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Influence of pH on the production of xylanases by Trichoderma reesei Rut C-30
Process Biochemistry 39 (2004) 729–733
Influence 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 24 December 2002; accepted 17 May 2003
Abstract Trichoderma reesei Rut C-30 was cultivated in bioreactors at different pH on a medium with lactose as the main carbon source. Compared to an earlier study, in which T. reesei Rut C-30 was 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 6.0 compared to pH 7.0 on 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 4 and 6 correlate with the pH activity profiles 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. © 2003 Elsevier Ltd. All rights reserved. Keywords: pH; Xylanase; Cellulase; Lactose; Trichoderma reesei; Filamentous fungi
1. Introduction Cellulases and xylanases are industrially important enzymes with applications in e.g. food, feed, textile and pulp and paper industries [1,2]. Industrially, these enzymes are produced mainly with filamentous fungi, particularly with different Trichoderma species. Trichoderma reesei (anamorph: Hypocrea jecorina) grows on simple carbon sources (e.g. cellulose and xylan) and secretes efficiently both cellulases and xylanases. T. reesei Rut C-30 is a widely studied mutant strain [3]. Compared to the wild-type and some other strains, Rut C-30 has improved enzyme production capabilities [4]. It is also less sensitive to glucose repression. Cellulose and xylan induce efficiently the production of the cellulolytic and xylanolytic enzymes of T. reesei. However, the high amount of insoluble material in cellulose- and xylan-based submerged bioreactor cultivations results in decreased 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: [email protected] (O. Turunen).
0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00178-X
materials, such as lactose and starch and wastepaper hydrolysates, are commonly used as the main carbon source [7,8]. pH is an important parameter in the production of enzymes by T. reesei. Earlier reports describe that the production of xylanases by T. reesei Rut C-30 on 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 5.5 and 9, respectively) are approximately 20 kDa proteins belonging to the family 11 of 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, 32 kDa) is a recently found, family 10 glycosyl hydrolase characterized from T. reesei PC-3-7 [11]. The pH optimum for xylanase III was observed at pH 6.0–6.5. Of the total xylanase activity in T. reesei PC-3-7 on a cellulose-based growth medium, xylanase III accounted for over 25%. Xylanase IV (pI 7.0, 43 kDa) 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-specific cellulases [13]. The isoelectric points of these cellulases were below 5.
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H. Xiong et al. / Process Biochemistry 39 (2004) 729–733
In this article, we studied the enzyme production by T. reesei Rut C-30 at different pH values. Compared to earlier studies, we present new results on xylanase production on a lactose-based growth medium. We also found that the different 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: 30 g/l lactose; 5 g/l (NH4 )2 SO4 ; 5 g/l KH2 PO4 ; 0.6 g/l MgSO4 ·7H2 O; 0.8 g/l CaCl2 ·2H2 O; 5.0 mg/l FeSO4 ·7H2 O; 1.6 mg/l MnSO4 ·H2 O; 1.4 mg/l ZnSO4 ·H2 O; 2.0 mg/l CoCl2 ·6H2 O; 0.2 ml/l Tween 80 (Fluka Chemie, Switzerland); 0.75 g/l peptone (Difco Laboratories, USA); and 0.3 g/l yeast extract (Lab M, International Diagnostics Group, UK). If otherwise not indicated, the components were purchased from Sigma-Aldrich Chemie, Germany. A stock solution of lactose was autoclaved separately (121 ◦ C, 20 min). T. reesei Rut C-30 was obtained from VTT, Finland. Dry powder spores were suspended in sterile 20% (v/v) glycerol and the suspension inoculated on potato dextrose agar (PDA) slants (Difco Laboratories, USA). The PDA slants were incubated at 30 ◦ C for 7 days and then stored at 4 ◦ C. The formed spores were collected by washing the slant with 3 ml sterile culture medium. The spore concentrate was pipetted into a 250-ml shake flasks containing 100 ml culture medium and incubated on a rotary shaker (200 rpm) at 30 ◦ C. After 36 h growth the medium was used as the inoculum for a bioreactor cultivation. Batch cultivations were carried out in 2-l glass-vessel bioreactors (Biostat MD system, B. Braun Biotech International, Germany). The cultivation parameters were as follows: temperature 28 ◦ C, agitation 400 rpm (tip speed 1.1 m/s, two Rushton type impellers), aeration 1 vvm and