y Hatada T Kobayashi S Ito HTakami and K Horikoshi 2000 Novel oxidatively sta-ble subtilisin-like serine proteases from alkaliphilicbacillus spp Enzymatic properties sequences and evo-lutionary relationships Biochem Biophys Res Com-mun 279313ndash319

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Saul D J L C Williams R A Reeves M D Gibbs andP L Bergquist 1995 Sequence and expression of a xyla-nase gene from the hyperthermophile Thermotoga spstrain Fjss3-B1 and characterization of the recombinantenzyme and its activity on kraft pulp Appl EnvironMicrobiol 614110ndash4113

Saunders C W and M S Guyer 1986 The Production ofHuman Serum Albumin in Bacillus New Gene FusionsPlasmids and Bacillus Strains Useful in Production ofHuman Serum Albumin Economically by CultivationPatent EP 0229712a2 13

Schmid A J S Dordick B Hauer A Kiener M Wubboltsand B Witholt 2001 Industrial biocatalysis today andtomorrow Nature 409258ndash268

Schrag J D Y Li M Cygler D Lang T Burgdorf H JHecht R Schmid D Schomburg T J Rydel J DOliver L C Strickland C M Dunaway S B Larson JDay and A McPherson 1997 The open conformationof a pseudomonas lipase Structure 5187ndash202

Schroen C G S Vandewiel P J Kroon E Devroom A EJanssen and J Tramper 2000 Equilibrium positionkinetics and reactor concepts for the adipyl-7-Adca-hydrolysis process [in process citation] Biotech-nol Bioengin 70654ndash661

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Shendye A and M Rao 1993 Cloning and extracellularexpression in Escherichia coli of xylanases from an alka-liphilic thermophilic bacillus sp Ncim-59 FEMS Micro-biol Lett 108297ndash302

Shibuya Y K Matsumoto and T Fujii 1981 Isolation andProperties of 7β-(4-carboxybutanamido) cephalospo-ranic acid acylase-producing bacteria Agric BiolChem 451561ndash1567

Simons P C M H A J Versteegh A V Jongbloed P AKemme P Skump K D Bos M G E Wolters R FBeudeker and G Verschoor 1990 Improvement ofphosphorus availability by microbial phytase in broilersand pigs Br J Nutr 64525ndash540


Sio C F A M Riemens J M van der Laan R M Verhaertand W J Quax 2002 Directed evolution of a glutarylacylase into an adipyl acylase Eur J Biochem 2694495ndash4504

Spezio M D B Wilson and P A Karplus 1993 Crystalstructure of the catalytic domain of a thermophilic endo-cellulase Biochemistry 329906ndash9916

Stemmer W P 1994 Rapid evolution of a protein in vitro byDNA shuffling Nature 370389ndash391

Stephenson K and C R Harwood 1998a Influence of acell-wall-associated protease on production of alpha-amylase by Bacillus subtilis Appl Environ Microbiol642875ndash2881

Stephenson K N M Carter C R Harwood M FPetitglatron and R G Chambert 1998b The influenceof protein folding on late stages of the secretion ofalpha-amylases from Bacillus subtilis FEBS Lett430385ndash389

Tamuri M M Kanno and Y Ishii 1997 Heat and Acid-stable Alpha-amylase Enzymes and Processes for Pro-ducing the Same US Patent 4283722

Tawfik D S and A D Griffiths 1998 Man-made cell-likecompartments for molecular evolution Nat Biotechnol16652ndash656

Tjalsma H M A Noback S Bron G Venema K Yamaneand J M van Dijl 1997 Bacillus subtilis contains fourclosely related Type I signal peptidases with overlappingsubstrate specificities Constitutive and temporally con-trolled expression of different Sip genes J Biol Chem27225983ndash25992

Van den Burg B G Vriend O R Veltman G Venema andV G Eijsink 1998 Engineering an enzyme to resistboiling Proc Natl Acad Sci USA 952056ndash2060

van der Laan J C Gerritse G Mulleners L J van derHoek R A Quax W J 1991 Cloning characterizationand multiple chromosomal integration of a Bacillusalkaline protease gene Appl Environ Microbiol57(4)901ndash909

