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Functional characterization of a recombinant thermostable xylanase from Pichia pastoris: A hybrid enzyme being suitable for xylooligosaccharides production
Biochemical Engineering Journal 48 (2009) 87–92
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Functional characterization of a recombinant thermostable xylanase from Pichia pastoris: A hybrid enzyme being suitable for xylooligosaccharides production He Jun a,b , Yu Bing a,b , Zhang Keying a,b , Chen Daiwen a,b,∗ a b
Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014, PR China Key Laboratory of Animal Disease-Resistance Nutrition, Ministry of Education, PR China
a r t i c l e
i n f o
Article history: Received 18 June 2009 Received in revised form 23 August 2009 Accepted 26 August 2009
Keywords: Xylanase Expression Characterization Thermostability Xylooligosaccharides
a b s t r a c t The hybrid xylanase gene Xyn2-A2 was previously constructed and expressed in Pichia pastoris. In this study, the hybrid recombinant enzyme (PTXC2) was prepared from a 2-L bioreactor and subsequently characterized. The produced xylanase was estimated by SDS-PAGE to be 26 kDa and practically free of cellulolytic activity. The optimal temperature and pH of PTXC2 was 65 ◦ C and pH 6.0, respectively. The hybrid enzyme was stable at 60 ◦ C and retained more than 85% of its activity after 30 min incubation at this temperature. The specificity of PTXC2 towards different natural substrates was evaluated. PTXC2 was highly specific towards xylans tested but exhibited low activities towards cellulosic substrates, such as gellan gum (9.7%), Avicel (1.5%) and carboxymethylcellulose (CMC, 1.2%). The apparent Km values of oat-spelt xylan and birchwood xylan was 1.6 and 1.8 mg/mL, respectively. Analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as an endo-xylanase with xylotriose as the main hydrolysis products. These attributes should make the enzyme an attractive applicant for various applications, such as large-scale production of xylooligosaccharides. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Xylan, an abundant component of plant cell wall, has a relatively complex structure based on a nonbranched -1,4-glycosidically linked xylose backbone. Depending on the origin, the backbone structure is substituted to various degrees with acetyl, l-arabinofuranosyl, glucuronyl and 4-O-methylglucuronyl groups [1]. Complete degradation of xylans requires the synergistic actions of several enzymes of which EC3.2.1.8-endo-beta-1,4-xylanases are the crucial enzymes for depolymerization [2]. Xylanases are believed to have considerable potential in various industrial applications, such as in biobleaching, paper making and in the food and animal feed industries. However, these applications are usually performed at high temperatures (over 60 ◦ C) [3–5]. Various microorganisms, such as bacteria, yeasts, and filamentous fungi were found to naturally secrete xylanases. But most xylanases present an optimum activity around 50–60 ◦ C and a half-life of about 1 h at 50 ◦ C [6]. Thus, highly thermostable xylanases may be particularly interesting for industrial applications. Currently, there are two approaches have been successfully tried to produce xylanases: one approach is to isolate enzymes from
∗ Corresponding author at: Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014, PR China. Tel.: +86 835 2885106; fax: +86 835 2885106. E-mail address: [email protected] (C. Daiwen). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.08.010
natural microorganisms and the other is to create enzymes by heterologous expression [7]. The filamentous fungus Trichoderma reesei is one of the most efficient xylanase producers. The highest total beta-xylanase activity obtained in shake flasks for hyperproducing mutant T. reesei Rut C-30 was up to 5400 nkat/mL [8]. The expression level is higher than levels obtained from most of natural microorganisms [6]. The two major endo-xylanases produced by this fungus are Xyn1 and Xyn2. Previous study indicated that Xyn2 represents more than 50% of the total xylanolytic activity of this fungus [9], suggesting that this enzyme will be a useful model for applied research. Xyn2 is a low-molecular-mass (21 kDa) hemicellulolytic enzyme with an alkaline isoelectric point (pI 9.0) and an activity optimum at pH 4–6. Xyn2 catalyzes the hydrolysis of xylan by cleaving specifically the endo-1,4--glycosidic bond between the subunits of the xylan polymer chain. But the enzyme rapidly loses its activity when incubated at temperatures over 50 ◦ C [10]. Therefore, the thermostability of Xyn2 needs to be improved. The production level of xylanase by Thermotoga maritima is lower than T. reesei. However, T. maritima is the most thermophilic xylan-degrading bacteria currently known capable of growth at temperatures up to 90 ◦ C [11]. The T. maritima XynA is an extremely thermostable modular enzyme with five domains (A1-A2-B-C1C2). The N-terminal domain, especially the domain A2 has been found to be responsible for enzyme thermostability [12–14]. In previous study, we have engineered the thermostabilizing domain A2 into the N-terminal region of T. reesei Xyn2 [15]. Our goal was to
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enhance the capability of the T. reesei Xyn2 withstanding inactivation at high temperatures. Though the hybrid xylanase gene was successfully expressed in P. pastoris, the catalytic properties of the recombinant enzyme have not been investigated in detail. In this study, we report the enzyme production by P. pastoris as well as the catalytic properties of the produced enzyme using various natural substrates. The main end products of xylan hydrolysis are also determined. 2. Materials and methods 2.1. Strains and media P. pastoris X-33 was cultivated in YPD medium (1% yeast extract, 2% peptone, 2% glucose). Recombinant plasmids were constructed and amplified in Escherichia coli DH5␣ cultivated at 37 ◦ C in Luria-Bertani liquid medium or Luria-Bertani agar. Plasmid vector pPICZ␣A (Invitrogen, USA) was used for the heterologous expression of the xylanase gene. All the recombinant DNA techniques were performed as described by Sambrook et al. [16]. 2.2. Cloning and expression of hybrid xylanase gene in P. pastoris The full-length of the hybrid xylanase gene (T. reesei Xyn2 fused in frame with the domain A2, 749 bp) supplied with the EcoRI and NotI restriction sites, was synthesized by TaKaRa Biotechnology (Dalian, China) Co., Ltd. The hybrid xylanase gene was cloned into pPICZ␣A and this plasmid (after linearization) was then transformed into P. pastoris by electroporation methods as described by the manufacturer (Bio-Rad, USA). The transformed cells were cultured by the method of Liu and Liu [17]. 2.3. Preparation of hybrid xylanase in a 2 L bioreactor A large-scale production was performed in a 2 L bioreactor (B. Braun Sartorius Ltd.). Recombinant P. pastoris was grown in 200 mL BMGY medium. Temperature and pH were maintained at 30 ◦ C and 6.0, respectively, throughout growth phase for 16–18 h. Agitation was kept within the range of 300–400 rpm. To induce enzyme secretion, cell pellets were resuspended in 800 mL of BMMY medium containing 1% methanol. The cultivation was maintained for 6 days by adding absolute methanol to a final concentration of 1% every day. Relative percentage of dissolved oxygen was maintained above 30% via adjusting agitation rate. Supernatants were collected every day and kept at −80 ◦ C before analysis. Cell pellets were washed, and dried at 80 ◦ C until constant weight was achieved.
