Influence of pH on the production of xylanases

Process Biochemistry 39(2004)

729–733

In?uence of pH on the production of xylanases

by Trichoderma reesei Rut C-30

Hairong Xiong,Niklas von Weymarn,Matti Leisola,Ossi Turunen ?

Laboratory of Bioprocess Engineering,Department of Chemical Technology,Helsinki University of Technology,

P .O.Box 6100,FIN-02015,Espoo,Finland

Received 24December 2002;accepted 17May 2003

Abstract

Trichoderma reesei Rut C-30was cultivated in bioreactors at different pH on a medium with lactose as the main carbon https://www.sodocs.net/doc/205694801.html,pared to an earlier study,in which T .reesei Rut C-30was cultivated using polysaccharides (cellulose or xylan)

as the main carbon sources,we now report a slightly lower pH value for maximal xylanase levels.The highest xylanase activity (IU/ml)on the lactose-based medium was observed at pH 6.0compared to pH 7.0on the polysaccharide-based media.When the pattern of different xylanases was analyzed by isoelectric focusing and activity zymogram,we observed that a low pH (4.0)favoured the production of xylanase I,whilst a high pH (6.0)favoured the production of xylanase III.Xylanase II was clearly produced at both pH values.The results at pH 4and 6correlate with the pH activity pro?les of xylanase I,II and III.Hence,the different T .reesei xylanases were produced according to which enzyme is most active in that particular environment.?2003Elsevier Ltd.All rights reserved.

Keywords:pH;Xylanase;Cellulase;Lactose;Trichoderma reesei ;Filamentous fungi

1.Introduction

Cellulases and xylanases are industrially important en-zymes with applications in e.g.food,feed,textile and pulp and paper industries [1,2].Industrially,these enzymes are produced mainly with ?lamentous fungi,particularly with different Trichoderma species.Trichoderma reesei (anamorph:Hypocrea jecorina )grows on simple carbon sources (e.g.cellulose and xylan)and secretes ef?ciently both cellulases and xylanases.T.reesei Rut C-30is a widely studied mutant strain [3].Compared to the wild-type and some other strains,Rut C-30has improved enzyme pro-duction capabilities [4].It is also less sensitive to glucose repression.

Cellulose and xylan induce ef?ciently the production of the cellulolytic and xylanolytic enzymes of T.reesei .How-ever,the high amount of insoluble material in cellulose-and xylan-based submerged bioreactor cultivations results in de-creased oxygen availability and consequently,more energy is required in the form of mixing [5,6].Instead,soluble raw

?

Corresponding author.Tel.:+358-9-451-2551;fax:+358-9-462-373.E-mail address:ossi.turunen@hut.?(O.Turunen).

materials,such as lactose and starch and wastepaper hy-drolysates,are commonly used as the main carbon source [7,8].

pH is an important parameter in the production of en-zymes by T.reesei .Earlier reports describe that the produc-tion of xylanases by T.reesei Rut C-30on cellulose-and xylan-based growth media was favoured by a high pH (7.0),whereas the production of cellulases was favoured by a low pH (4.0)[9].The xylanase activity of T.reesei is formed by xylanases I,II,III and IV ,and xylan-digesting cellulases [10–13].Xylanases I and II (pI 5.5and 9,respectively)are approximately 20kDa proteins belonging to the family 11of the glycosyl hydrolases [14].The pH optimum of xylanase I is at pH 4.0–4.5,whilst xylanase II is most active at a pH range of 4.0–6.0[10,15].Xylanase III (pI 9.1,32kDa)is a lanase III accounted for over 25%.Xylanase IV (pI 7.0,43kDa)is described in a recent patent application [12].Its pH optimum is 3.5–4.0.Besides the xylanases,xylan-digesting activity has also been observed in non-speci?c cellulases [13].The isoelectric points of these cellulases were below 5.