Van der Laan J M AV Teplyakov H Kelders K H KalkO Misset L S J M Mulleners and B W Dijkstra 1992Crystal structure of the high-alkaline serine protease-Pb92 from Bacillus alcalophilus Protein Engin 5405ndash411

Van Dijl J M A Dejong J Vehmaanpera G Venema andS Bron 1992 Signal peptidase-I of Bacillus subtilisPatterns of conserved amino acids in prokaryotic andeukaryotic Type-I signal peptidases EMBO J 112819ndash2282

Van Eekelen C A G J C van der Laan and L J S Mul-leners 1988 Molecular Cloning and Expression ofGenes Encoding Proteolytic Enzymes Patent EP0283075

Van Eekelen C A G L J S Mulleners J C van der LaanO Misset R A Cuperus and J H Alensink 1989Novel Proteolytic Enzymes and Their Use in Deter-gents Patent EP 0328229

van Leen R W Bakhuis J G van Beckhoven R F BurgerH Dorssers L C Hommes R W Lemson P JNoordam B Persoon N L Wagemaker G 1991 Pro-duction of human interleukin-3 using industrial micro-organisms Biotechnology 947ndash52

Van PouderoyenG T Eggert K E Jaeger and B W Dijk-stra 2001 The crystal structure of Bacillus subtilislipase A minimal alphabeta hydrolase fold enzyme JMolec Biol 309215ndash226

Van Solingen P D Meijer W A van der Kleij C BarnettR Bolle S D Power and B E Jones 2001 Cloning andexpression of an endocellulase gene from a novel strep-tomycete isolated from an East African soda lakeExtremophiles 5333ndash341

Van Straten N C R H I Duynstee E Devroom G A Vander Marel and J H van Boom 1997 Enzymatic cleav-age of N-phenylacetyl-protected ethanolamine phos-phates Liebigs Annalen 0 (6)1215ndash1220

Verhaert R M A M Riemens J M van der Laan J vanDuin and W J Quax 1997 Molecular cloning and anal-ysis of the gene encoding the thermostable penicillin gacylase from Alcaligenes faecalis Appl Environ Micro-biol 633412ndash3418

Verhaert R M J Beekwilder R Olsthoorn J van Duinand W J Quax 2002 Phage display selects for amylaseswith improved low pH starch-binding J Biotechnol96103ndash118

Wallerstein L 1939 Enzyme preparation from microorgan-isms Indust Engin Chem 311218ndash1224

Welker N E and L L Campbell 1967a Comparison of thealpha-amylase of Bacillus subtilis and Bacillus amy-loliquefaciens J Bacteriol 941131ndash1135

Welker N E and L L Campbell 1967b Unrelatedness ofBacillus amyloliquefaciens and Bacillus subtilis J Bac-teriol 941124ndash1130

Wind R D J C Uitdehaag R M Buitelaar B W Dijkstraand L Dijkhuizen 1998 Engineering of cyclodextrinproduct specificity and pH optima of the thermostablecyclodextrin glycosyltransferase from Thermoanaero-bacterium thermosulfurigenes Em1 J Biol Chem2735771ndash5779

Wong H C T Ting H-C Lin F Reichert K MyamboK W K Watt P L Toy and R J Drummond 1991Genetic organization and regulation of the xylose deg-radation genes in Streptomyces rubiginosum J Bacte-riol 1736849ndash6858

Xie Y S E van De T De Weerd and N H Wang 2001Purification of adipoyl-7-amino-3-deacetoxycepha-losporanic acid from fermentation broth using stepwiseelution with a synergistically adsorbed modulator JChromatogr A908273ndash291

Yamagata H and S Udaka 1994 Starch-processingenzymes produced by recombinant bacteria BioprocessTechnol 19325ndash340

Ye R Q J H Kim B G Kim S Szarka E Sihota andS L Wong 1999 High-level secretory production ofintact biologically active staphylokinase from Bacillussubtilis Biotechnol Bioengin 6287ndash96

Yuuki T T Nomura H Tezuka A Tsuboi H TsukagoshiN Yamagata and S Udaka 1985 Complete nucleotidesequence of gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis Comparison of theamino acid sequence of 3 bacterial liquefying alpha-amylases deduced from the DNA J Biochem 981147ndash1156

Zhao H M L Giver Z X Shao J A Affholter and F HArnold 1998 Molecular evolution by STaggered Exten-sion Process (STEP) in vitro recombination Nature Bio-technol 16258ndash261