photographed while raised approximately 12 in. (ca. 30 cm) above a black background. 2.5. Enzyme activity assays For enzyme characterization, the culture supernatant was filtered and concentrated (about 10-fold) in a Diaflo Ultrafilter PM10 concentrator (Amicon). Xylanase activity was assayed by the method described by Bailey et al. [21], with 1% oat-spelt xylan (Sigma) as the substrate at 50 ◦ C for 10 min. Appropriate dilutions of the recombinant protein (culture supernatant) in 50 mM sodium citrate buffer (pH 5.0) were used as the enzyme source. The amount of released sugar was determined by the dinitrosalicylic acid method described by Miller et al. [22]. One unit (nkat) of xylanase activity was defined as the quantity of enzyme that liberated reducing sugar at the rate of 1 nmol per second. The temperature optimum of the enzyme was measured by performing the enzyme activity assay at temperatures ranging from 20 to 90 ◦ C. Thermostability was tested by heating enzyme samples for different times at various temperatures, and the activity was assayed at 50 ◦ C for 10 min. The xylanase activity prior to the preincubations at different temperature was taken as 100%. Assays at different pH values were performed at the optimal temperature over a pH range of 3.0–9.0. The buffer used were 50 mM citrate (pH 3.0), 50 mM citrate phosphate (pH 4.0–7.0), 50 mM phosphate (pH 8.0) and 50 mM Boric Acid-Borax (pH 9.0), respectively. 2.6. Substrate specificity and kinetic parameters Substrate specificity of the enzyme was determined using different cellulose and hemicellulose substrates. The reaction was carried out in 50 mM citrate phosphate (pH 5.0) containing 2.0 mg/mL of each substrate at 50 ◦ C for 10 min. For each assay, six different substrate concentrations were prepared in 50 mM citrate phosphate (pH 5.0), and incubated with the purified enzyme at 50 ◦ C for 5 min. The Km and kcat values were calculated from the kinetics data as described by Jiang et al. [23]. 2.7. Polysaccharide-binding properties The polysaccharide-binding capacity was determined by the method described by Tenkanen et al. [24]. Purified Xyn2 (30 g) was incubated with different concentrations of Avicel or oat-spelt xylan in 50 mM citrate phosphate buffer (pH 5.0), at 4 ◦ C for 1 h with slow shaking. After centrifugation (10,000 × g, 5 min), the supernatant was collected and tested for its xylanase activity. Unbound enzyme was determined by measuring residual activity in the supernatant.
2.4. Analysis of the protein purity, concentration and xylanolytic activity
2.8. Analysis of hydrolytic capacity and products
The purity of the secreted enzyme protein, PTXC2 was monitored by SDS-PAGE analysis [18]. The protein fractions were boiled for 3 min and applied to the gel. Proteins were visualized by Coomassie brilliant blue R 250 staining. The protein concentration was determined by the Bradford assay using bovine serum albumin as a standard [19]. To determine the xylanolytic activity of the secreted protein, Zymogram analysis was performed as described by Royer and Nakas [20]. A native-PAGE was performed. Then the gel was washed twice with distilled water and overlaid on substrate gel (1.5% oat-spelt xylan, 1.5% agar in 50 mM sodium phosphate buffer, pH 5.0). The gels were next smoothed to remove bubbles, wrapped in plastic, and incubated at 50 ◦ C for 20 min. The gels were finally separated, and the substrate gels containing oatspelt xylan were immersed in 95% ethanol for 45 min and were
The hydrolysis of xylan-based substrates was carried out in 100 mL conical flask containing 50 mL citrate buffer (pH 5.0, 50 mM), 2.5 g xylan or wheat bran, 5 mg sodium azide and enzyme preparation (1000 nkat) as described by Adsul et al. [25]. The hydrolysis was performed for 6 days at 50 ◦ C, with a stirring rate of 150 rpm. The samples were analyzed for the reducing sugars after suitable time intervals. The hydrolyzed products of xylan was analyzed by the thin-layer chromatography (TLC) using silica gel Plates 60F 254 (E. Merck, Germany). Aliquots (100 l) of the samples were collected at 5, 10, and 30 min, 1, 2, 4, 8, and 12 h of the incubation period and 1 l of the aliquot was spotted on the TCL plates. The plates were subsequently developed with two runs of acetonitrile–water (85:15, v/v) followed by heating for a few minutes at 130 ◦ C in an oven after spraying the plates with a
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Fig. 1. SDS-PAGE and Zymogram analysis. (A) SDS-PAGE analysis of expressed product. M: protein marker; Lane 1–6: the culture supernatant containing recombinant xylanase on day 1, 2, 3, 4, 5, and 6. (B) Xylanolytic activity analysis (Zymogram). Lane 1: culture supernatant before induction; Lane 2–4: culture supernatant after 24, 48, and 72 h induction.
methanol–sulfuric acid mixture (95:5, v/v) [23]. A xylooligosaccharide mixture (Suntory Ltd., Japan) consisting of xylose, xylobiose, and xylotriose was used as the standard. 3. Results 3.1. Preparation of PTXC2 in 2 L bioreactor The recombinant P. pastoris was cultured in 2 L bioreactor. As we expected, the size of recombinant xylanase (PTXC2) determined by SDS-PAGE was 26 kDa (Fig. 1A). PTXC2 was the major protein (over 90% of total protein as detected by densitometer) secreted by P. pastoris into the culture medium. Therefore, the procedure for protein purification was not necessary. Both the highest xylanase activity (1484 nkat/mL) and protein concentration (0.26 mg/mL) in culture supernatant was recorded after 72 h induction (Fig. 2). However, the highest cell wet-weight (32 g/L) was obtained at 102 h. Both xylanase activity and cell wet-weight decreased slightly in the late induction period (after 120 h).
Fig. 2. Time course of xylanase activity produced by recombinant P. pastoris. () Biomass concentration; () xylanase activity.
