carotenoidluvialis wasr-like regionise gene wasornbardrnentlCZ reporter

re National(Grant no.

1076I/. Biopliys 297,

Bioteclinol 18,

7) J Bioi Chern

Pliysiol 125,

)98)Proc Natl

19 C K (2005)

19 C K (2006)

~03-2121994) Nucleic

eau Y, Peer Y«cids Res 30,

'l Pliysiol 32,

& Tseng C K

, (1994) Plant

.inozaki K &ad Sci (USA)

, Sakuma Y,~ Yamaguchi-

'7, /73-1821 R E (1996)

oi 30, 77-96fJlall! Cell 12,

Purification and characterization of trehalose-6-phosphate synthase fromSaccharomycopsis fibuligera A 11

Likun Liang, Zhenming Chi*, Lingmei Gao and Liyan MaUNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road. No.5. Qingdao, China

Received J 3 March 2006; revised 2 September 2006

Mutant A II, a mutant of Saccharoniycopsis [ibuligera Sdu with low acid and neutral trehalase was found toaccumulate over 18% (w/w) trehalose from starch in its cells. In this study, uchalosc-o-phosphatc synthase (Tps I) waspurified to homogeneity from this mutant, with a 30-fold increase in the specific enzyme activity, as compared to theconcentrated cell-free extract, from initial cells. The molecular mass of the purified enzyme as determined by SOS-PAGEwas 66 kDa. The optimum pH and temperature of the purified enzyme were 6.6 and 37°C, respectively. The enzyme wasactivated by Ca2+, K+ and Mg2+, with K+ showing the highest activation at 35 mM. On l'1le other hand, Mn2+, Cu2+, Fe3+,Hg2+ and Co2: inhibited the enzyme. The enzyme was also strongly inhibited by protease inhibitors such as iodoacetic acid.EOTA and PMSF.

Keywords: Saccharouiycopsis [ibuligera, Trehalose-6-phopshate synthase. Enzyme purification, Characterization of Tps I

Indian Journal of Biochemistry & BiophysicsVoL 43. October 2006, pp 289-294

Trehalose, a disaccharide formed by the two glucoseunits linked in an a,a-l,l-glycosidic linkage is widelypresent in yeasts, fungi and plants'. It has beenreceiving much attention due to its great importancein nature and industry. In nature, it plays a number ofroles from being an energy source to a stressprotectant, enhancing the resistance of cellularcomponents against different abiotic stresses, such ashigh temperature, freezing, high osmotic pressure,dehydration, and high ethanol concentration. Inindustry, it can be used as a cryoprotectant for cells inmedicine and microbiology, as an effectivecomponent in cosmetics, as a stabilizer for clinicalreagents and bioprod ucts, and as a preservati ve forfresh foodstuffs!".

Trehalose production has been reported throughf . 6-9 . f Iermentation , enzymatic process rom stare 1,

sucrose and maltose'l'":', and extraction fromtransformed plantsI4.IS. So far, two pathways fortrehalose biosynthesis have been reported inf ·1 101617 TI f I . I .. I' dungr: , '. re irst pat rway IS t rat It IS synt resizein a two-step process in Candida albicans,Aspergillus niger, Saccharomyces cerevisiae,

*Corrcsponding authorTeL and Fax: +86-532-82032266E-mail: zhenming@sdu.edu.cnAbbrevialiolls: OTT. dithiothrcitol: PAGE, polyacrylamide gelelectrophoresis; PMSF, phcnylmcthyl sulfonyl fluoride; SOS,sodium dodccyl sulphate; Tps I, trehalose-6-phosphate synthase;Tps2, trehalosc-o-phosphaiase.

Schizosacch aroniyces ponibe, Zygosacchrontycesrouxii, and others. First, trehalose-6-phosphate isformed from UDP-glucose and glucose-6-phosphateby trehalose-6-phosphate synthase (Tpsl( Second,this compound is dephosphorylated into trehalose bytrehalose-6-phosphatase (Tps2)IO. Both tps 1 and tps2genes, encoding Tpsl and Tps2, respectively, havebeen cloned in A. niger, A. nidulans C. albicans,Hansenula polymorpha, Kluyveromyces lactis, S.cerevisiae, S. ponibe, Yarrowia lipolytica. and Z.rouxii'i'", The second pathway is that trehalosephosphorylase or trehalose synthase (TSase) inAgaricus bisporus and Grifola frotulosa catalyzes thereversible reaction of degradation and synthesis oftrehalose from o-gl ucose and a-o-gl ucose-l-phosphateIO.17.