0032-9592/$–see front matter ?2003Elsevier Ltd.All rights reserved.doi:10.1016/S0032-9592(03)00178-X

730H.Xiong et al./Process Biochemistry39(2004)729–733

In this article,we studied the enzyme production by T. reesei Rut C-30at different pH https://www.sodocs.net/doc/205694801.html,pared to earlier studies,we present new results on xylanase production on a lactose-based growth medium.We also found that the dif-ferent xylanases are produced as a function of pH.The type of xylanase produced corresponded to the pH optimum of the particular enzyme.

2.Materials and methods

2.1.Cultivation conditions

The composition of the culture medium was as follows: 30g/l lactose;5g/l(NH4)2SO4;5g/l KH2PO4;0.6g/l MgSO4·7H2O;0.8g/l CaCl2·2H2O;5.0mg/l FeSO4·7H2O;

1.6mg/l MnSO4·H2O; 1.4mg/l ZnSO4·H2O;

2.0mg/l CoCl2·6H2O;0.2ml/l Tween80(Fluka Chemie,Switzer-land);0.75g/l peptone(Difco Laboratories,USA);and0.3 g/l yeast extract(Lab M,International Diagnostics Group, UK).If otherwise not indicated,the components were pur-chased from Sigma-Aldrich Chemie,Germany.A stock solution of lactose was autoclaved separately(121?C,20 min).

T.reesei Rut C-30was obtained from VTT,Finland.Dry powder spores were suspended in sterile20%(v/v)glycerol and the suspension inoculated on potato dextrose agar(PDA) slants(Difco Laboratories,USA).The PDA slants were in-cubated at30?C for7days and then stored at4?C.The formed spores were collected by washing the slant with3ml sterile culture medium.The spore concentrate was pipetted into a250-ml shake?asks containing100ml culture medium and incubated on a rotary shaker(200rpm)at30?C.After 36h growth the medium was used as the inoculum for a bioreactor cultivation.

Batch cultivations were carried out in2-l glass-vessel bioreactors(Biostat MD system,B.Braun Biotech Interna-tional,Germany).The cultivation parameters were as fol-lows:temperature28?C,agitation400rpm(tip speed1.1 m/s,two Rushton type impellers),aeration1vvm and culti-vation time5days.Foam was controlled by automatic addi-tion of10%(v/v)silicone antifoaming agent(BDH Labora-tories Supplies,UK).The pH was controlled by automatic addition of12.5%(v/v)ammonia water or10%(v/v)sulfu-ric acid.The bioreactor working volume was1-l.

2.2.Enzyme assays

Xylanase activity was analyzed with the3,5-dinitrosalicylic acid(DNS)method by assaying the reducing sugars re-leased during a10min reaction(1%(w/v)xylan,0.05 M citrate-phosphate buffer,pH5.0,50?C)[16].Cellulase activity was analyzed with?lter paper according to the method of Ghose[17].A Whatman No.1?lter paper(～50 mg,Whatman International,UK)was incubated at50?C for1h in1ml of0.05M Na-citrate buffer solution(pH 4.8)supplemented with0.5ml of enzyme solution.The liberated sugars were analyzed by the DNS method.The protein concentrations were determined by the method of Lowry et al.[18].

2.3.Isoelectric focusing

For isoelectric focusing(IEF)analysis,the proteins in the culture supernatants were collected by ammonium sulphate (65%of saturation)precipitation.The precipitates were dis-solved in distilled water and dialysed against distilled water at4?C overnight.The protein concentration was adjusted to 2g/l.The IEF was performed using polyacrylamide gel(Am-pholine PAG plate,Amersham Pharmacia Biotech,Sweden) with a pH range of3.5–9.5.Samples were focused at2500 V h and the end voltage was1500V.Proteins were stained with Coomassie blue(Bio-Rad Laboratories,USA).Puri-?ed samples of xylanase I and II were used as controls.The xylanase I sample was kindly provided by T.Reinikainen (VTT,Finland).The xylanase II sample was puri?ed by crystallisation from a commercial enzyme product(GC140, Genencor International,USA)[19].