Fig. 3. Characterization of the recombinant PTXC2. (A) Effect of temperature on the activity of PTXC2. (B) Effect of pH on the activity of PTXC2. (C) The thermostability of PTXC2 at different temperatures was determined by preincubating the enzyme at these temperatures in the absence of substrate for 5, 10, 15, 20, 25, and 30 min before measuring its activity [() 50 ◦ C; () 60 ◦ C; (䊉) 70 ◦ C]. The xylanase activity prior to the preincubations at different temperature was taken as 100%.
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3.2. Determination of xylanolytic activity (Zymogram) When the culture medium (after induction) were subjected to native-PAGE, xylanolytic activity was detected using oat-spelt xylan as substrate; Immersion of the substrate gel in ethanol resulted in precipitation of undegraded xylan, which enhanced contrast and revealed only one clear band against a black background (Fig. 1B). The result confirmed the xylanolytic activity of the hybrid enzyme. 3.3. Effect of pH and temperature on xylanase activity PTXC2 has an optimal activity at 65 ◦ C (Fig. 3A). Activity decreased rapidly with temperature, i.e., at 80 ◦ C, activity of the enzyme represented only 45% of the optimum. Concerning with the effect of the pH, the enzyme showed an optimal activity at approximately pH 6.0 (Fig. 3B). When the pH was below 3.0 and above 9.0, only 30–40% of the maximum activity was reached. PTXC2 was capable of withstanding a temperature up to 60 ◦ C, and the total activity of the enzyme retained more than 85% after 30 min incubating at this temperature (Fig. 3C).
Fig. 4. Effect of different concentrations of Avicel and oat-spelt xylan on the binding ability of PTXC2. Xylanase (25 g) was incubated with 1–24 mg/mL Avicel or oatspelt xylan in 50 mM citrate phosphate (pH 5.0) at 4 ◦ C [Avicel (); Oat-spelt xylan ()].
3.4. Substrate specificity and kinetic parameters The hydrolytic activity of the PTXC2 on various substrates was determined (Table 1). The highest activity (14,980 nkat/mL) was observed with the oat-spelt xylan followed by the birchwood xylan (13,800 nkat/mL) and beechwood xylan (12,490 nkat/mL). The enzyme exhibited low activities towards cellulosic substrates, such as Gellan gum (1450 nkat/mL), Avicel (220 nkat/mL) and CMC (180 nkat/mL). The determined Km and kcat were 1.5 mg/mL and 193.6 s−1 for oat-spelt xylan, and 1.7 mg/mL and 168.2 s−1 for birchwood xylan, respectively. 3.5. Polysaccharide-binding analysis The polysaccharide-binding capacity of PTXC2 was determined by incubating the enzyme with Avicel or oat-spelt xylan. As shown in Fig. 4, PTXC2 could not bind to Avicel, as about 94% enzyme activity still remained in supernatant. However, the enzyme did show its capacity to bind to xylan and 63% of the enzyme activity remained in the supernatant. 3.6. Analysis of hydrolytic capacity and products PTXC2 has been used to hydrolyze substrates at 5% concentration. As shown in Fig. 5, the yield of enzymatic hydrolysis was highest when oat-spelt xylan was used as the substrate (56.4%) followed by birchwood xylan (51%) and beechwood xylan (43%). The wheat bran hydrolysis was lowest (only 31%), but the hydrolysis for both xylans and wheat bran linearly increased with the
Fig. 5. The hydrolysis yield of Oat-spelt xylan (), Birchwood xylan (), Beechwood xylan () and Wheat bran (♦) using PTXC2. Substrates (2.5 g) were incubated with 1000 nkat of the enzyme in 50 mL 50 mM citrate buffer (pH 5.0) and the reaction was carried out at 50 ◦ C with shaking at 150 rpm.
increasing of reaction time. The products of hydrolysis of oatspelt xylan were analyzed by TLC. The predominant hydrolysis end product of oat-spelt xylan was xylotriose (Fig. 6). The xylotriose was produced within 5 min of the reaction period. As the reaction time increased, both the xylotriose and xylobiose concentration increased. These results confirmed that the recombinant Xyn2 was an endo-xylanase. 4. Discussion In recent years, xylanases has attracted considerable research interests because of their use in various applications [3–5]. To create a novel xylanase free of cellulolytic activity and improve the
Table 1 Substrate specificity and kinetic constants of Xyn2 and PTXC2 (all the values are the means of three replications). Substrate
Oat-spelt xylan Birchwood xylan Beechwood xylan Gellan gum Avicel CMC a b c
kcat (s−1 )
Activity (nkat/mL)
Relative activity (%)
Km (mg/mL)
Xyn2a
PTXC2b
Xyn2
PTXC2
Xyn2
PTXC2
Xyn2
PTXC2
875 988 702 143 24 15
14,980 13,800 12,490 1,450 220 180
100 113 80 16 3 2
100 92.1 83.3 9.7 1.5 1.2
3.1 ± 0.2 4.1 ± 0.3 2.7 ± 0.2 NAc NA NA
1.5 ± 0.03 1.7 ± 0.05 1.9 ± 0.02 NA NA NA
87.1 ± 5.4 106.3 ± 8.8 75.4 ± 6.3 NA NA NA
193.6 ± 12.1 168.2 ± 10.5 145.1 ± 9.4 NA NA NA
Xyn2 were obtained from recombinant E. coli. PTXC2 were obtained from P. pastoris. NA, not available.
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Fig. 6. Analysis of the hydrolyzed products. Oat-spelt xylan (40 mg) was incubated with 1000 nkat of the enzyme in 2 mL 50 mM citrate phosphate (pH 5.0) and the reaction was performed at 50 ◦ C for 12 h and then the hydrolyzates were analyzed by TLC.