The starch is considered as best substrate forproduction of trehalose, due to its low cost and easy

libili 1113 I . d 8 f d Iava I I ity , . n our previous stu y , we oun t ratSaccliaromycopsis fibuligera Sdu can use solublestarch as sole carbon and energy source and producesignificant amounts of trehalose (a yield of 18.0%,w/w, cell dry wt basis within 48 h of fermentation). Inanother study:', a mutant with low acid and neutraltrehalase activities of S. fibuligera Sdu was found toaccumulate more trehalose from soluble starch thanits parent strain, when grown in YPS medium.

In this study, attempts have been made to purifyand characterize trehalose-producing enzyme frommutant All of S. fubuligera Sdu, which has been

',I.

290 INDIAN J. BIOCI-IEM. BIOPHYS, VOL. 43, OCTOBER 2006

reported to produce a large amount of trehalose fromstarch3,8.

Materials and MethodsYeast strain

All, a mutant of S. fibuligera Sdu with low acidand neutral trehalase activities was used in the presentstudy. Earlier,· it was reported that this mutantproduced higher amounts of trehalose from solublestarch in YPS medium than its parent strain '. Themutant was maintained at 4°C on YPS mediumcontaining 1.0% yeast extract, 2.0% polypeptone,2.0% soluble starch, and 2.0% agar.

Preparation of cell-free extractThe cells of mutant All were cultivated in liquid

YPS medium at 30°C by shaking for 9-10 handcollected by centrifugation at 7227 g and 4°C for 5 minand washed 3··times with ice-cold distilled water. Thepellet was, suspended in 2 ml of ice-cold celldisintegration buffer containing 1.619 gNa2HPO.j.7H20, 0.55 g NaH2PO.j.H20, 0.075 g KCI,0.0246 g MgSOcl.7H20, 0.27 ml ~-mercaptothanol, 100ml distilled water, pH 7.0 to make a thick paste. Thepaste was homogenized in an electric glasshomogenizer (DY&9-1 Type, Xinzhi, Zhejiang, China)for 1 h on ice. The cell debris was removed bycentrifugation at 16260 g and 4°C fo'r 30 min and about50 ml of the supernatant was concentrated to 3-4 ml byultrafiltration (10 kDa cut-oft) with a Labscale" TFFsystem (Millipore, USA). The concentrated cell-freeextract was used as the enzyme preparation.

Enzyme and protein assayTpsl activity in the enzyme preparation was

measured using the assay described previously".Briefly, 0.4 ml of the reaction mixture containing 0.05mM HEPES-KOH (pH 7.0), 5 mM UDP-glucose, 10mM glucose-o-phosphate, 25 mM MgCh, 0.1 ml theenzyme preparation and a suitable amount of distilledwater, was incubated at 37°C for 20 min. The reactionwas terminated in 100°C water bath for 5 min. Aftercooling, the mixture was centrifuged at 2823 g for 5

'min. The concentration of UDP formed in thesupernatant was determined according to the decrease

, in OD3.jo I1Ill in 0.5 nil of the mixture containing 0.14 MHEPES-KOH (pH 7.6); 2 mM phosphoenolpyruvate,0.3 mM NADH, 5U lactic dehydrogenase and 5 Upyruvate kinase. .

Protein concentration was measured by the methodof Bradford using bovine serum albumin served as

·tf

standard". One unit of enzyme activity was definedas the amount of enzyme that produced 1.0 ~LM ofNAD+ per min at 37°C and pH 7.0.