cultivation time 5 days. Foam was controlled by automatic addition of 10% (v/v) silicone antifoaming agent (BDH Laboratories Supplies, UK). The pH was controlled by automatic addition of 12.5% (v/v) ammonia water or 10% (v/v) sulfuric acid. The bioreactor working volume was 1-l. 2.2. Enzyme assays Xylanase activity was analyzed with the 3,5-dinitrosalicylic acid (DNS) method by assaying the reducing sugars released during a 10 min reaction (1% (w/v) xylan, 0.05 M citrate-phosphate buffer, pH 5.0, 50 ◦ C) [16]. Cellulase activity was analyzed with filter paper according to the method of Ghose [17]. A Whatman No. 1 filter paper (∼50 mg, Whatman International, UK) was incubated at 50 ◦ C for 1 h in 1 ml of 0.05 M Na-citrate buffer solution (pH
4.8) supplemented with 0.5 ml 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 dissolved in distilled water and dialysed against distilled water at 4 ◦ C overnight. The protein concentration was adjusted to 2 g/l. The IEF was performed using polyacrylamide gel (Ampholine PAG plate, Amersham Pharmacia Biotech, Sweden) with a pH range of 3.5–9.5. Samples were focused at 2500 V h and the end voltage was 1500 V. Proteins were stained with Coomassie blue (Bio-Rad Laboratories, USA). Purified 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 purified 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 parts 95% (v/v) ethanol and one part 0.05 M acetate buffer (pH 5.4). 2.5. Zymogram analysis after SDS-PAGE Samples for IEF were prepared as described above. After adjusting the protein concentration to 2 g/l, IEF was performed using agarose gel (Agarose IEF, Amersham Pharmacia Biotech) with a pH range of 3.5–9.5 using Ampholine Preblended solution (Amersham Pharmacia Biotech). The IEF parameters were otherwise as described above. After running the agarose IEF, the part close to pI 9.1 was cut out of the gel and smashed in 0.05 M citrate–phosphate buffer (pH 5). The mixture was frozen and thawed twice to transfer the proteins into the buffer solution. A centrifugal filter device (Centriprep YM-3, Millipore, USA) was used to concentrate the sample solution to about 2 g protein/l. The protein concentrate was run in a SDS-PAGE gel containing 0.1% (v/v) xylan at 4 ◦ C and 100 V. SDS was washed out using a 2.5% (v/v) Triton X-100 solution (Sigma-Aldrich Chemie, Germany), where after the gel was incubated in 0.05 M acetate buffer (pH 5.4) at 50 ◦ C for 20 min. Bio-Rad Coomassic blue was used to stain the pro-
H. Xiong et al. / Process Biochemistry 39 (2004) 729–733
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tein standard (SDS-PAGE low range standard LS1610305, Bio-Rad Laboratories, USA). The gel was soaked in 0.2% (w/v) NaOH for 30 min. After removal of NaOH, 0.1% (v/v) Congo-Red solution was added to stain xylan (20 min incubation). Finally, 1 M 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 cellulases. The highest xylanase and cellulase activities were observed at pH 6.0 and 4.0–4.5, respectively, (Table 1). However, the cellulase activities did not change much at a pH range of 4.0–5.5. The highest concentration of soluble protein was observed at pH 4.5 and 5.0. Both xylanase and cellulase activities as well as the soluble protein concentrations decreased significantly at pH 3.5 and 7.5. Fig. 1a and Fig. 1b show the cultivation data at pH 4.0 and 6.0. At both pH values the lactose was depleted in less than 3 days. Similar soluble protein concentration levels were produced at both pH values. However, the enzyme activity profiles differed significantly. At a low pH cellulase was prevalent and at a high pH xylanase was prevalent. The activities increased slowly after lactose depletion. 3.2. Identification of pH-regulated enzymes by IEF, SDS-PAGE and zymogram analysis Samples from the two T. reesei cultivations, performed at pH 4 and pH 6, were analyzed by IEF (Fig. 2a). Xylanase I (XYNI) and xylanase II (XYNII) were identified in the IEF gel using purified xylanase samples as standards. The Table 1 Xylanase and cellulase activities (IU/ml), and soluble protein concentrations (g/l) in batch cultivations of T. reesei Rut C-30 at 28 ◦ C after 5 days as a function of pH pH
Xylanase (IU/ml)
Cellulase (IU/ml)
Soluble protein (g/l)
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
34.5a
3.0a
56.2 ± 2.1 61.2 ± 1.4 72.2 ± 4.2 86.8 ± 3.3 94.7 ± 4.8 67.9 ± 1.6 59.1a 28.5a
4.2 ± 0.4 4.3 ± 0.1 4.1 ± 0.0 3.9 ± 0.1 3.6 ± 0.3 2.9 ± 0.0 2.2a 0.9a
1.9a 2.9 ± 0.4 3.5 ± 0.2 3.4 ± 0.1 2.9 ± 0.4 2.8 ± 0.4 2.6 ± 0.3 1.9a 1.4a
The initial lactose concentration was 30 g/l. a Only one cultivation.