thermostability, we have previously expressed a hybrid Xyn2 gene in P. pastoris. The thermostabilizing domain (A2) from T. maritima has been successfully introduced into the N-terminal region of T. reesei Xyn2. As a result, the thermostability of the enzyme was improved [15]. In this study, the hybrid enzyme (PTXC2) was prepared from 2 L bioreactor and the enzymatic properties of PTXC2 have been investigated in detail. As shown in Fig. 1A, PTXC2 is the predominant protein (over 90% of purity) in extracellular medium. The molecular mass is 26 kDa as estimated by SDS-PAGE. There is thus a 5-kDa difference in the molecular mass of the xylanase secreted by T. reesei (Table 2). This is caused by the N-terminal fusion of domain A2. As shown in Fig. 1B, this large, fusion protein exhibited a high xylanolytic activity. The highest xylanase activity (1484 nkat/mL) was obtained in 2 L bioreactor after 72 h fermentation (Fig. 2). The expression level is higher than that of levels obtained from recombinant Saccharomyces cerevisiae strain [26] or any E. coli expression system [27,28]. As an unoptimized production level this is still lower than a recombinant T. reesei production system [29]. However, optimization is likely to further improve the production efficiency. For functional characterization, an unpurified enzyme was used and results indicated that even the non-purified enzyme shows highly specific xylanase activity. Therefore, PTXC2 produced in P. pastoris has an advantage of showing xylanase activity that is practically free of harmful side activities. The T. reesei Xyn2 has been reported to have an acidic pH optima (5.0) and a moderate temperature optima (50–55 ◦ C) [10]. Our data indicates that PTXC2 exhibited optimum xylanase activity close to neutral pH value (pH 6.0). It is also noteworthy that PTXC2 was active (over 60% activity retained) over a more wide pH range (3.5–8.0) than T. reesei Xyn2 (4.5–5.5). The altered pH range may be ascribed to the introduc-
Table 2 Comparison of molecular characteristics of PTXC2 and native Xyn2. Property
Native Xyn2a
PTXC2
References
Molecular mass (kDa) Optimum pH pH stabilityb Optimum temp (◦ C) Temp stability (30 min, 60 ◦ C)
21 5.0 4.5–5.5 55–60c NAd
26 6.0 3.5–8.0 65 85e
[10] [10] [10] [4,10] [10]
a b c d e
Produced by T. reesei. More than 60% of maximal activity retained. Temperature optimum of -xylanases produced by T. reesei, not only Xyn2. NA, not available. 85% of total activity retained.
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tion of the domain A2 which stabilizes the molecular structure of the protein and increases the capability of withstanding the harsh conditions (as shown by the improved thermostability). However, future study should be performed to confirm this hypothesis. The temperature optima of PTXC2 was 65 ◦ C, which was 10 ◦ C higher than T. reesei Xyn2. Previous study indicated that T. reesei Xyn2 is not capable of withstanding a temperature over 60 ◦ C [10]. However, the introduction of domain A2 significantly improved the thermostability of T. reesei Xyn2 and the hybrid enzyme retained more than 85% of its total activity after 30 min incubation at 60 ◦ C (Table 2). Our results are in good agreement with previous study which confirmed the thermostabilizing function of domain A2 in T. maritima XynA [4]. The improved thermostability may also be attributed to the N-terminal fusion itself. We have previously expressed the T. reesei Xyn2 gene in E. coli and obtained a recombinant Xyn2 with a fusion peptide (51 amino acids) in N-terminus. When the fusion peptide was deleted, the thermostability of the enzyme acutely decreased [30]. Like with the E. coli produced Xyn2, PTXC2 exhibited a high activity for xylans, but a low activity for cellulosic substrates (Table 1). These results were in good agreement with results from polysaccharide-binding analysis (Fig. 4). When incubating the enzyme with Avicel, more than 94% unbound enzyme was detected in the supernatant. It is noteworthy that the Km values of PTXC2 were lower than E. coli produced Xyn2 when oat-spelt xylan and birchwood xylan was used as the substrate. The results are in accordance with our previous findings that the introduction of domain A2 improved not only the thermostability but also the substrate-binding capacity of T. reesei Xyn2. However, no matter what substrate used, the activity of PTXC2 was always higher than E. coli produced Xyn2 (Table 1). The hydrolytic capacity of PTXC2 on different substrates was also determined. More than 50% hydrolysis was obtained when oat-spelt xylan and birchwood xylan were used as the substrate (Fig. 5). Because of its high purity and enhanced thermostability, PTXC2 might be an attractive enzyme for use in the paper industry. Previous study has already indicated the endo-acting nature of the native Xyn2 [10]. In this study, the products of hydrolysis of oat-spelt xylan (using PTXC2) were analyzed by TLC. Xylotriose was produced within 5 min of the reaction period (Fig. 6) and it was the predominant end product yielded. There were also smaller amount of xylobiose and xylose. The result confirmed the endoacting nature of PTXC2. The xylooligosaccharides (i.e. xylotriose and xylobiose) has been found to be helpful for the maintenance of a healthy intestinal microflora [23]. But, the production of xylooligosaccharides is remaining a time consuming and expensive process. Therefore, PTXC2 may be also useful for the large-scale production of xylooligosaccharides. 5. Conclusions A hybrid xylanase from P. pastoris has been prepared and characterized in this study. The molecular mass of the enzyme was found to be 26 kDa. The enzyme was practically free of cellulolytic activity and showed optimal xylanase activity at pH 6.0 and 65 ◦ C. The enzyme was effectively degrading both oat-spelt xylan and birchwood xylan. Xylotriose was the main end products of oat-spelt xylan hydrolysis. These properties indicated the potential application of the enzyme for xylooligosaccharides production, as well as biobleaching in paper and pulp industry. Acknowledgements This work was granted by Feed Biotechnology Project of Sichuan Province of China with grant No. 2007Z06-050 and Program for