To measure trehalose synthesis from cx-D-glucose-Lphosphate and gl ucose, 0.1 ml of the reactionmixture containing 100 mM cx-D-glucose-l-phosphate, 100 mM glucose, 100 mM HEPES buffer(pH 7.0) and 40 III of the enzyme preparation wasincubated at 35°C for 3 h and the reaction wasstopped by boiling for 5 min'". The inorganicphosphate liberated was determined by ammonium

70molybdate method- .

Purification of TpslPurification of Tpsl

AKTATM prime withBiosciences, Sweden).

was carried out usi ng'I'MHitrap , (Amersharn,

ovalbukDa).

Effects (The

determpH 4.Cbuffers0.01 MHCI bu30 milappropiconcernto 9.0rneasunstandan

Thewas detand 52'tempersdetermitemperameasuriabove. 'referenc

Effect ofTo e

Tps l acreactionmetal ioIons US(

KCI,MJthe abse

Effect of IThe e

iodoacetmM, resthe reacMethodsthe respethe staru

Purificatio

Cell extrd;Ultra-filtr,SepharoseDEAE-SerFast-Flow

Sepharose CL-4B gel filtraiion chromatographyThe 3 ml of the enzyme preparation was loaded on

a Sepharose CL-4B gel filtration column (16 x 100ern) equilibrated and eluted with the buffer containing50 mM Tris-HCI (pH 7.5), 1 mM DTT, 1 mM PMSFand 2 mM EDT A with a flow rate of 0.5 rnl/rnin and 3ml of fraction was collected in each tube. Tps Iactivity and protein content in each tube weredetermined by using the methods as described above.The fractions showing the Tps 1 activity were pooled.

DEAE-Sep/wrose Fast Flow anion exchange chromatographyThe pooled fractions showing the Tpsl activities

were applied to DEAE-Sepharose fast flow anion-exchange column (16 x 25 ern) equilibrated with 20mM Tris-HCI buffer (pH 7.8). After washing thecolumn (by eluting with 20 mM Tris-HCI buffer, pH7.8), the proteins were eluted with a linear gradient of0-1.0 M NaCI in the same buffer in a total volume of250 ml with a flow rate of 1.0 ml/rnin and 2 ml of theelute was collected in each tube. Tpsl activity andprotein content in each tube were determined by usingthe methods as described above. The fractionsshowing the Tpsl activity were concentrated to 100 ~liby ultrafiltration (3 kDa cut-oft).

Gel electrophoresisThe purity. and molecular mass of Tps l in the

concentrated fractions showing the activity wereanalyzed in non-continuous denaturing SDS-PAGE,Iwith a 2-D. electrophoresis system (Arnersharn,Biosciences, Sweden) and by silver staining". Themolecularrnassstandards used were ~-galactosidase(116.2 kDa), bovine serum albumin (66.2 kDa).

edof

se-on-1-'fer

vasvasnicum

.mgam,

j on100

nll1g

.VISF

.nd 3IpslwereJove.,led.

phy

viticsnion-th 20g the.r, pHent ofme ofIof the.y and. usingictions100 ~ll

in the, were'AGE:!I

.rsham.2(,. The

.osidase; kDa),

LIANGel al : TREHALOSE-6-PHOSPHATE SYNTHASE FROM S. FIBULIGERA A II 291

ovalbumin (45 kDa) and lactate dehydrogenase (35kDa).

Effects of pH and temperature onTpsl activityThe effect of pH on the enzyme activity was

determined by incubating the purified enzyme betweenpH 4.0-9.0 using the standard assay conditions. Thebuffers used were 0.01 M acetate buffer (pH 4.0-5.0),om M phosphate. buffer (PH 6.0-7.0) and 0.01 M Tris-HCI buffer (pH 7.5-9.0). Tile pH stability was tested by30 min pre-incubation of the purified enzyme inappropriate buffers that had the same ionicconcentrations at different pH values ranging from 4.0to 9.0 at 4°C. The residual activities of Tpsl weremeasured immediately after this treatment with thestandard method as mentioned above.

The optimal temperature for activity of the enzymewas determined at O°C, 19°C, 28°C, 37°C, 42°C, 47°Cand 52°C in the same buffer as described above. Thetemperature stability of the purified enzyme wasdetermined by pre-incubating the enzyme at different..temperatures ranging from O°C to 52°C for 30 min, andmeasuring residual activity immediately, as describedabove. The pre-incubated sample at 4°C was used asreference to calculate the residual activity.