2.4.Zymogram analysis after IEF

The zymogram analysis was performed according to Biely et al.[20].Remazol Brilliant Blue-Xylan(RBB-xylan)was used as the soluble substrate for the xylanases.The IEF gel was overlapped onto an agar-RBB-xylan gel.The gels were incubated at room temperature until the enzyme zones became clearly visible on the agar-RBB-xylan gel.The IEF gel was removed and the enzyme-degraded substrate zones on the agar-RBB-xylan gel were destained with a solution comprising two parts95%(v/v)ethanol and one part0.05 M acetate buffer(pH5.4).

2.5.Zymogram analysis after SDS-PAGE

Samples for IEF were prepared as described above.After adjusting the protein concentration to2g/l,IEF was per-formed using agarose gel(Agarose IEF,Amersham Pharma-cia Biotech)with a pH range of3.5–9.5using Ampholine Preblended solution(Amersham Pharmacia Biotech).The IEF parameters were otherwise as described above.After running the agarose IEF,the part close to pI9.1was cut out of the gel and smashed in0.05M citrate–phosphate buffer (pH5).The mixture was frozen and thawed twice to trans-fer the proteins into the buffer solution.A centrifugal?l-ter device(Centriprep YM-3,Millipore,USA)was used to concentrate the sample solution to about2g protein/l. The protein concentrate was run in a SDS-PAGE gel containing0.1%(v/v)xylan at4?C and100V.SDS was washed out using a 2.5%(v/v)Triton X-100solution (Sigma-Aldrich Chemie,Germany),where after the gel was incubated in0.05M acetate buffer(pH5.4)at50?C for20 min.Bio-Rad Coomassic blue was used to stain the pro-

H.Xiong et al./Process Biochemistry 39(2004)729–733731

tein standard

(SDS-PAGE low

range standard LS1610305,Bio-Rad Laboratories,USA).The gel was soaked in 0.2%(w/v)NaOH for 30min.After removal of NaOH,0.1%(v/v)Congo-Red solution was added to stain xylan (20min incubation).Finally,1M NaCl solution was used to remove unbound Congo-Red.

If otherwise not stated,all data are given as mean values and standard deviations of two independent experiments.

3.Results and discussion

3.1.Total xylanase and cellulase activities at different pH values

Lactose was used as the main carbon source since it is known to induce the production of both xylanases and cellu-lases.The highest xylanase and cellulase activities were ob-served at pH 6.0and 4.0–4.5,respectively,(Table 1).How-ever,the cellulase activities did not change much at a pH range of 4.0–5.5.The highest concentration of soluble pro-tein was observed at pH 4.5and 5.0.Both xylanase and cel-lulase activities as well as the soluble protein concentrations decreased signi?cantly at pH 3.5and 7.5.

Fig.1a and Fig.1b show the cultivation data at pH 4.0and 6.0.At both pH values the lactose was depleted in less than 3days.Similar soluble protein concentration levels were produced at both pH values.However,the enzyme activity pro?les prevalent The activities 3.2.Identi?cation of pH-regulated enzymes by IEF ,SDS-PAGE and zymogram analysis

Samples from the two T .reesei cultivations,performed at pH 4and pH 6,were analyzed by IEF (Fig.2a).Xylanase I (XYNI)and xylanase II (XYNII)were identi?ed in the IEF gel using puri?ed xylanase samples as standards.The

Table 1

Xylanase and cellulase activities (IU/ml),and soluble protein concentra-tions (g/l)in batch cultivations of T .reesei Rut C-30at 28?C after 5days as a function of pH pH Xylanase (IU/ml)Cellulase (IU/ml)Soluble protein (g/l)3.534.5a

3.0a

1.9a

4.056.2±2.1 4.2±0.4 2.9±0.44.561.2±1.4 4.3±0.1 3.5±0.2

5.072.2±4.2 4.1±0.0 3.4±0.15.58

6.8±3.3 3.9±0.1 2.9±0.46.094.7±4.8 3.6±0.3 2.8±0.46.56

7.9±1.6 2.9±0.0 2.6±0.37.059.1a 2.2a 1.9a 7.5

28.5a

0.9a

1.4a

The initial lactose concentration was 30g/l.a Only one cultivation.