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[15] J. He, B. Yu, K.Y. Zhang, X.M. Ding, D.W. Chen, Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2, New Biotechnol. (2009), doi: 10.1016/j.nbt.2009.04.002. [16] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning—A Laboratory Manual, second ed., Cold Spring Harbour Laboratory, New York, 1989. [17] M.Q. Liu, G.F. Liu, Expression of recombinant Bacillus licheniformis xylanse A in Pichia pastoris and xylooligosaccharides released from xylans by it, Protein Expr. Purif. 57 (2008) 101–107. [18] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (London) 227 (1970) 680–685. [19] M.M. Bradford, A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1796) 248–254. [20] J.C. Royer, J.P. Nakas, Simple, sensitive zymogram technique for detection of xylanase activity in polyacrylamide gels, Appl. Environ. Microbiol. 56 (1990) 1516–1517. [21] M.J. Bailey, P. Biely, K. Poutanen, Interlaboratory testing of methods for assay of xylanase activity, J. Biotechnol. 23 (1992) 257–270. [22] G.L. Miller, R. Blum, W.E. Glennon, A.L. Burton, Measurement of carboxymethylcellulase activity, Anal. Biochem. 2 (1960) 127–132. [23] Z.Q. Jiang, W. Deng, Y.P. Zhu, L.T. Li, Y.J. Sheng, K. Hayashi, The recombinant xylanase B of Thermotoga maritima is highly xylan specific and produces exclusively xylobiose from xylans, a unique character for industrial applications, J. Mol. Catal. B: Enzym. 27 (2004) 207–213. [24] M. Tenkanen, J. Buchert, L. Uiikari, Binding of hemicellulases on isolated polysaccharide substrates, Enzyme Microb. Technol. 17 (1995) 499–505. [25] M.G. Adsul, K.B. Bastawde, A.J. Varma, D.V. Gokhale, Strain improvement of Penicillium janthinellum NCIM1171 for increased cellulase production, Bioresour. Technol. 98 (2007) 1437–1473. [26] D.C. La Grange, I.S. Pretorius, W.H. Van Zyl, Expression of a Trichoderma reesei -xylanase gene (XYN2) in Saccharomyces cerevisiae, Appl. Environ. Microbiol. 62 (1996) 1036–1044. [27] Z. Chenyan, B. Jiangyu, D. Shanshan, W. Jin, Z. Jie, W. Minchen, W. Wu, Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli, Bioresour. Technol. 99 (2008) 831–838. [28] H. Junli, W. Guixue, X. Li, Cloning, sequencing and expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli, Bioresour. Technol. 97 (2006) 802–808. [29] M. Paloheimo, A. Mantyla, J. Kallio, T. Puranen, P. Suominen, Increased production of xylanase by expression of a truncated version of the xyn11A gene from Nonomuraea flexuosa in Trichoderma reesei, Appl. Environ. Microbiol. 73 (2007) 3215–3224. [30] J. He, B. Yu, K.Y. Zhang, X.M. Ding, D.W. Chen, Expression of a Trichoderma reesei -xylanase gene in Escherichia coli and activity of the enzyme on fiber-bound substrate, Protein Expr. Purif. 67 (2009) 1–6.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Functional characterization of a recombinant thermostable xylanase from Pichia pastoris: A hybrid enzyme being suitable for xylooligosaccharides production He Jun a,b , Yu Bing a,b , Zhang Keying a,b , Chen Daiwen a,b,∗ a b
Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014, PR China Key Laboratory of Animal Disease-Resistance Nutrition, Ministry of Education, PR China
a r t i c l e
i n f o
Article history: Received 18 June 2009 Received in revised form 23 August 2009 Accepted 26 August 2009
Keywords: Xylanase Expression Characterization Thermostability Xylooligosaccharides
a b s t r a c t The hybrid xylanase gene Xyn2-A2 was previously constructed and expressed in Pichia pastoris. In this study, the hybrid recombinant enzyme (PTXC2) was prepared from a 2-L bioreactor and subsequently characterized. The produced xylanase was estimated by SDS-PAGE to be 26 kDa and practically free of cellulolytic activity. The optimal temperature and pH of PTXC2 was 65 ◦ C and pH 6.0, respectively. The hybrid enzyme was stable at 60 ◦ C and retained more than 85% of its activity after 30 min incubation at this temperature. The specificity of PTXC2 towards different natural substrates was evaluated. PTXC2 was highly specific towards xylans tested but exhibited low activities towards cellulosic substrates, such as gellan gum (9.7%), Avicel (1.5%) and carboxymethylcellulose (CMC, 1.2%). The apparent Km values of oat-spelt xylan and birchwood xylan was 1.6 and 1.8 mg/mL, respectively. Analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as an endo-xylanase with xylotriose as the main hydrolysis products. These attributes should make the enzyme an attractive applicant for various applications, such as large-scale production of xylooligosaccharides. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Xylan, an abundant component of plant cell wall, has a relatively complex structure based on a nonbranched -1,4-glycosidically linked xylose backbone. Depending on the origin, the backbone structure is substituted to various degrees with acetyl, l-arabinofuranosyl, glucuronyl and 4-O-methylglucuronyl groups [1]. Complete degradation of xylans requires the synergistic actions of several enzymes of which EC3.2.1.8-endo-beta-1,4-xylanases are the crucial enzymes for depolymerization [2]. Xylanases are believed to have considerable potential in various industrial applications, such as in biobleaching, paper making and in the food and animal feed industries. However, these applications are usually performed at high temperatures (over 60 ◦ C) [3–5]. Various microorganisms, such as bacteria, yeasts, and filamentous fungi were found to naturally secrete xylanases. But most xylanases present an optimum activity around 50–60 ◦ C and a half-life of about 1 h at 50 ◦ C [6]. Thus, highly thermostable xylanases may be particularly interesting for industrial applications. Currently, there are two approaches have been successfully tried to produce xylanases: one approach is to isolate enzymes from
∗ Corresponding author at: Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014, PR China. Tel.: +86 835 2885106; fax: +86 835 2885106. E-mail address: [email protected] (C. Daiwen). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.08.010
natural microorganisms and the other is to create enzymes by heterologous expression [7]. The filamentous fungus Trichoderma reesei is one of the most efficient xylanase producers. The highest total beta-xylanase activity obtained in shake flasks for hyperproducing mutant T. reesei Rut C-30 was up to 5400 nkat/mL [8]. The expression level is higher than levels obtained from most of natural microorganisms [6]. The two major endo-xylanases produced by this fungus are Xyn1 and Xyn2. Previous study indicated that Xyn2 represents more than 50% of the total xylanolytic activity of this fungus [9], suggesting that this enzyme will be a useful model for applied research. Xyn2 is a low-molecular-mass (21 kDa) hemicellulolytic enzyme with an alkaline isoelectric point (pI 9.0) and an activity optimum at pH 4–6. Xyn2 catalyzes the hydrolysis of xylan by cleaving specifically the endo-1,4--glycosidic bond between the subunits of the xylan polymer chain. But the enzyme rapidly loses its activity when incubated at temperatures over 50 ◦ C [10]. Therefore, the thermostability of Xyn2 needs to be improved. The production level of xylanase by Thermotoga maritima is lower than T. reesei. However, T. maritima is the most thermophilic xylan-degrading bacteria currently known capable of growth at temperatures up to 90 ◦ C [11]. The T. maritima XynA is an extremely thermostable modular enzyme with five domains (A1-A2-B-C1C2). The N-terminal domain, especially the domain A2 has been found to be responsible for enzyme thermostability [12–14]. In previous study, we have engineered the thermostabilizing domain A2 into the N-terminal region of T. reesei Xyn2 [15]. Our goal was to