Effect of different metal ions on Tps l activityTo examine the effect of different metal ions on

Tpsl activity, the enzyme assay was performed in thereaction mixture as described above, with variousmetal ions at a final concentration of 25 mM. The metalions used were CuSO.j.5H:!O, MgSO.j, FeCI), CaCh,KCI, MrrCl-, HgCl2 and CoCl2. The activity assayed inthe absence of metal ions served as control.

Effect of protein inhibitors on Tps l activityThe effect of protease inhibitors (EDTA, PMSF and

iodoacetic acid at a final concentration of 1.0 and 5.0mM, respectively) on Tpsl activity was measured inthe reaction mixture as described in 'Materials andMethods'. The purified enzyme was pre-incubated withthe respective inhibitors for 10 min at 4°C, followed bythe standard enzyme assay as described above. The

activity assayed in the absence of the, protein inhibitorsserved as control.

ResultsDetermination of pathway for trehalose synthesis in mutantAll

The biosynthesis of trehalose in yeasts occurspredominantly via the trehalose synthase complex I.

and this pathway has been studied extensively. Analternative pathway for trehalose synthesis anddegradation is provided by trehalose phosphorylase inGrifola [rondosa and Agaricus bisporuslO.17 In orderto know the biosynthesis of trehalose in mutant A I I.in the present study, Tpsl and trehalosephosphorylase activities were determined in the cellextract. Although Tpsl activity was detected in thecell extract, no trehalose phosphorylase activity wasfound (data not shown).

Purification of Tps lTpsl was purified from the concentrated cell

extract, prepared from the cells cultivated for 9-10 hat 30°C. Elution profile of Sepharose CL-4B gelfiltration chromatography (Fig. lA) shows that peak 2with Tpsl activity from 150 to 240 min displayed asingle peak. Therefore, the fractions were collectedand the pooled fractions were applied to DEAE-Sepharose fast-flow anion-exchange column. Theelution profile (Fig. IB) shows that peak 4 with Tpslactivity displayed a single peak, when NaCiconcentration reached 0.69 M. Therefore, the fractionswere collected and concentrated by the ul trafi Itration.The results in Table 1 show that the enzyme waspurified to homogeneity. with a 30-fold increase inthe specific activity and a yield of about 61 %.compared to the concentrated cell-free extract.

Gel electrophoresisSDS-PAGE was used to determine protein purity

and molecular mass of the finally concentrated elute asdescribed by Laemmh". The results in Fig. 2 showonly a single protein band from the finally concentratedelute with a relative molecular mass of 66 kDa. Thus, itmay be concluded that Tpsl from mutant All was

"7composed of monomer- .

Puritication steps

Table I-Summary of purification procedure of trchalosc-o-phosphatc synthase

Total protein Total activity Specific activity Purification(mg) (U/min) (U/min.mg) fold21.5 51.9 2.4 I8.5 40.8 4.8 2

4.48 34.6 7.7 3.2

Cell extractUltra-n ItrationScpharoscCL-4BDEAE-SepharoseFast-Flow 0.44 31.7 72 30 61

Yield(%)

1007966

292 INDIAN J. BIOCHEM. BIOPI-IYS .. VOL. 43. OCTOBER 2006

---ManualRun7:1 UII --MllnIA1Run7:1 COMmAu ---lVIonual Run7:( eo", --- ManualRun1J]l1ICtiollS 1I.B~

A mAu B100 120

400

100

80

300 80

60 604

20040

4l

20

100

20

.20

00

0 50 300 min

-ManuelRun8;l UV -'-'-ManUllRun8:1 Cond---Manual Run8:1)rections --~MI1l\ll&.lR\I\ S:l:Con~

4

.', 100'I

/1J

/l'

/.'I

Ir

/ r'I i

I ,/ iI ,.

I ;3 I iI .I .'

