Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression, purification and enzymatic characterization in Pichia pastoris

Process Biochemistry 45 (2010) 75–80

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Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression, purification and enzymatic characterization in Pichia pastoris§ Xingzhou Chen, Shunqing Xu, Maosheng Zhu, Luosheng Cui, Hui Zhu, Yunxiang Liang, Zhongming Zhang * State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 June 2009 Received in revised form 5 August 2009 Accepted 10 August 2009

Xylanase is an important industrial enzyme. In this research, to improve the thermostability and biochemical properties of a xylanase from Aspergillus niger F19, five arginine substitutions and a disulfide bond were introduced by site-directed mutagenesis. The wild-type gene xylB and the mutant gene xylCX8 were expressed in Pichia pastoris. Compare to those of the wild-type enzyme, the optimal reaction temperature for the mutant enzyme increased from 45 8C to 50 8C, the half-life of the mutant enzyme extended from 10 min to 180 min, and the specific activity increased from 2127 U/mg to 3330 U/mg. However, the Vmax and Km of the mutant xylanase decreased. The enzyme activity in broth obtained from shake flask cultures could be induced to 1850 U/mL in 7 days, which is higher than results reported previously. Furthermore, the highest achievable enzyme activity was 7340 U/mL from 140 g/L of biomass in a 3 L fermentor used in our study. ß 2009 Elsevier Ltd. All rights reserved.

Keywords: Xylanase Aspergillus niger Site-directed mutagenesis Pichia pastoris Thermostability Bioreactor

1. Introduction Xylan is the main component of hemicellulose in the plant cell walls. Since hemicellulose is the most abundant polysaccharide fibers in nature that is secondary to cellulose, it is an attractive research subject for the development of reusable agricultural residues [1–3]. Xylanases (EC 3.2.1.18) are a set of glycoside hydrolases that randomly hydrolyze a variety of xylans into xylooligosaccharides or D-xylose. Endoxylanase cleaves the internal b-1, 4-bonds of xylan [4]. Based on hydrophobic cluster analysis and amino acid sequence similarity among their catalytic domains, xylanases are classified into family 10 and family 11 (family F and family G in previous references, respectively) [3]. The two families of xylanases differ in molecular weight, electric charge, and enzymatic properties. Generally, members in family 10 are active on aryl cellobiosides. In contrast, members in family 11 have a lower catalytic versatility and are exclusively active on substrates containing sole D-xylose [3]. However, xylanases in both families hydrolyze aryl #b-glycosides of xylobiose and xylotriose at the aglyconic bond [3].

§ This work was supported by the National Natural Science Foundation of China (Grant No. 30870186 to Z.Z.), by the National Basic Research Program (973) of China (Grant No. 2009CB724700 and Grant No. 2010CB126502 to Z.Z.). * Corresponding author. Tel.: +86 27 87281687; fax: +86 27 8728 0670. E-mail addresses: [email protected] (X. Chen), [email protected] (Z. Zhang).

1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.08.009

Xylanases not only have a wide array of biotechnological and industrial applications in processes such as food, animal feed, paper pulp bleach and functional xylooligosaccharides production [1,5–9], but also have great potentials in the bioconversion from lignocellulosic feedstocks to fuel-grade ethanol, which have attracted tremendous interests in the past few years. Different forms of xylanases have been isolated and characterized from various microorganisms including bacteria and fungi [2,4,5,7]. However, native xylanases isolated from their original hosts are limited in functionalities to meet all industrial requirements due to their drawbacks in thermostability, proteolysis susceptibility, reactivity to metal ions, acidity and alkalinity, and so on [10]. To further increase the potentials of xylanases in industrial applications, some molecular biology methods are introduced to improve their enzymatic structures and activities. Heterologous recombinant protein expression is an efficient strategy to obtain purified xylanases with high enzymatic activities, especially for the production of xylanase without cellulose activity. Escherichia coli and Pichia pastoris are the most commonly used heterologous protein expression systems. Up to now, some recombinant xylanase have been expressed in both systems [11–16]. However, enzyme production at a commercially compatible scale had only been carried out in yeast systems [15,17]. A hyperthermostable variant EvXyn11TS that contains seven mutations is obtained by screening a shuffling library. This variant has similar catalytic properties, but a higher melting temperature (Tm) of 25 8C than its parental enzyme. Similarly, 15 thermostable mutants of