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enhance the capability of the T. reesei Xyn2 withstanding inactivation at high temperatures. Though the hybrid xylanase gene was successfully expressed in P. pastoris, the catalytic properties of the recombinant enzyme have not been investigated in detail. In this study, we report the enzyme production by P. pastoris as well as the catalytic properties of the produced enzyme using various natural substrates. The main end products of xylan hydrolysis are also determined. 2. Materials and methods 2.1. Strains and media P. pastoris X-33 was cultivated in YPD medium (1% yeast extract, 2% peptone, 2% glucose). Recombinant plasmids were constructed and amplified in Escherichia coli DH5␣ cultivated at 37 ◦ C in Luria-Bertani liquid medium or Luria-Bertani agar. Plasmid vector pPICZ␣A (Invitrogen, USA) was used for the heterologous expression of the xylanase gene. All the recombinant DNA techniques were performed as described by Sambrook et al. [16]. 2.2. Cloning and expression of hybrid xylanase gene in P. pastoris The full-length of the hybrid xylanase gene (T. reesei Xyn2 fused in frame with the domain A2, 749 bp) supplied with the EcoRI and NotI restriction sites, was synthesized by TaKaRa Biotechnology (Dalian, China) Co., Ltd. The hybrid xylanase gene was cloned into pPICZ␣A and this plasmid (after linearization) was then transformed into P. pastoris by electroporation methods as described by the manufacturer (Bio-Rad, USA). The transformed cells were cultured by the method of Liu and Liu [17]. 2.3. Preparation of hybrid xylanase in a 2 L bioreactor A large-scale production was performed in a 2 L bioreactor (B. Braun Sartorius Ltd.). Recombinant P. pastoris was grown in 200 mL BMGY medium. Temperature and pH were maintained at 30 ◦ C and 6.0, respectively, throughout growth phase for 16–18 h. Agitation was kept within the range of 300–400 rpm. To induce enzyme secretion, cell pellets were resuspended in 800 mL of BMMY medium containing 1% methanol. The cultivation was maintained for 6 days by adding absolute methanol to a final concentration of 1% every day. Relative percentage of dissolved oxygen was maintained above 30% via adjusting agitation rate. Supernatants were collected every day and kept at −80 ◦ C before analysis. Cell pellets were washed, and dried at 80 ◦ C until constant weight was achieved.
photographed while raised approximately 12 in. (ca. 30 cm) above a black background. 2.5. Enzyme activity assays For enzyme characterization, the culture supernatant was filtered and concentrated (about 10-fold) in a Diaflo Ultrafilter PM10 concentrator (Amicon). Xylanase activity was assayed by the method described by Bailey et al. [21], with 1% oat-spelt xylan (Sigma) as the substrate at 50 ◦ C for 10 min. Appropriate dilutions of the recombinant protein (culture supernatant) in 50 mM sodium citrate buffer (pH 5.0) were used as the enzyme source. The amount of released sugar was determined by the dinitrosalicylic acid method described by Miller et al. [22]. One unit (nkat) of xylanase activity was defined as the quantity of enzyme that liberated reducing sugar at the rate of 1 nmol per second. The temperature optimum of the enzyme was measured by performing the enzyme activity assay at temperatures ranging from 20 to 90 ◦ C. Thermostability was tested by heating enzyme samples for different times at various temperatures, and the activity was assayed at 50 ◦ C for 10 min. The xylanase activity prior to the preincubations at different temperature was taken as 100%. Assays at different pH values were performed at the optimal temperature over a pH range of 3.0–9.0. The buffer used were 50 mM citrate (pH 3.0), 50 mM citrate phosphate (pH 4.0–7.0), 50 mM phosphate (pH 8.0) and 50 mM Boric Acid-Borax (pH 9.0), respectively. 2.6. Substrate specificity and kinetic parameters Substrate specificity of the enzyme was determined using different cellulose and hemicellulose substrates. The reaction was carried out in 50 mM citrate phosphate (pH 5.0) containing 2.0 mg/mL of each substrate at 50 ◦ C for 10 min. For each assay, six different substrate concentrations were prepared in 50 mM citrate phosphate (pH 5.0), and incubated with the purified enzyme at 50 ◦ C for 5 min. The Km and kcat values were calculated from the kinetics data as described by Jiang et al. [23]. 2.7. Polysaccharide-binding properties The polysaccharide-binding capacity was determined by the method described by Tenkanen et al. [24]. Purified Xyn2 (30 g) was incubated with different concentrations of Avicel or oat-spelt xylan in 50 mM citrate phosphate buffer (pH 5.0), at 4 ◦ C for 1 h with slow shaking. After centrifugation (10,000 × g, 5 min), the supernatant was collected and tested for its xylanase activity. Unbound enzyme was determined by measuring residual activity in the supernatant.
2.4. Analysis of the protein purity, concentration and xylanolytic activity
2.8. Analysis of hydrolytic capacity and products
The purity of the secreted enzyme protein, PTXC2 was monitored by SDS-PAGE analysis [18]. The protein fractions were boiled for 3 min and applied to the gel. Proteins were visualized by Coomassie brilliant blue R 250 staining. The protein concentration was determined by the Bradford assay using bovine serum albumin as a standard [19]. To determine the xylanolytic activity of the secreted protein, Zymogram analysis was performed as described by Royer and Nakas [20]. A native-PAGE was performed. Then the gel was washed twice with distilled water and overlaid on substrate gel (1.5% oat-spelt xylan, 1.5% agar in 50 mM sodium phosphate buffer, pH 5.0). The gels were next smoothed to remove bubbles, wrapped in plastic, and incubated at 50 ◦ C for 20 min. The gels were finally separated, and the substrate gels containing oatspelt xylan were immersed in 95% ethanol for 45 min and were
The hydrolysis of xylan-based substrates was carried out in 100 mL conical flask containing 50 mL citrate buffer (pH 5.0, 50 mM), 2.5 g xylan or wheat bran, 5 mg sodium azide and enzyme preparation (1000 nkat) as described by Adsul et al. [25]. The hydrolysis was performed for 6 days at 50 ◦ C, with a stirring rate of 150 rpm. The samples were analyzed for the reducing sugars after suitable time intervals. The hydrolyzed products of xylan was analyzed by the thin-layer chromatography (TLC) using silica gel Plates 60F 254 (E. Merck, Germany). Aliquots (100 l) of the samples were collected at 5, 10, and 30 min, 1, 2, 4, 8, and 12 h of the incubation period and 1 l of the aliquot was spotted on the TCL plates. The plates were subsequently developed with two runs of acetonitrile–water (85:15, v/v) followed by heating for a few minutes at 130 ◦ C in an oven after spraying the plates with a
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Fig. 1. SDS-PAGE and Zymogram analysis. (A) SDS-PAGE analysis of expressed product. M: protein marker; Lane 1–6: the culture supernatant containing recombinant xylanase on day 1, 2, 3, 4, 5, and 6. (B) Xylanolytic activity analysis (Zymogram). Lane 1: culture supernatant before induction; Lane 2–4: culture supernatant after 24, 48, and 72 h induction.
