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Li, F., Yang, S., Zhao, L., Li, Q., and Pei, J. (2012). "Synonymous condon usage bias and overexpression of a synthetic xynB gene from Aspergillus niger NL-1 in Pichia pastoris," BioRes. 7(2), 2330-2343.


To further improve the expression level of recombinant xylanase in Pichia pastoris, the xynB gene, encoding the mature peptide from Aspergillus niger NL-1, was designed and synthesized based on the synonymous condon bias of P. pastoris and optimized G+C content. 155 nucleotides were changed, and the GC content decreased from 57.7% to 43.6%. The synthetic xynB was inserted into the pPICZaA and then integrated into P. pastoris GS115. The activity of the recombinant xylanase reached 1414.7 U/mL, induced with 0.8% methanol after 14-day cultivation at a temperature of 28oC in shake flasks, which was 267% higher than that of the native gene. Furthermore, the maximum xylanase activity of 20424.2 U/mL was obtained by high-density fermentation in a 5-L fermenter, which was the highest xylanase expression in P. pastoris yet reported. The recombinant xylanase had its optimal activity at a pH of 5.0 and temperature of 50oC. The recombinant xylanase was stable over a pH range of 4.5 to 8.0. Thus, this report provides an industrial means to produce the recombinant xylanase in P. pastoris.

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Fei Li,a,b Shiyi Yang,a,b Linguo Zhao,a,b,* Qi Li,a,b and Jianjun Pei a,b

To further improve the expression level of recombinant xylanase in Pichia pastoris, the xynB gene, encoding the mature peptide from Aspergillus niger NL-1, was designed and synthesized based on the synonymous condon bias of P. pastoris and optimized G+C content. 155 nucleotides were changed, and the GC content decreased from 57.7% to 43.6%. The synthetic xynB was inserted into the pPICZaA and then integrated into P. pastoris GS115. The activity of the recombinant xylanase reached 1414.7 U/mL, induced with 0.8% methanol after 14-day cultivation at a temperature of 28oC in shake flasks, which was 267% higher than that of the native gene. Furthermore, the maximum xylanase activity of 20424.2 U/mL was obtained by high-density fermentation in a 5-L fermenter, which was the highest xylanase expression in P. pastoris yet reported. The recombinant xylanase had its optimal activity at a pH of 5.0 and temperature of 50oC. The recombinant xylanase was stable over a pH range of 4.5 to 8.0. Thus, this report provides an industrial means to produce the recombinant xylanase in P. pastoris.

Keywords: Endoxylanase xynB; Synonymous condon; Synthesis; Over expression; Aspergillus niger

Contact information: a: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China; b: Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, Jiangsu, 210037, China; *Corresponding author:


Lignocellulosic biomass is considered a prime alternative to fossil fuels and a source for many of our fuel and chemical feedstock needs. Xylans, as the most abundant hemicellulose of the plant cell wall, consists of a -1,4-linked D-xylose polymer with arabinosyl-, acetyl-, and/or 4-0-methylglucurosyl side branches (Li and Ljungdahl 1994), which represent a major renewable carbon resource in nature (Haki and Rakshit 2003). Among xylan-degrading enzymes, endo-1,4--D-xylanases (EC are key enzymes for random cleavage of the xylan backbone (Huang et al. 2006) and hence have broad industrial significance because they have broad uses and potential applications, including biopulping and biobleaching in the pulp and paper industry, bioconversion of lignocellulose material to fermentative products, feed additive of animals, and use in the brewing industry (Liu and Liu 2008; Qiu et al. 2010; Khandeparker and Numan 2008). Therefore, the topic of producing high-activity and low-cost xylanase has become hot in these fields.

Xylanases are mainly produced by bacteria and fungi (Sunna and Antranikian 1997). Aspergillus niger is a well known fungus that produces multiple xylanases with different physicochemical properties (Krengel and Dijkstra 1996). In a previous study conducted in our laboratory, a specific endo--1,4-xylanase obtained from A. niger NL-1 was identified as an excellent feed additive for broilers (Li et al. 2010). However, the application of the xylanase was hampered by the presence of several other enzymes, such as cellulase, -glucosidase, and -xylosidase, as well as the products of the enzymes. Large-scale production of recombinant xylanase has been facilitated with the advent of genetic engineering. Genes encoding xylanases have been cloned in homologous and heterologous hosts with the objectives of overproducing the enzyme (Baba et al. 1994; Ahmed et al. 2009).