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native xylanases have Tms that are 1–8 8C higher than their parental enzymes [18]. Using forced protein evolution, a treble mutant of xylanase (D262N/A80T/R347C) of family 10 has been generated from Cellvibrio japonicus. In the absence of calcium, the mutant is more thermostable than the wild-type CjXyn10A [19]. Besides thermostability, enzymatic activity and optimum pH and temperature of xylanases can also be improved by some critical amino acids substitutions [20–22]. Two mutant xylanases from Bacillus circulans, Q167M and R73V that have higher optimum pH than the wild type, have been generated by molecular modeling [23]. These facts suggest that the substitutions of amino acid residues and the introduction of disulfide bonds are useful in changing the enzymatic activity or/and catalytic properties of xylanases. In this present research, a xylanase gene xylB was cloned from Aspergillus niger F19 strain. To improve the thermostability and biochemical properties of the xylanase, seven amino acid residues were substituted by site-directed mutagenesis. The wild-type gene xylB and the mutant gene xylCX8 were then introduced and expressed in P. pastoris. Enzymatic characterization indicated that the mutant xylanase was improved in optimal reaction temperature, half-life, specific activity and proteolysis properties. 2. Materials and methods 2.1. Plasmids, microorganisms and culture conditions A. niger F19 (a present from professor Liang Yunxiang, Huazhong Agricultural University) was cultured in liquid potato dextrose (PD) medium on a rotary shaker (200 rpm) at 28 8C. E. coli strain DH10B was used as a host for gene cloning and plasmid propagation. The host of heterologous expression, P. pastoris GS 115 (Invitrogen, USA) was grown in YPD (peptone, 2%; glucose, 2%; and yeast extract, 1%). The cloning vector pMD18-T (TaKaRa, Japan) was used for cloning and sequencing of PCR fragments. Yeast expression vector pPIC9K (Invitrogen, USA) was used for heterologous expression of xylanases in P. pastoris. All oligonucleotides used in this research were synthesized by Beijing AuGCT biotechnology Co., Ltd., China. 2.2. Total RNA and genomic DNA extraction from A. niger Total RNA from A. niger F19 was extracted using Trizol (Invitrogen, USA) according to the laboratory manual of molecular cloning [24] and the supplier’s recommendations. Genomic DNA was isolated according to the molecular cloning manual [24]. Briefly, Aspergillus spores were eluted and suspended in sterile water. Spore suspension (1 mL) was inoculated in 100 mL liquid PD medium and cultured for approximately 36 h at 200 rpm. To induce and enhance the transcription of the xylanase B gene, 1.5% oat spelt xylan was added to the culture medium (Sigma, German). Mycelial pellets were collected by filtration after approximately 36 h of incubation. 2.3. Cloning and sequence analysis of the xylanase B gene First strand cDNA was synthesized from 1 mg of total RNA using RNA PCR Kit (AMV) Ver. 3.0 (TaKaRa, Japan). According to the endoxylanases gene sequence of A. niger in GeneBank (AY551187), a forward primer P1F (50 -GAATTCATGTCGACCCCGAGCTCGAC-30 ) and a reverse primer P2R (50 -CTCGAGTCACTGAACAGTAATGGAGGAAG-30 ) were designed for the synthesis of the second strand cDNA. P1F was located upstream of the coding region without signal sequence because the xylanase gene was designed to fusing with the yeast a-factor for secretion. A 567bp and a 634-bp PCR fragments were amplified with P1F and P2R using cDNAs and genomic DNA as the template, respectively. The two fragments were then cloned into pMD18-T and sequenced (AuGCT, Beijing, China). 2.4. Site-directed mutagenesis of the xylanase B gene The analogous three-dimensional structure (3-D structure) of the xylanase was obtained online (http://swissmodel.expasy.org/) [25]. The surface amino acid residues were analyzed with biology software (RasMol Version 2.7.3.1). Another Software (Disulfide by Design V1.20) was used to analyze the feasibility of introducing a disulfide bond in the 3-D structure of xylanase. These analyses suggested that a disulfide bond could be introduced by substituting two amino acid residues (S33C, T186C). Five additional amino acid residues were substituted with arginine (S39R, S54R, T101R, T105R and T150R) to improve the thermostability of the xylanase. These mutations were generated by overlapping-PCR. The mutant xylCX-8 gene with the mutations was then cloned into pMD18-T and sequenced to verify the targeted substitutions.