I I

II

II

150

;111111111

111111111HI~lvl~ Waste

t I_.~. I I

~·-·-·-·-I-·-·200 :DO min2j)50 100

Fig. I A-Elution profile of Scpharose CL-4B gcl filtration

chromatography

Fig I B-Elution profile of DEAE-Sepharosc fast-flow anion-exchange chromatography

.-- n6kD

120

?j 100 .----~

~ 80

~ 60~ 40+:IIII

~ 200~--~==~~-~~~~~-4~4-~

o

+--'- 66.2kD

101-45kD

20 30 40Tem peratu re

50

1-35kDFig. 3-EITect 01' temperature on activity 01' trehalosc-6-phosphatcsynthase [(~), thermal stability of the enzyme; and (.), optimaltemperature. Values are given as mean ± SO, n = 3)

activity profile of the enzyme was stable just aroundthe opti mal pH, but the enzyme acti vi ty decreasedsignificantly at pH value lower or higher than 6.6(Fig. 3). These results suggested that the enzyme wasvery sensitive to change of pH. Tpsl activity,measured as a function of temperature from 0 to S2°eshowed the highest activity at 37°C (Fig. 4). Theenzyme was stable up to 19°C, but inactivated rapidlyat temperature above this and was completely

2

Fig. 2-SDS-PAGE analysis 0[" irehalose-o-phosphatc synthasefro m mutant A II [Lane I. purified sample; and lane 2, standardprotein markers]

Effect of pH and temperature onTpsl activityTpsl activity was measured at various pHs in

buffers with the same ionic concentrations. Themaximum activity was observed at pH 6.6. The

Effect of pr

The efshown ininhibitedpurified ewas identPMSF atenzyme acresidues \However,(data not s

120:;;100~80.~~ 60(j)~ 40~ 20a:::

o

60Fig. 4-synthaseValues ar

inactivaunstable

20

Effect of,Fig.

acti vatinhighestF 3+e , Hfwith Hg:study, thenzyme,Mg2+ weabove, f(optimal,found to(results "1indicatedacids in tl

60

DiscussiorThe res

formed frcby trehalosthat the tnAll was t

LIANG 1'/ al.: TREHALOSE-6-PHOSPHATE SYNTHASE FROM S. FIBULIGERA A II

120

~100o

~80.~g 60QJ~ 40\1lQi 20a::

o o 9

\I.1l

100

80 2 3 4 5pH

6 7 8

roFig. 4-EITcct of pH on activity of trchalosc-o-phosphatesynthase [(A), pH stability of thc enzyme; and (+) optimal pH.Values are gi vcn as mean ± SO. n = 3]

41 inactivated at 28°C, indicating that Tpsl became veryunstable at temperature higher than 19°C.

20

Ellcct of different cations on Tps l activity. C"+ + "+ I dFig. 5 shows that a:", K and Mg" ra an

activating effect 0)1 the enzyme, with K+ showing thehighest (188%) acti vation. However, Mn2+, Cu2+,Fe3+, Hg2+ and C02+ inhibited the enzyme activity,with Hg2+ showing the highest inhibition (3.76%). Tostudy, the optimal concentrations for activation of theenzyme, the different concentrations of Ca2+, K+ andMg2+were added to the reaction mixture as describedabove, followed by the standard enzyme assays. The

. I . f C?+ K+ d M ?+optima concentrations 0 a-, an g- werefound to be 10 mM, 35 mM and 35 mM, respectively(results not shown). The inhibition by Hg2+ ionsindicated the possible role of thiol-containing aminoacids in the enzyme functiorr". "

Lon-

-'60

Effect of protease inhibitors on activity of TpslThe effect of protease inhibitors of the enzyme is

shown in Table 2. The chelating agent EDT A stronglyinhibited the enzyme activity, indicating that therurified enzyme was a metallo-enzyrne'". This result!Vasidentical to that of Fig. 5. Iodoacetic acid andPMSF at I and 5 mM also strongly inhibited theenzyme activity, suggesting that Cys residues and Serresidues were essential for the enzyme activity".However, the enzyme could be protected by DTT(datanot shown).