Fig. 1. The time course of xylanase and cellulase production by T. reesei Rut C-30 grown on lactose at (a) pH 4.0 and (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 containing agarose gel. Xylanase II activity was detected in both samples, whilst xylanase I activity was clearly higher in the pH 4.0 sample (Fig. 2b). The sample from the pH 6.0 cultivation 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 xylanase III (XYNIII). Based on an earlier work, we knew that the pI of this enzyme is 9.1 [11]. Using an SDS-PAGE zymogram of the excised pI 9.1 region (Fig. 3), the size of the enzyme was shown to be approximately 32 kDa, 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 typically less than 5 [13]. Hence, these cellulases were detected in the samples from both pH 4.0 and pH 6.0 cultivations (Fig. 2a). The pH-dependent xylanase activity profiles of T. reesei (purified enzymes) correspond with the pH-dependent production 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 results, the production of xylanase I was favoured by a low pH (4.0). In pH 6.0 cultivation, only weak xylanase I activity was detected. Xylanase II activity, on the other hand, was detected in both cultivations. The xylanase III activity was significantly higher at pH 6.0.
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Fig. 2. IEF, (a) IEF gel stained by Coomassie blue; (b) xylanase activity zymogram after IEF. Purified 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. Purified xylanase II (XYNII, ∼20 kDa) 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 significant part of the total xylanase activity in the pH 6 sample was detected in the cathodic part (high pI) 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 Biochemistry 39 (2004) 729–733
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 pI. It appears that xylanase II (family 11) and xylanase III (family 10) represent together the main xylan-digesting activity, when T. reesei Rut C-30 cells are grown at a high pH (pH ∼6), whilst xylanase I (family 11) does the same, together with xylanase II, at a low pH (pH ∼4).
4. Conclusions The filamentous fungus T. reesei Rut C-30 reacts to the pH of the growth environment by modifying its enzyme production patterns. The pH-dependent activity profiles of the purified T. reesei xylanases and the pH-dependent production 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. Financial support from the Research Foundation of Helsinki University of Technology is gratefully acknowledged. References [1] Nakari-Setälä T, Penttilä 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. Production of fungal xylanases. Bioresource Technol 1996;58:137–61. [3] Montenecourt BS, Eveleigh DE. Selective screening methods for the isolation of high yielding cellulase mutants of Trichoderma reesei. Adv Chem Ser 1979;181:289–301.