2.5. Construction and transformation of the yeast shuttle vector for xylanase heterogeneous expression Wild-type gene xylB and the mutant gene xylCX8 were subcloned into the shuttle vector pPIC9K, respectively, resulting in two yeast expression plasmids pPIC9K-xylB and pPIC9K-xylCX8. The expression plasmids (10 mg each) were linearized using salI and introduced into P. pastoris strain GS115 according to the supplier’s protocol (Invitrogen, USA). The recombinant yeasts were selected on RDB plates without arginine [18.6% sorbitol, 1.34% YNB (Sigma, USA), 2% dextrose, 0.4 ppm biotin, amino acid mixture (0.005% Glu, Met, Lys, Leu and Ile each), and 2% agar]. The methanol utilization phenotype of the transformants was identified on MD plates (1.34% YNB, 2% dextrose, 0.4 ppm biotin, and 2% agar) and MM plates (1.34% YNB, 0.5% [v/v] methanol, 0.4 ppm biotin, and 2% agar). Transformants with similar growth rates on MD and MM plates had a fast methanol utilization phenotype (Mut+), while transformants with different growth rates on MD and MM plates had a slow methanol utilization phenotype (Mut). To verify the integration of the gene of interest into the host genomic DNA, PCR analysis was carried out using total DNA of the transformants as templates. The total DNA of the transformants was isolated according to the standard glass beads method [24]. The activity of the recombinant xylanase was detected on MM plates with xylan. 2.6. Xylanase activity assays and determination of protein concentrations Xylanolytic activity was quantified using 3,5-dinitrosalicyclic acid (DNS) method as described by Bailey et al. [26]. Pure xylose (Sigma, USA) was used as the standard reducing sugar to generate the standard curve. As the substrate for enzymatic reactions, 1% oat spelt xylan (w/v) (Sigma, USA) was dissolved in sodium citrate buffer (50 mM, pH 5.0) at 50 8C with agitation for 10 min before each assay. The amount of hydrolytic reducing sugar was determined by the absorption of visible light at 540 nm using the protein analyzer DU800 (Beckman, USA). One international unit of enzyme activity was defined as the amount of the enzyme required for releasing 1 mmol xylose or equivalent reducing sugar per minute. Specific activity was defined as the amount of enzymatic units in each mg of enzyme protein (U/mg). Protein concentration was determined using Bradford assay [27], and bovine serum albumin (BSA) was used as the standard protein sample for the standard curve. 2.7. Expression and purification of recombinant xylanases in P. pastoris Several transformants were cultured in 10 mL liquid YPD medium at 200 rpm and 30 8C. For small-scale xylanase production, cells were collected by centrifugation (3500 rpm, 3 min) at OD600 2–6 and transferred into 10 mL liquid BMMY medium (10 g/L yeast extract, 20 g/L peptone, 13.4 g/L YNB, 0.0004 g/L biotin, 5 mL/ L methanol, and 0.1 M phosphate buffer pH 7.0). The induction was lasted for 72 h with the addition of 0.5% (v/v) methanol every 24 h. The amount of enzymatic activity units in broth was measured in order to screen for the recombinant yeast strains that produced xylanases with high activities. Crude enzyme was obtained by ammonium sulfate sedimentation. The expression of secreted proteins was analyzed by SDS-PAGE gel electrophoresis [24]. Based on the xylan-binding site in the xylanase 3-D structure, the procedure of recombinant xylanase purification was carried out using the xylan-binding method described previously with modifications [28]. Oat spelt xylan was dissolved in low salt buffer (20 mM Tris–Cl, 50 mM NaCl, pH 8.0) at 10% final concentration. The isometric crude protein and xylan solution were mixed to bind for more than 1 h on ice. Xylanase was transiently bind to xylan, and other proteins and free target enzymes were removed from the crude protein with low salt buffer. The pellet was collected by centrifugation for 5 min (5000 rpm, 4 8C) and eluted with high salt buffer (20 mM Tris–base, 100 mM NaCl, pH 8.0). The eluate was dialysed to concentrate the enzyme. Concentrated xylanase was then characterized. 2.8. Characterization of enzymatic properties To determine the optimum temperature for hydrolysis, the activities of the purified wild type and mutant xylanases were measured individually with the xylan substrate under standard pH (5.0) after 10 min of reaction at different temperatures ranging from 30 8C to 80 8C. The temperature at which peak xylanase activity was detected was the optimal temperature. The thermostability of xylanase was determined by measuring the residual enzymatic activity after incubating an aliquot of the enzyme at 50 8C for different time periods without substrate. The longest incubation time was 5 h. The residual activity was measured according to the standard method at the optimal temperature. The kinetic parameters Km and Vmax were deduced according to Michaelis– Menten equation using the Lineweaver–Burk double-reciprocal plots. The initial reaction velocity of the enzyme was deduced using the enzymatic activities at the optimal temperature corresponding to a series of different substrate concentrations (oat spelt xylan) ranging from 2 mg/mL to 50 mg/mL. To evaluate the hydrolyzing properties of enzymes, hydrolysates were analysed according to the relative accumulation of the reducing sugar. Aliquots of xylan were