atenal

nded1.6'as Discussion

The results showed that trehalose-o-phosphate isformed from UDP-glucose and glucose-o-phosphateDY trehalose-6-phosphate synthase (Tps 1), suggesti ngthat the trehalose synthesis pathway in the mutant~Ilwas the si mi lar to that inS. cerevisiae. Stress

ty,)CheIlysly

293

250'0'~ 200z-:~ 150g~ 100.~

~ 50

o eEoo

r-:

u'"u

N

U<=:~

u 0 ~ 0:><: r/J U r/J= Q) OJ)U 1.1.. ~

Different ions (25 mM)

N

UOJ)::r:

N

UoU10

Fig. 5-EITcct or different ions on activity of trchalosc-o-phosphate synthase [Values are given as mean ± SO, n = 31

Table 2-EITcct of protein inhibitors on the enzyme activity

[Values represent the average of three independent experiments]

Inhibitors Concentrations Relative activity Inhibition(mM) (%)

Control 100EOTA I 55 ++

5 0 +++PMSF I 33 ++

5 0 +++Iodoacetic acid I 0 +++

5 0 +++++. Inhibition; +++.complete inhibition: control was expressed as100% of relative activity obtained in thc absence of any addedprotease inhibitor.

treatments, such as heat and ethanol shock causedincrease in trehalose-o-phosphare, Tpsl and mRNAencoding Tpsl and trehalose content in S. cerevisiaecells, due to the presence of a heat shock element inthe promoter of tps I gene.'. However, our previousresults showed that heat shock did not result inincrease in trehalose content in cells of S. fibuligera',Also, under the normal growth conditions, the mutantAll accumulates larger amounts of trehalose fromstarch than S. cerevisiae. This implies that regulationof tpsI gene in S. fibuligera appears to be differentfrom that in S. cerevisiae. But, the relationshipbetween the synthesis of trehalose and stress responsein this mutant can be explained, only after the geneencoding Tpsl is cloned.

Although Tpsl obtained from different sourcescatalyze the same reaction, it has shown variation inthe structure from different organisms. Tps I frommutant All is composed of monomer (Fig. 3). Incontrast, in S. cerevisiae, Tpsl (56 kDa) is a subunitof the trehalose-6-phosphate synthase-complex,having a high molecular mass (600-800 kDa) and

294 INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, OCTOBER 2006 IndiarVol. 4

contains three other proteins Tps2, Tsll and Tps31. InE. coli, Tpsl is not part of a complex and isindependent of the phosphatase activity. InSelagiuella lepidophylla, trehalose-6-phosphatesynthase complex is a multi-protein complex (440kDa) with three subunits of 50, 67, and 115 kDa(Tpsl)14. In Z. rouxii, full-length Zr tpsl cDNA iscomposed of 1476 nucleotides, encoding a protein of492 amino acids with a molecular mass of 56 kDaiG

.

Sequence analysis of H. polymorpha tps 1 revealed anopen-reading-frame of 1, 428 bp coding for a putativeprotein of 476 amino acids (54.4 kDa)22.

Tpsl from this mutant was stable only at lowtemperature and in the narrow range of pH (Fig. 3),suggesting that the intracellular enzyme was verysensitive to the change of temperature and pH. Theresults in Fig. 5 demonstrated that Ca2+, K+ and Mg2+had an activating effect on the enzyme. These ionswere also found to be the activators of Tpsl inSelaginella lepidophylla'", and the maximal effectswere observed at 1 mM, 50 mM and 5 mM,respectively. Tpsl from some other organisms isactivated by K+ at concentrations up to 0.4 M14.However, Ca2+ does not affect E. coli Tpsl.

In conclusion, the results of the present studydemonstrated that Tps 1 of the mutant A 11 wassignificantly different from that in other fungi.However, to understand its gene structure andregulation, the attempts are being made to clone tps 1gene from the mutant.

AcknowledgementThe authors thank Natural Science Foundation of

Shandong Province for its financial support (Grant no.Z2003DOl).

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(2006) Vaccine 24, 6046-6052

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Amylthe pioriginmicroproduand tlin indiand dtto bestarch:

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*Author.Tel: (084EmaiI: v