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[4] Ilmén C, Thrane C, Penttilä M. The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full length and a truncated mutant form. Mol Gen Genet 1996;251:451–60. [5] Domingues FC, Queiroz JA, Cabral JMS, Fonseca LP. The influence of culture conditions on mycelial structure and cellulase production by Trichoderma reesei Rut C-30. Enzyme Microb Technol 2000;26:394–401. [6] Ju LK, Afolabi OA. Wastepaper hydrolysate as soluble inducing substrate for cellulase production in continuous culture of Trichoderma reesei. Biotechnol Prog 1999;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 Biotechnol 1993;40:224–9. [10] Tenkanen M, Puls J, Poutanen K. Two major xylanases of Trichoderma reesei. Enzyme Microb Technol 1992;14:566–74. [11] Xu J, Takakuwa N, Nogawa M, Okada H, Morikawa Y. A third xylanase from Trichoderma reesei PC-3-7. Appl Microbiol Biotechnol 1998;49:718–24. [12] Clarkson K, Siika-Aho M, Tenkanen M, Bower BS, Penttilä ME, Saloheimo M. Trichoderma reesei xylanase. Patent application WO0149859 2001. [13] Biely P, Markovic O. Resolution of glycanases of Trichoderma reesei with respect to cellulose and xylan degradation. Biotechnol Appl Biochem 1988;10:99–106. [14] Törrönen A, Rouvinen J. Structural and functional properties of low molecular weight endo-1,4-b-xylanases. J Biotechnol 1997;57:137– 49. [15] Törrönen A, Mach RL, Messner R, Gonzalez R, Kalkkinen N, Harkki A, et al. The two major xylanases from Trichoderma reesei: characterization of both enzymes and genes. Bio/Technology 1992;10:1461–5. [16] Turunen O, Etuaho K, Fenel F, Vehmaanperä J, Wu X, Rouvinin J, et al. Combination of weakly stabilizing mutations with a disulfide-bridge in the ␣-helix region of Trichoderma reesei endo-1,4--xylanase II increases the thermal stability through synergism. J Biotechnol 2001;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 measurement with the Folin-phenol reagent. J Biol Chem 1951;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.
Influence 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 24 December 2002; accepted 17 May 2003
Abstract Trichoderma reesei Rut C-30 was cultivated in bioreactors at different pH on a medium with lactose as the main carbon source. Compared to an earlier study, in which T. reesei Rut C-30 was 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 6.0 compared to pH 7.0 on 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 4 and 6 correlate with the pH activity profiles 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. © 2003 Elsevier Ltd. All rights reserved. Keywords: pH; Xylanase; Cellulase; Lactose; Trichoderma reesei; Filamentous fungi
1. Introduction Cellulases and xylanases are industrially important enzymes with applications in e.g. food, feed, textile and pulp and paper industries [1,2]. Industrially, these enzymes are produced mainly with filamentous fungi, particularly with different Trichoderma species. Trichoderma reesei (anamorph: Hypocrea jecorina) grows on simple carbon sources (e.g. cellulose and xylan) and secretes efficiently both cellulases and xylanases. T. reesei Rut C-30 is a widely studied mutant strain [3]. Compared to the wild-type and some other strains, Rut C-30 has improved enzyme production capabilities [4]. It is also less sensitive to glucose repression. Cellulose and xylan induce efficiently the production of the cellulolytic and xylanolytic enzymes of T. reesei. However, the high amount of insoluble material in cellulose- and xylan-based submerged bioreactor cultivations results in decreased 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: [email protected] (O. Turunen).
0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00178-X
materials, such as lactose and starch and wastepaper hydrolysates, are commonly used as the main carbon source [7,8]. pH is an important parameter in the production of enzymes by T. reesei. Earlier reports describe that the production of xylanases by T. reesei Rut C-30 on 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 5.5 and 9, respectively) are approximately 20 kDa proteins belonging to the family 11 of 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, 32 kDa) is a recently found, family 10 glycosyl hydrolase characterized from T. reesei PC-3-7 [11]. The pH optimum for xylanase III was observed at pH 6.0–6.5. Of the total xylanase activity in T. reesei PC-3-7 on a cellulose-based growth medium, xylanase III accounted for over 25%. Xylanase IV (pI 7.0, 43 kDa) 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-specific cellulases [13]. The isoelectric points of these cellulases were below 5.