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Table 1 Induction media for optimal xylanase production. Medium type

N BN NT BNT YP BYP YPT BYPT YPN BYPN YPNT BMMY

Component

Potassium phosphate

Yeast extract

Peptone

YNB

Biotin

Methanol

Abbreviation Amount

B 0.1 M  +  +  +  +  +  +

Y 1%     + + + + + + + +

P 2%     + + + + + + + +

N 1.34% + + + +     + + + +

T 4  105%   + +   + +   + +

M 0.5% + + + + + + + + + + + +

Note: +, the presence of the ingredients in the medium; , the absence of the ingredients in the medium. hydrolyzed by the wild type and mutant xylanases at the optimal temperature, respectively for different time periods ranging from 5 min to 60 min. The amount of accumulated reducing sugar after 5 min of reaction by the wild type or mutant enzyme was defined as the standard amount for evaluation of hydrolytic quality, respectively. Hydrolysate accumulation was plotted against the corresponding amount of reducing sugar at different time period. 2.9. Large-scale production of recombinant xylanase in P. Pastoris For large-scale production of the recombinant xylanase, the recombinant P. pastoris was grown in various isometric media (Table 1) and the enzyme activities in culture broth were measured in order to determine the optimal production medium. Larger scale production was carried out in a 3 L fermenter (New Brunswick Scientific BioFlo 110) using BYPN medium (for ingredient see Table 1). The culture was initially grown in 2 L YPD medium at 30 8C with agitation at 500 rpm for 16–20 h. Cells were then pelleted, re-suspended in 2 L BYPN medium, and inoculated into the bioreactor with batch feedings of absolute methanol. The feeding rate was 1 mL/h/L initial fermentation volume during the first 2–3 h and increased to 2 mL/h/L for the following 2 h. The feeding rate was further increased to 4 mL/h/L for the remainder of the fermentation. The induction period lasted for 150 h during which the temperature and pH were maintained at 30 8C and 5.5, respectively. Agitation rate was kept at 600–800 rpm to allow a relative percentage of dissolved oxygen (DO) of above 20%. If DO decreased to lower than 20%, the methanol feeding stopped. The DO was maintained above 20% by increasing agitation or pressure. Every 12 h, 10 mL fermentation solution was collected and centrifuged for 1 min at 12,000 rpm, and cell pellets were weighed to evaluate wet cell mass over time. The broth was kept at 4 8C for analysis.

3. Results 3.1. Sequence analysis and site-directed mutagenesis of xylB Using DNA alignment (DNAMAN, http://www.bioguider.com), it was revealed that the PCR fragment amplified from genomic DNA

of A. niger F19 was 67-bp longer than the cDNA. The size difference indicated that the coding region was interrupted by a 67-bp intron. Sequence of the xylanase gene was deposited in GeneBank (EU430370). The analogous 3-D structure analysis showed that the catalytic center of the enzyme was located in a cleft formed in the middle of the overall-sandwich structure. Several hydrophilic amino acid residues (threonine and serine) were found on the surface of the protein. According to the analysis, five arginine substitutions (S39R, S54R, T101R, T105R and T150R) were introduced by overlapping-PCR. In addition, consideration with the energy distribution and the effect on the xylanase conformation caused by introduction of disulfide bonds, a disulfide bond was introduced by two amino acid residue substitutions (S33C and T186C). The analogous 3-D structure of the mutated protein CX8 indicated that these modifications were unlikely to cause conformational changes (data not shown). 3.2. Expression and purification of the wild type and recombinant xylanase B in P. pastoris To screen the recombinant yeast for xylanase gene integrations, PCR was carried out using genomic DNA of yeast transformants as the templates. Mutant xylCX8 gene was integrated into the genomic DNA of the host strain (Fig. 1B). In addition, semitransparent hydrolysis halo around colonies of the transformants was observed on the MM plate containing xylan, which suggested that recombinant xylanases were active in P. pastoris and secreted into the medium (Fig. 1A). SDS-PAGE analysis indicated that the