Zhu G P D Luo Y F Cai X Y Zhu M K Teng andY Z Wang 2000 Mutations of Q20l and G247dimproved the specific-activity and optimum pH of glu-cose isomerase Sheng Wu Gong Cheng Xue Bao16469ndash473


Fig 1 A copy of the original patent by Roumlhm which describes for the first time the use of proteases as a cleaning aid






Starch




















O

OH CH3OH

CH2OH CH2OHCH3

OH

OH OH

OH OHOH

OH

O

O O

O OOO

O













Detergents




120

rela

tive

activ

ity (

)

100 80 60 40

1 2 3 4 5 6 7 8 9

Time (days)

10 11 121314

K253RWild Type

20 0







Outer membrane

Periplasm TUVW

A S

R

Xcp machinery

Ch

Sec

QP

X

Y

Z






Food Processing











Textiles




Fine Chemicals



Amidases










H2N H2N SH

NH

OO

O

N

N

H2N

S

S

CH3

CH3

CH3

CH3

CH3

NH2

NHHO

CH3

CH3

CH3

NH2

NH

OO

N

S

O

O

O

O

O

OO

O

OO N

N

S

S

COOH

CH3

H2NCOOH

COOHCOOH

COOH

COOH

COOH

HOOC

COOH

L-amino adipic acid



Clutaryl - acylase

Penicillin G

6-APA

7-ACA

COOH



Cephalosporin C


Glutaryl-7-ACA








CH3

CH3

CH3

N

N

S

S

O

O

O

O

O

O

HO

Adipyl-penicillin




NH

New process

NHHO

COOH

CH3

CH3N

S

O

O

Penicillin G


Current process

NH

COOH

CH3

CH3

N

S

N

S

O

O

O

NH

H2N

COOH

COOH

penG acylase

7-ADCA

Cephalosporin G

E coli∆ G

COOH

CH3

N

S

O

N2H

COOH

7-ADCA







Areas of Research

Feed






Paper and Pulp

























Prospects

Extremophiles



Directed Evolution





Literature Cited



































































































































































Starch




















O

OH CH3OH

CH2OH CH2OHCH3

OH

OH OH

OH OHOH

OH

O

O O

O OOO

O













Detergents




120

rela

tive

activ

ity (

)

100 80 60 40

1 2 3 4 5 6 7 8 9

Time (days)

10 11 121314

K253RWild Type

20 0







Outer membrane

Periplasm TUVW

A S

R

Xcp machinery

Ch

Sec

QP

X

Y

Z






Food Processing











Textiles




Fine Chemicals



Amidases










H2N H2N SH

NH

OO

O

N

N

H2N

S

S

CH3

CH3

CH3

CH3

CH3

NH2

NHHO

CH3

CH3

CH3

NH2

NH

OO

N

S

O

O

O

O

O

OO

O

OO N

N

S

S

COOH

CH3

H2NCOOH

COOHCOOH

COOH

COOH

COOH

HOOC

COOH

L-amino adipic acid



Clutaryl - acylase

Penicillin G

6-APA

7-ACA

COOH



Cephalosporin C


Glutaryl-7-ACA








CH3

CH3

CH3

N

N

S

S

O

O

O

O

O

O

HO

Adipyl-penicillin




NH

New process

NHHO

COOH

CH3

CH3N

S

O

O

Penicillin G


Current process

NH

COOH

CH3

CH3

N

S

N

S

O

O

O

NH

H2N

COOH

COOH

penG acylase

7-ADCA

Cephalosporin G

E coli∆ G

COOH

CH3

N

S

O

N2H

COOH

7-ADCA







Areas of Research

Feed






Paper and Pulp

























Prospects

Extremophiles



Directed Evolution





Literature Cited





































































































































































O

OH CH3OH

CH2OH CH2OHCH3

OH

OH OH

OH OHOH

OH

O

O O

O OOO

O













Detergents




120

rela

tive

activ

ity (

)

100 80 60 40

1 2 3 4 5 6 7 8 9

Time (days)