In this study, the xynB gene was optimized and synthesized to match the codon preference of Pichia pastoris, and the recombinant enzyme was characterized. The induction conditions of expression and fermentation strategy were also discussed.


Bacterial Strains, Plasmids, and Growth Media

A. niger NL-1 was isolated from a soil sample collected in a suburb of Nanjing for its activity of hemicellulase, which was preserved by our laboratory and was grown at 30oC in YPD medium. Escherichia coli TOP10F’ and the vector pMD-19T (TaKaRa, Japan) were used for general DNA manipulations and for DNA sequencing. P. pastoris GS115 (his4) and the expression vector pPICZA (Invitrogen, USA) were used for the heterologous expression of the optimized xylanase xynB. The recombinant strain, pPICzαA-xynB, was constructed as described in a previously published paper by our laboratory (Li et al. 2010).

The DNA purification kit, restriction endonucleases, T4 DNA ligase, and Primerstar DNA polymerase were purchased from TaKaRa. Birchwood xylan was obtained from Sigma Chemical Company (USA). All other chemicals were of analytical grade. GenScript synthesized the primers.

Luria-Bertani (LB) medium, YPDS medium, minimal dextrose (MD) medium, minimal methanol (MM) medium, buffered glycerol complex (BMGY) medium, fermentation basal salts medium (BSM), and PTM1 trace salts solution were prepared according to the manual of Pichia Expression Kit (Invitrogen 2002).

Optimization and Oligonucleotide Design for Synthesis of Xylanase Gene

The gene sequence of xylanase xynB obtained by our laboratory (Li et al. 2010) was selected and optimized according to codon usage bias of P. pastoris, GC content, repeat sequence, and RNA instability motif ( About 28 oligomers, with overlap regions with their neighbors, were designed to synthesize the optimized xynB gene based on DNAWorks (Hoover and Lubkowski 2002; Rouillard et al. 2004), as shown in Table 1. Overlap melting temperatures were designed to be 55 to 60oC. The P1 and P28 oligomer were designed to contain an EcoR I site and an Xba I restriction site, respectively.

Table 1 Sequence of Oligomers Used in Synthesis of Codon-Optimized xynB

Synthesis of xynB Gene and Construction of the Expression Vector

The 624 bp mature peptide domain of xynB was reconstructed in a two-step procedure. The initial primer extension step was performed in 50 L reaction volumes containing 5 L oligonucleotides P1 to P28 (100 nM each) mixture, 10 L 10 × ExTaq DNA polymerase buffer, 2.5 U ExTaq polymerase, and 200 M each of dNTPs and 2 mM MgCl2 under the following PCR conditions: a hot start at 94oC for 5 min, 18 cycles of 94oC for 30 seconds, 59oC for 30 seconds and 72oC for 50 seconds, followed by one cycle of 72oC for 10 min. To amplify the target fragment, 2 L of the product resulting from the first PCR was used for the second step with forward primer P1 and the reverse primer P28. The concentration of ExTaq polymerase was as above. The PCR parameters were: denaturation at 94oC for 5 min first; 30 cycles of (30 seconds at 94oC, 30 seconds at 64oC, 50 seconds at 72oC); followed by 10 min at 72oC. The PCR product was gel purified and cloned into pMD-19T vector, and the resulting plasmid pMD-19T-SxynB was then transformed into E. coli TOP10F′cells for sequencing.

For the expression of syn-xynB in P. pastoris, the synthetic xynB sequence was amplified by PCR from the cloning plasmid pMD-19T-SxynB using the primers P1 and P28. The PCR product was gel purified and digested with EcoR I and Xba I before cloning into the plasmid pPICZA vector at restriction sites EcoR I and Xba I, resulting in the recombinant plasmid pPICZA-SxynB. After being transformed into E. coli TOP10F’, the transformants were cultured in LLB medium containing 25 g/mL Zeocin. The positive recombinant vector containing the syn-xynB fragment was confirmed via resequencing.