Fig. 1. P. pastoris transformants confirmation, xylanase expression and purification. (A) Expression and enzyme activity of recombinant xylanase in P. pastoris on MM plate containing xylan. I: xylCX8 transformant; II: xylB transformant; III: pPIC9K transformant; IV: P. pastoris wild-type strain; (B) PCR analysis of yeast transformants. Xylanase genes were amplified from pPIC9K-xylB (xylB, lane 1), pPIC9K-xylCX8 (xylCX8, lane 2), no template control (lane 3), P. pastoris wild-type strain (lane 4), pPIC9K transformant (lane 5), pPIC9K-xylB transformant (lane 6), and pPIC9K-xylCX8 transformant (lane 7). M: DNA marker; (C) SDS-PAGE of the recombinant xylanases. Lanes show proteins from P. pastoris wild-type strain culture broth (lane 1), proteins from pPIC9K transformant culture broth (lane 2), crude xylB protein (lane 3), protein from xylB transformant culture broth (lane 4), purified xylB protein (lane 5), crude xylCX8 protein (lane 6), purified xylCX8 protein (lane 7), and protein from the xylCX8 mutant culture broth (lane 8). M: protein molecular marker.

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Fig. 2. (A) The optimum temperature of the wild type and mutant xylanases. The optimum temperatures of the wild type and the mutant xylanase were 45 8C and 50 8C, respectively; (B) thermostabilities of the wild type and mutant xylanases. Half-life of the two enzymes was shorter than 10 min and 3 h, respectively; (C) exponential accumulation of reducing sugar during hydrolysis catalyzed by mutant xylanase at optimal temperature. The accumulation of reducing sugar resulted from wild-type xylanase activity increased gradually in the first 20 min of reaction. (D) Highest enzymatic activity in shake flask was induced in BYPN medium.

molecular weight of the mutant xylanase protein was approximately 21 kDa, which was identical to the predicted value (Fig. 1C, lanes 6–8). The wild-type xylanase gene xylB was also integrated (Fig. 1B, lane 6) and expressed as expected (Fig. 1C, lanes 3–5). On the contrary, the host strain and transformants containing pPIC9K did not express protein of the same size (Fig. 1C, lanes 1 and 2). The wile-type and mutant xylanase crude proteins were a quite purity after initial xylan-binding purification and concentrated by ammonium sulfate precipitation (Fig. 1C, lanes 3 and 6, respectively). In order to evaluate the enzyme characteristics, the wild type and recombinant xylanases were further purified using the xylan-binding method (Fig. 1C, lanes 5 and 7 respectively). 3.3. Enzymatic characterization of the recombinant xylanase The optimal reaction temperature of the wild type and recombinant xylanases were 45 8C and 50 8C, respectively (Fig. 2A), which indicated that the thermotolerance of the recombinant xylanase was increased. The activity of the wild-type enzyme decreased rapidly during the reaction, and was lost completely after 20 min (Fig. 2B). On the contrary, the activity of the recombinant enzyme decreased at a lower rate under the same condition, and 27.7% of the activity remained after 300 min (Fig. 2B). The activity of the wild-type enzyme decreased to 39.9% after 10 min, while 50% of the activity of the recombinant enzyme retained after 180 min of reaction (Fig. 2B). Therefore, the half-life of the recombinant xylanase was approximately 180 min, while that of the wild-type enzyme was