methanol–sulfuric acid mixture (95:5, v/v) [23]. A xylooligosaccharide mixture (Suntory Ltd., Japan) consisting of xylose, xylobiose, and xylotriose was used as the standard. 3. Results 3.1. Preparation of PTXC2 in 2 L bioreactor The recombinant P. pastoris was cultured in 2 L bioreactor. As we expected, the size of recombinant xylanase (PTXC2) determined by SDS-PAGE was 26 kDa (Fig. 1A). PTXC2 was the major protein (over 90% of total protein as detected by densitometer) secreted by P. pastoris into the culture medium. Therefore, the procedure for protein purification was not necessary. Both the highest xylanase activity (1484 nkat/mL) and protein concentration (0.26 mg/mL) in culture supernatant was recorded after 72 h induction (Fig. 2). However, the highest cell wet-weight (32 g/L) was obtained at 102 h. Both xylanase activity and cell wet-weight decreased slightly in the late induction period (after 120 h).
Fig. 2. Time course of xylanase activity produced by recombinant P. pastoris. () Biomass concentration; () xylanase activity.
Fig. 3. Characterization of the recombinant PTXC2. (A) Effect of temperature on the activity of PTXC2. (B) Effect of pH on the activity of PTXC2. (C) The thermostability of PTXC2 at different temperatures was determined by preincubating the enzyme at these temperatures in the absence of substrate for 5, 10, 15, 20, 25, and 30 min before measuring its activity [() 50 ◦ C; () 60 ◦ C; (䊉) 70 ◦ C]. The xylanase activity prior to the preincubations at different temperature was taken as 100%.
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3.2. Determination of xylanolytic activity (Zymogram) When the culture medium (after induction) were subjected to native-PAGE, xylanolytic activity was detected using oat-spelt xylan as substrate; Immersion of the substrate gel in ethanol resulted in precipitation of undegraded xylan, which enhanced contrast and revealed only one clear band against a black background (Fig. 1B). The result confirmed the xylanolytic activity of the hybrid enzyme. 3.3. Effect of pH and temperature on xylanase activity PTXC2 has an optimal activity at 65 ◦ C (Fig. 3A). Activity decreased rapidly with temperature, i.e., at 80 ◦ C, activity of the enzyme represented only 45% of the optimum. Concerning with the effect of the pH, the enzyme showed an optimal activity at approximately pH 6.0 (Fig. 3B). When the pH was below 3.0 and above 9.0, only 30–40% of the maximum activity was reached. PTXC2 was capable of withstanding a temperature up to 60 ◦ C, and the total activity of the enzyme retained more than 85% after 30 min incubating at this temperature (Fig. 3C).
Fig. 4. Effect of different concentrations of Avicel and oat-spelt xylan on the binding ability of PTXC2. Xylanase (25 g) was incubated with 1–24 mg/mL Avicel or oatspelt xylan in 50 mM citrate phosphate (pH 5.0) at 4 ◦ C [Avicel (); Oat-spelt xylan ()].
3.4. Substrate specificity and kinetic parameters The hydrolytic activity of the PTXC2 on various substrates was determined (Table 1). The highest activity (14,980 nkat/mL) was observed with the oat-spelt xylan followed by the birchwood xylan (13,800 nkat/mL) and beechwood xylan (12,490 nkat/mL). The enzyme exhibited low activities towards cellulosic substrates, such as Gellan gum (1450 nkat/mL), Avicel (220 nkat/mL) and CMC (180 nkat/mL). The determined Km and kcat were 1.5 mg/mL and 193.6 s−1 for oat-spelt xylan, and 1.7 mg/mL and 168.2 s−1 for birchwood xylan, respectively. 3.5. Polysaccharide-binding analysis The polysaccharide-binding capacity of PTXC2 was determined by incubating the enzyme with Avicel or oat-spelt xylan. As shown in Fig. 4, PTXC2 could not bind to Avicel, as about 94% enzyme activity still remained in supernatant. However, the enzyme did show its capacity to bind to xylan and 63% of the enzyme activity remained in the supernatant. 3.6. Analysis of hydrolytic capacity and products PTXC2 has been used to hydrolyze substrates at 5% concentration. As shown in Fig. 5, the yield of enzymatic hydrolysis was highest when oat-spelt xylan was used as the substrate (56.4%) followed by birchwood xylan (51%) and beechwood xylan (43%). The wheat bran hydrolysis was lowest (only 31%), but the hydrolysis for both xylans and wheat bran linearly increased with the
Fig. 5. The hydrolysis yield of Oat-spelt xylan (), Birchwood xylan (), Beechwood xylan () and Wheat bran (♦) using PTXC2. Substrates (2.5 g) were incubated with 1000 nkat of the enzyme in 50 mL 50 mM citrate buffer (pH 5.0) and the reaction was carried out at 50 ◦ C with shaking at 150 rpm.
increasing of reaction time. The products of hydrolysis of oatspelt xylan were analyzed by TLC. The predominant hydrolysis end product of oat-spelt xylan was xylotriose (Fig. 6). The xylotriose was produced within 5 min of the reaction period. As the reaction time increased, both the xylotriose and xylobiose concentration increased. These results confirmed that the recombinant Xyn2 was an endo-xylanase. 4. Discussion In recent years, xylanases has attracted considerable research interests because of their use in various applications [3–5]. To create a novel xylanase free of cellulolytic activity and improve the
Table 1 Substrate specificity and kinetic constants of Xyn2 and PTXC2 (all the values are the means of three replications). Substrate
Oat-spelt xylan Birchwood xylan Beechwood xylan Gellan gum Avicel CMC a b c
kcat (s−1 )
Activity (nkat/mL)
Relative activity (%)
Km (mg/mL)
Xyn2a
PTXC2b
Xyn2
PTXC2
Xyn2
PTXC2
Xyn2
PTXC2
875 988 702 143 24 15
14,980 13,800 12,490 1,450 220 180
100 113 80 16 3 2
100 92.1 83.3 9.7 1.5 1.2
3.1 ± 0.2 4.1 ± 0.3 2.7 ± 0.2 NAc NA NA
1.5 ± 0.03 1.7 ± 0.05 1.9 ± 0.02 NA NA NA
87.1 ± 5.4 106.3 ± 8.8 75.4 ± 6.3 NA NA NA
193.6 ± 12.1 168.2 ± 10.5 145.1 ± 9.4 NA NA NA
Xyn2 were obtained from recombinant E. coli. PTXC2 were obtained from P. pastoris. NA, not available.