Expression of syn-xynB in P. pastoris and Mut Phenotype Selection

The recombinant plasmid pPICZA-SxynB was linearized using BstX I and transformed into P. pastoris GS115 competent cells by electroporation according to the Pichia expression vectors manual (Invitrogen 2002). The transformed cells were selected on the basis of Zeocin resistance using YPDS plates containing 500 g/mL of this antibiotic at 28oC until colonies appeared. The single colonies were picked and transferred to MM and MD plates, respectively, to identify the Mut phenotype using GS115/Muts Ablumin and GS115/pPICZ/lacZ Mut+ as parallel strains.

The selected clones were tested for expression of xylanase in a shake flask with 30 mL BMGY medium at 28oC with constant shaking at 180 rpm according to the manufacturer instructions (Invitrogen). To maintain induction, 100% methanol was added to the culture to a final concentration of 0.5% (v/v) every 24 hours. The xylanase activity was measured every 24 hours according to standard methods. The recombinant strain with the best expression performance was then used for further studies.

Optimization of Syn-xylanase Expression in BMGY Medium

The recombinant strain was incubated in 10 mL of BMGY medium for 48 hours in 28oC with constant shaking at 180 rpm. When OD600 reached between 2 to 6, the cultures were harvested and resuspended in 30 mL of BMGY until an OD600 of 1.0 was reached for the shaking culture at 28oC with a constant shaking at 180 rpm. Methanol (100%) was added daily (final concentration 0.5% (v/v)) to maintain induction. The samples were centrifuged at 12,000 rpm for 5 minutes. The supernatant was stored at 4oC to determine enzyme activity.

Maintaining all factors at constant levels, except for the one being studied, the culture medium and culture condition were optimized for xylanase production. The expression at a different initial pH (ranging from 3.0 to 8.0) was set. Induction was continued with the addition of methanol to achieve concentrations ranging from 0.5% to 1.5% (v/v) at every 24 hours to sustain the expression after incubation for 48 hours. The effect of different histidine concentrations (0.05%, 0.1%, 0.2%, 0.3%, and 0.5%) on expression of xylanase was studied.

High-Cell-Density Fermentation in a 5 L Fermenter

Larger scale production was carried out in a 5 L fermenter using BSM medium. A 300 mL inoculum in BMGY medium was used to inoculate the 5 L fermenter of BSM medium with 13 mL PTM1 trace salts solution at 28oC. Before inoculation, the pH was adjusted to 6.0 with concentrated ammonium hydroxide, and 3% histidine, and 18 mL biotin (0.02%) was supplemented. The initial cultivation continued until the glycerol had been consumed (about 27 hours), and was followed by the glycerol-fed phase at a continuous feeding rate of 0.6 mL/min/L and initial fermentation volume of 50% (v/v) glycerol supplemented with 3% histidine and 12 mL/L glycerol PTM1 trace salts solution until cell density reached 240 g/L wet weight. Before starting the methanol induction phase, the glycerol feed was completely stopped and the dissolved oxygen level kept towards 100% for 2 hours to avoid repression of the AOX promoter. The methanol feeding was started at 3.6 mL/h/L fermentation volume containing 3% histidine and PTM1 trace salts solution (12 mL/L methanol) during the first 2 to 4 hours, and increased up to 7.2 mL/h/L for the following 2 hours. The feeding rate was further increased to 10.9 mL/h/L for the remainder of the fermentation. During the induction period, the pH was kept at 5.0. Pure oxygen was supplemented to keep the DO level above 20% during the whole fermentation phase. Samples were collected every 12 hours for optical density measurement, cellular wet weight, xylanase activity assay, and determination of total soluble protein and SDS-PAGE analysis.

Enzyme and Protein Assays

Xylanase activity was measured using 1% birchwood xylan as a substrate in 50 mM sodium citric acid buffer, pH 5.0, at 50oC, for 30 min. The liberation of reducing sugar was estimated by the dinitrosalicylic acid (DNS) method, using xylose as a standard (Miller 1959). One unit of xylanase activity was defined as the amount of enzyme that liberated 1mol of reducing sugar from the substrate solution per minute.