less than 10 min (Fig. 2B). Thus the thermostability of the xylanase was significantly improved after site-directed mutagenesis. The Kms of the wild type and recombinant xylanases were 56.9 mg/mL and 37.2 mg/mL, respectively. The Vmax of the wild type and recombinant xylanases were 82.9 mmoL/mL/min and 27.0 mmoL/mL/min, respectively (Table 2). Specific activity of the xylanase was also increased from 2127 U/mg to 3330 U/mg after mutations (Table 2). 3.4. Improvement of catalytic property of the recombinant xylanase During hydrolysis reaction catalyzed by the wild-type xylanase, the relative accumulation level of the reducing sugar increased up to 136.9% at 20 min, and remained unchanged thereafter due to the complete loss of enzyme activity (Fig. 2C). Compared to the wild-type xylanase, the reducing sugar level resulted from the recombinant enzyme-mediated hydrolysis increased exponentially throughout the entire reaction period to 376.3% (Fig. 2C). Therefore, the catalytic property of the Table 2 Properties of the wild type and mutant xylanases. Properties

Wild type

Mutant CX8

Topt (8C) t1/2 (min) Specific activity (U/mg) Km (mg/mL) Vmax (mmol/mL min mg)

45 <10 2127.9  83.9 56.9 82.9

50 180 3330.9  173.4 37.2 27.0

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recombinant xylanase was improved after site-directed mutagenesis. 3.5. Large-scale production of the recombinant xylanase in P. pastoris Preliminary experiments indicated that the BYPN medium was the optimal inducing medium, because highest xylanase activity of 208 U/mL was detected in the broth (Fig. 2D). In shake flask cultures, the recombinant xylanase production was induced and the enzyme activity was 1850 U/mL at 7 days post inoculation. During fed-batch cultivation, the cell density increased 3-fold from 45 g to 140 g (wet weight)/L after 24 h of cultivation with methanol feeding. The induction phase lasted for 150 h and the highest xylanase activity obtained was 7340 U/mL, which was almost 4 times higher than that of the shake flask cultures.

4. Discussion Xylanase isolation and purifications have attracted increasing attentions in the past two decades. Because the current production model failed to meet the requirement of the increasing demands in quantity and a wider range of industrial processes, heterologous expression becomes an alternative method to produce industrial enzymes. To date, E. coli and P. pastoris are the most common expression platforms for industrial enzyme productions. Generally, bacteria are not perfect expression systems due to complicated downstream processing and high purification cost. In contrast, recombinant proteins expressed in P. pastoris are secreted into the medium and can be purified easily at a lower cost. In addition, P. pastoris enables some post-translation modifications including the assembly of disulfide bonds, the exclusion of signal peptides, and glycosylation, etc. Therefore, P. pastroris is a preferable production platform, provided enough expression level. In this research, both the wild-type xylB and the mutant xylCX8 genes were expressed at high levels in P. pastoris. SDS-PAGE analysis indicated that the recombinant xylanases were secreted into medium as a major protein (Fig. 1B), and the expression level and purification strategy are appropriate for industrial production of the enzymes. Previous studies have demonstrated that substitutions of amino acids on the surface of some enzymes with arginines lead to the increase in thermotolerance of the protein without causing conformational changes [20,21,30,31,32]. According to structural analysis, a few arginine substitutions on the surface likely have similar effect on the xylanase from A. niger. On the other hand, favorable disulfide bond assembly also has an important effect on protein thermostability [29,31–33]. Aberrant disulfide bond assembly could change molecular conformation and result in decreased enzymatic activity. Our data showed that the recombinant enzyme has improved thermotolerance and thermostability (Figs. 2A and 2B) due to five arginine substitutions and the introduction of a disulfide bond. The improvement of the thermotolerance and thermostability resulted in an increased specific activity of the purified recombinant enzyme from 2127 U/mg to 3330 U/mg. To our knowledge, the activity of the recombinant xylanase reported here is higher than those of a majority of the recombinant xylanases reported previously [11–16]. On the other hand, the inducing level of the recombinant enzyme in shake flask cultures reaches 1850 U/ml under optimal condition and is also higher than those reported previously [11–16]. Although the wet cell weight of the yeast cells was only 140 g/L in our first trial when cells were grown in the fermentor, the yield of the recombinant enzyme were 7340 U/mL. Since a 500 g/L biomass can be achieved with express high-density