Fig.1.The time course of xylanase and cellulase production by T.reesei Rut C-30grown on lactose at (a)pH 4.0and (b)pH 6.0.The error bars represent standard deviations of two independent experiments.

enzyme activities of the different bands in the IEF gel were detected by overlapping the IEF gel on an RBB-xylan con-taining agarose gel.Xylanase II activity was detected in both samples,whilst xylanase I activity was clearly higher in the pH 4.0sample (Fig.2b).

The sample from the pH 6.0cultivation had an additional band at pI >9(Fig.2a).This band region showed xylanase activity (Fig.2b),which lead us to suspect that it was xy-lanase III (XYNIII).Based on an earlier work,we knew that the pI of this enzyme is 9.1[11].Using an SDS-PAGE zy-mogram of the excised pI 9.1region (Fig.3),the size of the enzyme was shown to be approximately 32kDa,which also corresponded to the earlier xylanase III data.Xylanase IV ,which should be located at pI 7.0,was not detected.The pI values of the xylan-digesting cellulases are typi-cally less than 5[13].Hence,these cellulases were detected in the samples from both pH 4.0and pH 6.0cultivations (Fig.2a).

The pH-dependent xylanase activity pro?les of T .reesei (puri?ed enzymes)correspond with the pH-dependent pro-duction levels in culture conditions.The pH optima of T .reesei xylanases are as follows:xylanase I,4.0–4.5;xylanase II,4.0–6.0;and xylanase III,6.0–6.5[10,11,14].In our re-sults,the production of xylanase I was favoured by a low pH (4.0).In pH 6.0cultivation,only weak xylanase I ac-tivity was detected.Xylanase II activity,on the other hand,was detected in both cultivations.The xylanase III activity was signi?cantly higher at pH 6.0.

732H.Xiong et al./Process Biochemistry 39(2004)

729–733

Fig.2.IEF,(a)IEF gel stained by Coomassie blue;(b)xylanase activity zymogram after IEF.Puri?ed xylanase I (XYNI,pI 5.5)and xylanase II (XYNII,pI 9)were used as controls.XYNIII =Xylanase III.

3.3.The levels of xylanase and cellulase activities in the cathodic and anodic parts of the IEF gel

The agarose IEF gel was cut into two halves,a cathodic and an anodic half.The xylanase and cellulase activities

in

Fig.3.Xylanase activity zymogram after SDS-PAGE.Puri?ed xylanase II (XYNII,～20kDa)was used as the control.MW Standard =SDS-PAGE low range standard LS1610305,Bio-Rad Laboratories.

each half are shown in Fig.4a and Fig.4b.A signi?cant part of the total xylanase activity in the pH 6sample was detected in the cathodic part (high p I )of the gel,i.e.,in the region containing xylanase II and III (Fig.4a).The anodic part,containing cellulases and xylanase I,comprised only 16%of the total xylanase activity at pH 6.At pH 4,the cathodic and the anodic parts contained approximately equal amounts

of

Fig.4.The activity levels of (a)xylanases and (b)cellulases in the anodic and cathodic halves of the IEF gel.