10 11 121314

K253RWild Type

20 0







Outer membrane

Periplasm TUVW

A S

R

Xcp machinery

Ch

Sec

QP

X

Y

Z






Food Processing











Textiles




Fine Chemicals



Amidases










H2N H2N SH

NH

OO

O

N

N

H2N

S

S

CH3

CH3

CH3

CH3

CH3

NH2

NHHO

CH3

CH3

CH3

NH2

NH

OO

N

S

O

O

O

O

O

OO

O

OO N

N

S

S

COOH

CH3

H2NCOOH

COOHCOOH

COOH

COOH

COOH

HOOC

COOH

L-amino adipic acid



Clutaryl - acylase

Penicillin G

6-APA

7-ACA

COOH



Cephalosporin C


Glutaryl-7-ACA








CH3

CH3

CH3

N

N

S

S

O

O

O

O

O

O

HO

Adipyl-penicillin




NH

New process

NHHO

COOH

CH3

CH3N

S

O

O

Penicillin G


Current process

NH

COOH

CH3

CH3

N

S

N

S

O

O

O

NH

H2N

COOH

COOH

penG acylase

7-ADCA

Cephalosporin G

E coli∆ G

COOH

CH3

N

S

O

N2H

COOH

7-ADCA







Areas of Research

Feed






Paper and Pulp

























Prospects

Extremophiles



Directed Evolution





Literature Cited










































































































































































Detergents




120

rela

tive

activ

ity (

)

100 80 60 40

1 2 3 4 5 6 7 8 9

Time (days)