The optimal temperature was determined by the standard activity assay at various temperatures from 30 to 70oC at pH 5.0. To estimate thermal stability, the enzyme was pre-incubated for 30 min at the different temperatures. The optimal pH was determined at 50oC for 10 min in a sodium citrate buffer at a pH range from 3.0 to 8.0. The pH stability of the enzyme was determined by examining the residual activities under standard conditions after a pre-incubation of the enzyme at room temperature for 30 min at various pH levels. The activity of the enzyme without pre-incubation was defined as 100%.

SDS-PAGE was performed in gel containing 12% (w/v) acrylamide and 0.1% SDS (w/v), using a Tris/glycin buffer system. Resolved proteins were visualized by staining with Coomassie Brilliant Blue R-250 (Laemmli 1970). Protein concentrations were determined using the Bradford method with bovine serum albumin as a standard (Bradford 1976).

Hydrolysis Products of Birchwood Xylan

The 1% (w/v) birchwood xylan in pH 5.0 and 50 mM sodium citrate buffer was incubated with syn-xylanase at 45oC. The aliquots were removed and extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1) at different time intervals, and standard xylose was analyzed by HPLC with Sugarpak I column, pure water as the mobile phase (0.5 mL/min), and an injection volume of 10 L.

Nucleotide Sequence Accession Number

The nucleotide sequence optimized xynB genes was deposited in the Genbank database under accession number HQ385274.


Synthesis of Codon-Optimized xynB Gene and Construction of the Expression Vector

P. pastoris is an efficient host for recombinant protein production with the advantages including growth to very high cell densities in a simple, defined medium and strongly inducible promoters (Zhang et al. 2000). However, the use of synonymous codons may vary widely between different genes and organisms. Like most living organisms, yeasts display usage bias towards codons (Nakamura et al. 1999). The presence of a large rare-codon in the heterogeneous gene expressed in P. pastoris may lead to compromising the host by depleting charged pools of the rare tRNA, and may lead to ribosome pausing that is deleterious to high levels of expression (Brandes et al. 1996). Therefore, the expression levels of xynB may be further improved through optimizing for codon usage, G+C content, as well as signal peptide (Teng et al. 2007). The optimization of codon coding target protein, by the codon bias of the host cell, can usually result in an average 10- to 50-fold increase of target protein production (Outchkourov et al. 2002; Sinclair and Choy 2002). Optimizing the conditions of the fermentation is another method that can increase the quantity of recombinant protein (Wang et al. 2007).

It was found that usage biases of codon in A. niger and P. pastoris have significant differences. The DNA sequence of the A. niger NL-1 xynB gene showed that some amino acid residues were encoded by codons that are rarely used in P. pastoris. To further improve the expression level of recombinant xylanase in P. pastoris, the xynB gene was optimized based on the codon usage bias of P. pastoris. At the same time, the mRNA secondary structure, GC content, and unfavorable peaks were optimized to prolong the half-life of the mRNA. The Stem-Loop structures, which impact ribosomal binding and stability of mRNA, were broken. Moreover, to avoid premature termination, changing AT-rich codons to AT-deficient ones eliminated AT-rich stretches. The synthetic gene sequence, in which a total of 155 nucleotides were changed, had 43.6% GC content compared to 57.7% GC content of the initial gene as shown in Fig 1, closer to the actual GC content in P. pastoris (Huang et al. 2008). The optimized gene without a signal peptide sequence was synthesized by overlapping extension PCR using 28 oligomers designed by the online program DNAWorks. The mature peptide sequence of the optimized xynB gene, with a length of 624 bp, was cloned into pMD-19T and sequenced. Then, the fragment was amplified from the cloning plasmid pMD-19T-SxynB and was introduced into the expression plasmid pPICZA to yield the recombinant plasmid pPICZA-SxynB.