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Fig. 6. Analysis of the hydrolyzed products. Oat-spelt xylan (40 mg) was incubated with 1000 nkat of the enzyme in 2 mL 50 mM citrate phosphate (pH 5.0) and the reaction was performed at 50 ◦ C for 12 h and then the hydrolyzates were analyzed by TLC.
thermostability, we have previously expressed a hybrid Xyn2 gene in P. pastoris. The thermostabilizing domain (A2) from T. maritima has been successfully introduced into the N-terminal region of T. reesei Xyn2. As a result, the thermostability of the enzyme was improved [15]. In this study, the hybrid enzyme (PTXC2) was prepared from 2 L bioreactor and the enzymatic properties of PTXC2 have been investigated in detail. As shown in Fig. 1A, PTXC2 is the predominant protein (over 90% of purity) in extracellular medium. The molecular mass is 26 kDa as estimated by SDS-PAGE. There is thus a 5-kDa difference in the molecular mass of the xylanase secreted by T. reesei (Table 2). This is caused by the N-terminal fusion of domain A2. As shown in Fig. 1B, this large, fusion protein exhibited a high xylanolytic activity. The highest xylanase activity (1484 nkat/mL) was obtained in 2 L bioreactor after 72 h fermentation (Fig. 2). The expression level is higher than that of levels obtained from recombinant Saccharomyces cerevisiae strain [26] or any E. coli expression system [27,28]. As an unoptimized production level this is still lower than a recombinant T. reesei production system [29]. However, optimization is likely to further improve the production efficiency. For functional characterization, an unpurified enzyme was used and results indicated that even the non-purified enzyme shows highly specific xylanase activity. Therefore, PTXC2 produced in P. pastoris has an advantage of showing xylanase activity that is practically free of harmful side activities. The T. reesei Xyn2 has been reported to have an acidic pH optima (5.0) and a moderate temperature optima (50–55 ◦ C) [10]. Our data indicates that PTXC2 exhibited optimum xylanase activity close to neutral pH value (pH 6.0). It is also noteworthy that PTXC2 was active (over 60% activity retained) over a more wide pH range (3.5–8.0) than T. reesei Xyn2 (4.5–5.5). The altered pH range may be ascribed to the introduc-
Table 2 Comparison of molecular characteristics of PTXC2 and native Xyn2. Property
Native Xyn2a
PTXC2
References
Molecular mass (kDa) Optimum pH pH stabilityb Optimum temp (◦ C) Temp stability (30 min, 60 ◦ C)
21 5.0 4.5–5.5 55–60c NAd
26 6.0 3.5–8.0 65 85e
[10] [10] [10] [4,10] [10]
a b c d e
Produced by T. reesei. More than 60% of maximal activity retained. Temperature optimum of -xylanases produced by T. reesei, not only Xyn2. NA, not available. 85% of total activity retained.
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tion of the domain A2 which stabilizes the molecular structure of the protein and increases the capability of withstanding the harsh conditions (as shown by the improved thermostability). However, future study should be performed to confirm this hypothesis. The temperature optima of PTXC2 was 65 ◦ C, which was 10 ◦ C higher than T. reesei Xyn2. Previous study indicated that T. reesei Xyn2 is not capable of withstanding a temperature over 60 ◦ C [10]. However, the introduction of domain A2 significantly improved the thermostability of T. reesei Xyn2 and the hybrid enzyme retained more than 85% of its total activity after 30 min incubation at 60 ◦ C (Table 2). Our results are in good agreement with previous study which confirmed the thermostabilizing function of domain A2 in T. maritima XynA [4]. The improved thermostability may also be attributed to the N-terminal fusion itself. We have previously expressed the T. reesei Xyn2 gene in E. coli and obtained a recombinant Xyn2 with a fusion peptide (51 amino acids) in N-terminus. When the fusion peptide was deleted, the thermostability of the enzyme acutely decreased [30]. Like with the E. coli produced Xyn2, PTXC2 exhibited a high activity for xylans, but a low activity for cellulosic substrates (Table 1). These results were in good agreement with results from polysaccharide-binding analysis (Fig. 4). When incubating the enzyme with Avicel, more than 94% unbound enzyme was detected in the supernatant. It is noteworthy that the Km values of PTXC2 were lower than E. coli produced Xyn2 when oat-spelt xylan and birchwood xylan was used as the substrate. The results are in accordance with our previous findings that the introduction of domain A2 improved not only the thermostability but also the substrate-binding capacity of T. reesei Xyn2. However, no matter what substrate used, the activity of PTXC2 was always higher than E. coli produced Xyn2 (Table 1). The hydrolytic capacity of PTXC2 on different substrates was also determined. More than 50% hydrolysis was obtained when oat-spelt xylan and birchwood xylan were used as the substrate (Fig. 5). Because of its high purity and enhanced thermostability, PTXC2 might be an attractive enzyme for use in the paper industry. Previous study has already indicated the endo-acting nature of the native Xyn2 [10]. In this study, the products of hydrolysis of oat-spelt xylan (using PTXC2) were analyzed by TLC. Xylotriose was produced within 5 min of the reaction period (Fig. 6) and it was the predominant end product yielded. There were also smaller amount of xylobiose and xylose. The result confirmed the endoacting nature of PTXC2. The xylooligosaccharides (i.e. xylotriose and xylobiose) has been found to be helpful for the maintenance of a healthy intestinal microflora [23]. But, the production of xylooligosaccharides is remaining a time consuming and expensive process. Therefore, PTXC2 may be also useful for the large-scale production of xylooligosaccharides. 5. Conclusions A hybrid xylanase from P. pastoris has been prepared and characterized in this study. The molecular mass of the enzyme was found to be 26 kDa. The enzyme was practically free of cellulolytic activity and showed optimal xylanase activity at pH 6.0 and 65 ◦ C. The enzyme was effectively degrading both oat-spelt xylan and birchwood xylan. Xylotriose was the main end products of oat-spelt xylan hydrolysis. These properties indicated the potential application of the enzyme for xylooligosaccharides production, as well as biobleaching in paper and pulp industry. Acknowledgements This work was granted by Feed Biotechnology Project of Sichuan Province of China with grant No. 2007Z06-050 and Program for
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