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fermentation technology, it is promising to produce the recombinant xylanase commercially. The Vmax of the wild-type xylanase was much faster than that of the mutant (Table 2). However, the accumulation of the relative reducing sugar in the mutant was significantly higher than that of the wild-type strain throughout the hydrolysis process. Apparently, the hydrolysis of mutant xylanase could be lasted for much longer time than the wild enzyme. Since the Km is defined as the concentration of the substrate when the current reaction rate reaches half of the Vmax, lower Km indicates lower remaining substrate concentrations at a given time during the reaction. It is suggested that the mutant xylanase hydrolyses xylan more efficiently than the wild-type xylanase. In conclusion, our results indicated that a series of site-directed mutagenesis resulted in the improvement of the thermostability, specific activity, and catalytic properties of xylanase from A. niger F19, which are important for increasing the utilization efficiency of hemicellulosic materials. Although the mechanism of the improvement is unknown, this study provides a prelude for large-scale commercial production of xylanases. Acknowledgements We thank Professor Yunxiang Liang for kindly providing A. niger strain F19. This work was supported by the National Natural Science Foundation of China (Grant No. 30870186 to Z.Z.), by the National Basic Research Program (973) of China (Grant No. 2009CB724700 and Grant No. 2010CB126502 to Z.Z.). References [1] Beg QK, Kapoor M, Mahajan L, Hoondal GS. Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 2001;56:326–38. [2] Fang HY, Chang SM, Lan CH, Fang TJ. Purification and characterization of a xylanase from Aspergillus carneus M34 and its potential use in photoprotectant preparation. Process Biochem 2008;43:49–55. [3] Collins T, Gerday C, Feller G. Xylanases, xylanase families and extre mophilic xylanases. FEMS Microbiol Rev 2005;29:3–23. [4] Kim DY, Han MK, Lee JS, Oh HW, Park DS, Shin DH, et al. Isolation and characterization of a cellulase-free endo-b-1,4-xylanase produced by an invertebrate-symbiotic bacterium, Cellulosimicrobium sp. HY-13. Process Biochem 2009;44:1055–9. [5] Katapodis P, Nerinckx W, Claeyssens M, Christakopoulos P. Purification and characterization of a thermostable intracellular b-xylosidase from the thermophilic fungus Sporotrichum thermophile. Process Biochem 2006;41:2402– 9. [6] Polizeli MLTM, Rizzatti ACS, Monti R. Xylanases from fungi properties and industrial applications. Appl Microbiol Biotechnol 2005;67:577–91. [7] Sandrima VC, Rizzattib ACS, Terenzib HF, Jorgeb JA, Milagresc AMF, Polizeli MLTM. Purification and biochemical characterization of two xylanases produced by Aspergillus caespitosus and their potential for kraft pulp bleaching. Process Biochem 2005;40:1823–8. [8] Savitha S, Sadhasivam S, Swaminathan K. Application of Aspergillus fumigatus xylanase for quality improvement of waste paper pulp. Bull Environ Contam Toxicol 2007;78:217–21. [9] Zhao J, Li XZ, Qu YB. Application of enzymes in producing bleached pulp from wheat straw. Bioresour Technol 2006;97:1470–6. [10] Iyer PV, Ananthanarayan L. Enzyme stability and stabilization—aqueous and non-aqueous environment. Process Biochem 2008;43:1019–32. [11] Chantasingh D, Pootanakit K, Champreda V, Kanokratana P, Eurwilaichitr L. Cloning expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris. Prot Expres Purif 2006;46:143–9. [12] Cheng YF, Yang CH, Liu WH. Cloning and expression of Thermobifida xylanase gene in the methylotrophic yeast Pichia pastoris. Enzyme Microb Technol 2005;37:541–6. [13] Deng P, Li D, Cao YH, Lu WQ, Wang CL. Cloning of a gene encoding an acidophilic endo-b-1,4-xylanase obtained from Aspergillus niger CGMCC1067 and constitutive expression in Pichia pastoris. Enzyme Microb Technol 2006;39:1096–102. [14] Huang JL, Wang GX, Xiao L. Cloning, sequencing and expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli. Bioresour Technol 2006;97:802–8. [15] Liu M, Weng XY, Sun JY. Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan. Protein Expr Purif 2006;48:292–9. [16] Wang YR, Zhang HL, He YZ, Luo HY, Yao B. Characterization, gene-cloning, and expression of a novel xylanase XYNB from Streptomyces olivaceoviridis A1. Aquaculture 2007;267:328–34.

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