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H. Xiong et al. / Process Biochemistry 39 (2004) 729–733
In this article, we studied the enzyme production by T. reesei Rut C-30 at different pH values. Compared to earlier studies, we present new results on xylanase production on a lactose-based growth medium. We also found that the different 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: 30 g/l lactose; 5 g/l (NH4 )2 SO4 ; 5 g/l KH2 PO4 ; 0.6 g/l MgSO4 ·7H2 O; 0.8 g/l CaCl2 ·2H2 O; 5.0 mg/l FeSO4 ·7H2 O; 1.6 mg/l MnSO4 ·H2 O; 1.4 mg/l ZnSO4 ·H2 O; 2.0 mg/l CoCl2 ·6H2 O; 0.2 ml/l Tween 80 (Fluka Chemie, Switzerland); 0.75 g/l peptone (Difco Laboratories, USA); and 0.3 g/l yeast extract (Lab M, International Diagnostics Group, UK). If otherwise not indicated, the components were purchased from Sigma-Aldrich Chemie, Germany. A stock solution of lactose was autoclaved separately (121 ◦ C, 20 min). T. reesei Rut C-30 was obtained from VTT, Finland. Dry powder spores were suspended in sterile 20% (v/v) glycerol and the suspension inoculated on potato dextrose agar (PDA) slants (Difco Laboratories, USA). The PDA slants were incubated at 30 ◦ C for 7 days and then stored at 4 ◦ C. The formed spores were collected by washing the slant with 3 ml sterile culture medium. The spore concentrate was pipetted into a 250-ml shake flasks containing 100 ml culture medium and incubated on a rotary shaker (200 rpm) at 30 ◦ C. After 36 h growth the medium was used as the inoculum for a bioreactor cultivation. Batch cultivations were carried out in 2-l glass-vessel bioreactors (Biostat MD system, B. Braun Biotech International, Germany). The cultivation parameters were as follows: temperature 28 ◦ C, agitation 400 rpm (tip speed 1.1 m/s, two Rushton type impellers), aeration 1 vvm and cultivation time 5 days. Foam was controlled by automatic addition of 10% (v/v) silicone antifoaming agent (BDH Laboratories Supplies, UK). The pH was controlled by automatic addition of 12.5% (v/v) ammonia water or 10% (v/v) sulfuric acid. The bioreactor working volume was 1-l. 2.2. Enzyme assays Xylanase activity was analyzed with the 3,5-dinitrosalicylic acid (DNS) method by assaying the reducing sugars released during a 10 min reaction (1% (w/v) xylan, 0.05 M citrate-phosphate buffer, pH 5.0, 50 ◦ C) [16]. Cellulase activity was analyzed with filter paper according to the method of Ghose [17]. A Whatman No. 1 filter paper (∼50 mg, Whatman International, UK) was incubated at 50 ◦ C for 1 h in 1 ml of 0.05 M Na-citrate buffer solution (pH
4.8) supplemented with 0.5 ml 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 dissolved in distilled water and dialysed against distilled water at 4 ◦ C overnight. The protein concentration was adjusted to 2 g/l. The IEF was performed using polyacrylamide gel (Ampholine PAG plate, Amersham Pharmacia Biotech, Sweden) with a pH range of 3.5–9.5. Samples were focused at 2500 V h and the end voltage was 1500 V. Proteins were stained with Coomassie blue (Bio-Rad Laboratories, USA). Purified 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 purified 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 parts 95% (v/v) ethanol and one part 0.05 M acetate buffer (pH 5.4). 2.5. Zymogram analysis after SDS-PAGE Samples for IEF were prepared as described above. After adjusting the protein concentration to 2 g/l, IEF was performed using agarose gel (Agarose IEF, Amersham Pharmacia Biotech) with a pH range of 3.5–9.5 using Ampholine Preblended solution (Amersham Pharmacia Biotech). The IEF parameters were otherwise as described above. After running the agarose IEF, the part close to pI 9.1 was cut out of the gel and smashed in 0.05 M citrate–phosphate buffer (pH 5). The mixture was frozen and thawed twice to transfer the proteins into the buffer solution. A centrifugal filter device (Centriprep YM-3, Millipore, USA) was used to concentrate the sample solution to about 2 g protein/l. The protein concentrate was run in a SDS-PAGE gel containing 0.1% (v/v) xylan at 4 ◦ C and 100 V. SDS was washed out using a 2.5% (v/v) Triton X-100 solution (Sigma-Aldrich Chemie, Germany), where after the gel was incubated in 0.05 M acetate buffer (pH 5.4) at 50 ◦ C for 20 min. Bio-Rad Coomassic blue was used to stain the pro-