H.Xiong et al./Process Biochemistry39(2004)729–733733

xylanase activity.A part of the xylanase activity detected in the anodic part of the IEF gel,at both pH values,was due to xylan-degrading cellulases(Fig.2b).Fig.4b shows that the bulk cellulase activity is present in the anodic part of the IEF gel.This correlates to the production of xylan-digesting cellulases at low p I.It appears that xylanase II(family11) and xylanase III(family10)represent together the main xylan-digesting activity,when T.reesei Rut C-30cells are grown at a high pH(pH～6),whilst xylanase I(family11) does the same,together with xylanase II,at a low pH(pH ～4).

4.Conclusions

The?lamentous fungus T.reesei Rut C-30reacts to the pH of the growth environment by modifying its enzyme pro-duction patterns.The pH-dependent activity pro?les of the puri?ed T.reesei xylanases and the pH-dependent produc-tion levels appear to be linked together.Hence,at a low pH, T.reesei produces xylanase I,which is most active at low pH values.At a high pH the fungus produces xylanase III, which is most active at high pH values.The study of the enzyme production patterns at different growth conditions is potentially useful in the planning of industrial production systems for T.reesei.

Acknowledgements

We thank Johanna Aura for technical assistance.Finan-cial support from the Research Foundation of Helsinki Uni-versity of Technology is gratefully acknowledged. References

[1]Nakari-Set?l?T,Penttil?M.Production of Trichoderma reesei

cellulases on glucose-containing media.Appl Environ Microbiol 1995;61:3650–5.

[2]Haltrich D,Nidetzky B,Kulbe KD,Steiner W,Zupancic S.Produc-

tion of fungal xylanases.Bioresource Technol1996;58:137–61. [3]Montenecourt BS,Eveleigh DE.Selective screening methods for the

isolation of high yielding cellulase mutants of Trichoderma reesei.

Adv Chem Ser1979;181:289–301.

[4]Ilmén C,Thrane C,Penttil?M.The glucose repressor gene cre1of

Trichoderma:isolation and expression of a full length and a truncated mutant form.Mol Gen Genet1996;251:451–60.

[5]Domingues FC,Queiroz JA,Cabral JMS,Fonseca LP.The in?u-

ence of culture conditions on mycelial structure and cellulase pro-duction by Trichoderma reesei Rut C-30.Enzyme Microb Technol 2000;26:394–401.

[6]Ju LK,Afolabi OA.Wastepaper hydrolysate as soluble inducing sub-

strate for cellulase production in continuous culture of Trichoderma reesei.Biotechnol Prog1999;15:91–7.

[7]Morikawa Y,Chen S,Wayman M.Novel inducers derived from

starch for cellulase production by Trichoderma reesei.Proc Biochem 1992;27:327–34.

[8]Morikawa Y,Ohashi T,Mantani O,Okada H.Cellulase induction

by lactose in Trichoderma reesei PC-3-7.Appl Microbiol Biotechnol 1995;44:106–11.

[9]Bailey MJ,Buchert J,Viikari L.Effect of pH on production of

xylanase by Trichoderma reesei on xylan-and cellulose-based media.

Appl Microbiol Biotechnol1993;40:224–9.

[10]Tenkanen M,Puls J,Poutanen K.Two major xylanases of Tricho-

derma reesei.Enzyme Microb Technol1992;14:566–74.

[11]Xu J,Takakuwa N,Nogawa M,Okada H,Morikawa Y.A third xy-

lanase from Trichoderma reesei PC-3-7.Appl Microbiol Biotechnol 1998;49:718–24.

[12]Clarkson K,Siika-Aho M,Tenkanen M,Bower BS,Penttil?ME,

Saloheimo M.Trichoderma reesei xylanase.Patent application WO01498592001.

[13]Biely P,Markovic O.Resolution of glycanases of Trichoderma reesei

with respect to cellulose and xylan degradation.Biotechnol Appl Biochem1988;10:99–106.

[14]T?rr?nen A,Rouvinen J.Structural and functional properties of low

molecular weight endo-1,4-b-xylanases.J Biotechnol1997;57:137–

49.