10 11 121314

K253RWild Type

20 0







Outer membrane

Periplasm TUVW

A S

R

Xcp machinery

Ch

Sec

QP

X

Y

Z






Food Processing











Textiles




Fine Chemicals



Amidases










H2N H2N SH

NH

OO

O

N

N

H2N

S

S

CH3

CH3

CH3

CH3

CH3

NH2

NHHO

CH3

CH3

CH3

NH2

NH

OO

N

S

O

O

O

O

O

OO

O

OO N

N

S

S

COOH

CH3

H2NCOOH

COOHCOOH

COOH

COOH

COOH

HOOC

COOH

L-amino adipic acid



Clutaryl - acylase

Penicillin G

6-APA

7-ACA

COOH



Cephalosporin C


Glutaryl-7-ACA








CH3

CH3

CH3

N

N

S

S

O

O

O

O

O

O

HO

Adipyl-penicillin




NH

New process

NHHO

COOH

CH3

CH3N

S

O

O

Penicillin G


Current process

NH

COOH

CH3

CH3

N

S

N

S

O

O

O

NH

H2N

COOH

COOH

penG acylase

7-ADCA

Cephalosporin G

E coli∆ G

COOH

CH3

N

S

O

N2H

COOH

7-ADCA







Areas of Research

Feed






Paper and Pulp

























Prospects

Extremophiles



Directed Evolution





Literature Cited




































































































































































Outer membrane

Periplasm TUVW

A S

R

Xcp machinery

Ch

Sec

QP

X

Y

Z






Food Processing











Textiles




Fine Chemicals



Amidases










H2N H2N SH

NH

OO

O

N

N

H2N

S

S

CH3

CH3

CH3

CH3

CH3

NH2

NHHO

CH3

CH3

CH3

NH2

NH

OO

N

S

O

O

O

O

O

OO

O

OO N

N

S

S

COOH

CH3

H2NCOOH

COOHCOOH

COOH

COOH

COOH

HOOC

COOH

L-amino adipic acid



Clutaryl - acylase

Penicillin G

6-APA

7-ACA

COOH



Cephalosporin C


Glutaryl-7-ACA








CH3

CH3

CH3

N

N

S

S

O

O

O

O

O

O

HO

Adipyl-penicillin




NH

New process

NHHO

COOH

CH3

CH3N

S

O

O

Penicillin G


Current process

NH

COOH

CH3

CH3

N

S

N

S

O

O

O

NH

H2N

COOH

COOH

penG acylase

7-ADCA

Cephalosporin G

E coli∆ G

COOH

CH3

N

S

O

N2H

COOH

7-ADCA







Areas of Research

Feed






Paper and Pulp

























Prospects

Extremophiles



Directed Evolution





Literature Cited



































































































































































Food Processing











Textiles




Fine Chemicals



Amidases










H2N H2N SH

NH

OO

O

N

N

H2N

S

S

CH3

CH3

CH3

CH3

CH3

NH2

NHHO

CH3

CH3

CH3

NH2

NH

OO

N

S

O

O

O

O

O

OO

O

OO N

N

S

S

COOH

CH3

H2NCOOH

COOHCOOH

COOH

COOH

COOH

HOOC

COOH

L-amino adipic acid



Clutaryl - acylase

Penicillin G

6-APA

7-ACA

COOH



Cephalosporin C


Glutaryl-7-ACA








CH3

CH3

CH3

N

N

S

S

O

O

O

O

O

O

HO

Adipyl-penicillin




NH

New process

NHHO

COOH

CH3

CH3N

S

O

O

Penicillin G


Current process

NH

COOH

CH3

CH3

N

S

N

S

O

O

O

NH

H2N

COOH

COOH

penG acylase

7-ADCA

Cephalosporin G

E coli∆ G

COOH

CH3

N

S

O

N2H

COOH

7-ADCA







Areas of Research

Feed






Paper and Pulp

























Prospects

Extremophiles



Directed Evolution





Literature Cited































































































































































Textiles




Fine Chemicals



Amidases










H2N H2N SH

NH

OO

O

N

N

H2N

S

S

CH3

CH3

CH3

CH3

CH3

NH2

NHHO

CH3

CH3

CH3

NH2

NH

OO

N

S

O

O

O

O

O

OO

O

OO N

N

S

S

COOH

CH3

H2NCOOH

COOHCOOH

COOH

COOH

COOH

HOOC

COOH

L-amino adipic acid



Clutaryl - acylase

Penicillin G

6-APA

7-ACA

COOH



Cephalosporin C


Glutaryl-7-ACA








CH3

CH3

CH3

N

N

S

S

O

O

O

O

O

O

HO

Adipyl-penicillin




NH

New process

NHHO

COOH

CH3

CH3N

S

O

O

Penicillin G


Current process

NH

COOH

CH3

CH3

N

S

N

S

O

O

O

NH

H2N

COOH

COOH

penG acylase

7-ADCA

Cephalosporin G

E coli∆ G

COOH

CH3

N

S

O

N2H

COOH

7-ADCA







Areas of Research

Feed






Paper and Pulp

























Prospects

Extremophiles



Directed Evolution





Literature Cited




































































































































































H2N H2N SH

NH

OO

O

N

N

H2N

S

S

CH3

CH3

CH3

CH3

CH3

NH2

NHHO

CH3

CH3

CH3

NH2

NH

OO

N

S

O

O

O

O

O

OO

O

OO N

N

S

S

COOH

CH3

H2NCOOH

COOHCOOH

COOH

COOH

COOH

HOOC

COOH

L-amino adipic acid



Clutaryl - acylase

Penicillin G

6-APA

7-ACA

COOH



Cephalosporin C


Glutaryl-7-ACA








CH3

CH3

CH3

N

N

S

S

O

O

O

O

O

O

HO

Adipyl-penicillin




NH

New process

NHHO

COOH

CH3

CH3N

S

O

O

Penicillin G


Current process

NH

COOH

CH3

CH3

N

S

N

S

O

O

O

NH

H2N

COOH

COOH

penG acylase

7-ADCA

Cephalosporin G

E coli∆ G

COOH

CH3

N

S

O

N2H

COOH

7-ADCA







Areas of Research

Feed






Paper and Pulp

























Prospects

Extremophiles



Directed Evolution





Literature Cited





































































































































































CH3

CH3

CH3

N

N

S

S

O

O

O

O

O

O

HO

Adipyl-penicillin




NH

New process

NHHO

COOH

CH3

CH3N

S

O

O

Penicillin G


Current process

NH

COOH

CH3

CH3

N

S

N

S

O

O

O

NH

H2N

COOH

COOH

penG acylase

7-ADCA

Cephalosporin G

E coli∆ G

COOH

CH3

N

S

O

N2H

COOH

7-ADCA







Areas of Research

Feed






Paper and Pulp

























Prospects

Extremophiles



Directed Evolution





Literature Cited


































































































































































Areas of Research

Feed






Paper and Pulp

























Prospects

Extremophiles



Directed Evolution





Literature Cited





















































































































































































Prospects

Extremophiles



Directed Evolution





Literature Cited

































































































































































Literature Cited






























































































































































the prokaryotes || bacterial enzymes

Documents