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tein standard (SDS-PAGE low range standard LS1610305, Bio-Rad Laboratories, USA). The gel was soaked in 0.2% (w/v) NaOH for 30 min. After removal of NaOH, 0.1% (v/v) Congo-Red solution was added to stain xylan (20 min incubation). Finally, 1 M 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 cellulases. The highest xylanase and cellulase activities were observed at pH 6.0 and 4.0–4.5, respectively, (Table 1). However, the cellulase activities did not change much at a pH range of 4.0–5.5. The highest concentration of soluble protein was observed at pH 4.5 and 5.0. Both xylanase and cellulase activities as well as the soluble protein concentrations decreased significantly at pH 3.5 and 7.5. Fig. 1a and Fig. 1b show the cultivation data at pH 4.0 and 6.0. At both pH values the lactose was depleted in less than 3 days. Similar soluble protein concentration levels were produced at both pH values. However, the enzyme activity profiles differed significantly. At a low pH cellulase was prevalent and at a high pH xylanase was prevalent. The activities increased slowly after lactose depletion. 3.2. Identification of pH-regulated enzymes by IEF, SDS-PAGE and zymogram analysis Samples from the two T. reesei cultivations, performed at pH 4 and pH 6, were analyzed by IEF (Fig. 2a). Xylanase I (XYNI) and xylanase II (XYNII) were identified in the IEF gel using purified xylanase samples as standards. The Table 1 Xylanase and cellulase activities (IU/ml), and soluble protein concentrations (g/l) in batch cultivations of T. reesei Rut C-30 at 28 ◦ C after 5 days as a function of pH pH
Xylanase (IU/ml)
Cellulase (IU/ml)
Soluble protein (g/l)
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
34.5a
3.0a
56.2 ± 2.1 61.2 ± 1.4 72.2 ± 4.2 86.8 ± 3.3 94.7 ± 4.8 67.9 ± 1.6 59.1a 28.5a
4.2 ± 0.4 4.3 ± 0.1 4.1 ± 0.0 3.9 ± 0.1 3.6 ± 0.3 2.9 ± 0.0 2.2a 0.9a
1.9a 2.9 ± 0.4 3.5 ± 0.2 3.4 ± 0.1 2.9 ± 0.4 2.8 ± 0.4 2.6 ± 0.3 1.9a 1.4a
The initial lactose concentration was 30 g/l. a Only one cultivation.
Fig. 1. The time course of xylanase and cellulase production by T. reesei Rut C-30 grown on lactose at (a) pH 4.0 and (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 containing agarose gel. Xylanase II activity was detected in both samples, whilst xylanase I activity was clearly higher in the pH 4.0 sample (Fig. 2b). The sample from the pH 6.0 cultivation 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 xylanase III (XYNIII). Based on an earlier work, we knew that the pI of this enzyme is 9.1 [11]. Using an SDS-PAGE zymogram of the excised pI 9.1 region (Fig. 3), the size of the enzyme was shown to be approximately 32 kDa, 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 typically less than 5 [13]. Hence, these cellulases were detected in the samples from both pH 4.0 and pH 6.0 cultivations (Fig. 2a). The pH-dependent xylanase activity profiles of T. reesei (purified enzymes) correspond with the pH-dependent production 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 results, the production of xylanase I was favoured by a low pH (4.0). In pH 6.0 cultivation, only weak xylanase I activity was detected. Xylanase II activity, on the other hand, was detected in both cultivations. The xylanase III activity was significantly higher at pH 6.0.
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Fig. 2. IEF, (a) IEF gel stained by Coomassie blue; (b) xylanase activity zymogram after IEF. Purified 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. Purified xylanase II (XYNII, ∼20 kDa) 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 significant part of the total xylanase activity in the pH 6 sample was detected in the cathodic part (high pI) 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.
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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 pI. It appears that xylanase II (family 11) and xylanase III (family 10) represent together the main xylan-digesting activity, when T. reesei Rut C-30 cells are grown at a high pH (pH ∼6), whilst xylanase I (family 11) does the same, together with xylanase II, at a low pH (pH ∼4).
4. Conclusions The filamentous fungus T. reesei Rut C-30 reacts to the pH of the growth environment by modifying its enzyme production patterns. The pH-dependent activity profiles of the purified T. reesei xylanases and the pH-dependent production 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.
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