[15]T?rr?nen A,Mach RL,Messner R,Gonzalez R,Kalkkinen N,Harkki

A,et al.The two major xylanases from Trichoderma reesei:character-ization of both enzymes and genes.Bio/Technology1992;10:1461–5.

[16]Turunen O,Etuaho K,Fenel F,Vehmaanper?J,Wu X,Rou-

vinin J,et https://www.sodocs.net/doc/205694801.html,bination of weakly stabilizing mutations with

a disul?de-bridge in the?-helix region of Trichoderma reesei

endo-1,4-?-xylanase II increases the thermal stability through syn-ergism.J Biotechnol2001;88:37–46.

[17]Ghose TK.Measurement of cellulase activities.Pure Appl Chem

1987;59:257–68.

[18]Lowry OH,Rosebrough NJ,Farr AL,Randall RJ.Protein measure-

ment with the Folin-phenol reagent.J Biol Chem1951;193:265–75.

[19]Finell J,Jokela J,Leisola M,Riekkola M-L.Total hydrolysis of

xylotetrose and xylobiose by soluble and cross-linked crystalline xylanase II from Trichoderma reesei.Biocatalysis Biotransform 2002;20:281–90.

[20]Biely P,Mislovicova D,Toman R.Remazol Brilliant Blue-Xylan:

a soluble chromogenic substrate for xylanases.Methods Enzymol

1988;160:536–41.

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●I wonder if it’s because I have been at school for so long that I’ve grown so crazy about going home. ●It is because she wasn’t well that she fell far behind her classmates this semester. ●I can well remember that there was a time when I took it for granted that friends should do everything for me. ●In order to make a difference to society, they spent almost all of their spare time in raising money for the charity. ●It’s no pleasure eating at school any longer because the food is not so tasty as that at home. ●He happened to be hit by a new idea when he was walking along the riverbank. ●I wonder if I can cope with stressful situations in life independently. ●It is because I take things for granted that I make so many mistakes. ●The treasure is so rare that a growing number of people are looking for it. ●He picks on the weak mn in order that we may pay attention to him. ●It’s no pleasure being disturbed whena I settle down to my work. ●I can well remember that when I was a child, I always made mistakes on purpose for fun. ●It’s no pleasure accompany her hanging out on the street on such a rainy day. ●I can well remember that there was a time when I threw my whole self into study in order to live up to my parents’ expectation and enter my dream university. ●I can well remember that she stuck with me all the time and helped me regain my confidence during my tough time five years ago. ●It is because he makes it a priority to study that he always gets good grades. ●I wonder if we should abandon this idea because there is no point in doing so. ●I wonder if it was because I ate ice-cream that I had an upset student this morning. ●It is because she refused to die that she became incredibly successful. ●She is so considerate that many of us turn to her for comfort. ●I can well remember that once I underestimated the power of words and hurt my friend. ●He works extremely hard in order to live up to his expectations. ●I happened to see a butterfly settle on the beautiful flower. ●It’s no pleasure making fun of others. ●It was the first time in the new semester that I had burned the midnight oil to study. ●It’s no pleasure taking everything into account when you long to have the relaxing life. ●I wonder if it was because he abandoned himself to despair that he was killed in a car accident when he was driving. ●Jack is always picking on younger children in order to show off his power. ●It is because he always burns the midnight oil that he oversleeps sometimes. ●I happened to find some pictures to do with my grandfather when I was going through the drawer. ●It was because I didn’t dare look at the failure face to face that I failed again. ●I tell my friend that failure is not scary in order that she can rebound from failure. ●I throw my whole self to study in order to pass the final exam. ●It was the first time that I had made a speech in public and enjoyed the thunder of applause. ●Alice happened to be on the street when a UFO landed right in front of her. ●It was the first time that I had kept myself open and talked sincerely with my parents. ●It was a beautiful sunny day. The weather was so comfortable that I settled myself into the

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