Transformation, Screen, and Expression of syn-xynB in P. pastoris

The recombinant plasmid pPICZA-SxynB was integrated into P. pastiris GS115 strain after being linearized using BstX I by electroporating. About 105 transformants were screened on a YPDS plate. The Mut phenotype was identified in MD and MM medium for the pPICZαA-SxynB recombinant cell. Colonies that grew normally in both of the two plates were of the Mut+type; however those that grew normally only in the MD plate but slowly in the MMH plate were of the MutS type. The results showed that all the transformants were Mut+ type. The colonies were selected for induction expression. After 14 days of induction in a shake flask, the colony with the highest xylanase activity, 1408 U/mL (Fig. 2a), was approximately 3-fold higher than that of the native recombinant (Li et al. 2010).

Fig. 1. Alignment of nucleotide sequence between the native and optimized xynB gene. The gene encoding 131 residues in xynB were optimized. The shadow indicates optimized residues of xynB.

Optimization of Syn-xylanase Expression in BMGY Medium

The transformant was used to study the effect of methanol concentration on the xylanase expression. P. pastoris is a kind of methylotrophic yeast, capable of metabo-lizing methanol as its sole carbon source. The promoter regulating the production of alcohol oxidase is the one used to drive heterologous protein expression in P. pastoris. So, methanol concentration in a P. pastoris process is extremely important (Sreekrishna et al. 1997), since high levels of methanol can be toxic to the cells and low levels of methanol may not be enough to initiate transcription. In this study, we can see that the optimal methanol concentration was 0.8%, with the maximum xylanase activity of 1418 U/mL, as shown in Fig. 2b. When the methanol concentration was above or below 0.8%, the activity decreased significantly.

Fig. 2. Expression of recombinant syn-xylanase in P. pastoris: (a) Syn-xylanase activity level of the transformant with the highest activity incubated at 28oC, 180 rpm, pH 6.0 in 30 mL BMGY medium. After 24 hours, 0.5% methanol was added every 24 hours to the induction expression. (b) Effect of methanol concentration on xylanase expression. The transformant with the highest activity was induced at pH 6.0 at different methanol concentrations with initial OD600 of 1.0. (c) Effect of pH values on xylanase expression. The transformant with the highest activity was induced at a different initial pH with initial OD600 of 1.0 and 0.5%, and methanol was added every 24 hours. (d) Effect of histidine concentration on xylanase expression. The transformant with the highest activity was induced at pH 6.0 at different histidine concentrations with initial OD600 of 1.0 and 0.5% methanol added every 24 hours with no histidine added as a control. The above mentioned were cultivated and inducted at 28oC, 180 rpm, in 30 mL BMGY medium.

The pH value has an influence on the growth and metabolism of P. pastoris in general. To study the effect of pH in the BMGY medium on xylanase expression, we incubated the recombinant in BMGY medium containing 50 mM of K2HPO4-KH2PO4 buffer. The cultures were prepared with initial pH values of 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0. The results showed that syn-xylanase production increased as the pH values increased within a pH range between 3.0 and 7.0, but the activity slightly decreased when the pH reached 8.0, as shown in Fig. 2c.

The P. pastoris GS115 has a mutation in the histidinol dehydrogenase gene (his4) that prevents it from synthesizing histidine. However, histidinol dehydrogenase gene was integrated into the genome of the recombinant yeast in the process of construction of recombinant protein, resulting in mutation defect complement, which was considered to be an effective way of screening phenotypes (Cregg et al. 2000). Because the histidine producing ability of the recombinant P. pastoris was not as good as the wild type, it would be a bottleneck of the heterologous expression. Extra histidine needs to be supplied to relieve the pressure of lack of histidine when the expressed heterologous protein was rich in histidine. To study the effect of histidine concentration on xylanase expression, different histidine concentrations of 0.05%, 0.1%, 0.3%, and 0.5% (v/v) were added to BMGY medium with no histidine added as a control. The result is shown in Fig. 2d. With increasing histidine concentration, the xylanase activity was enhanced. The histidine concentration of 0.3% led to the highest activity with 1538 U/mL. When the histidine concentration increased to 0.5%, protein yields began to decrease, which was the result of the metabolism of the recombinant being feedback-restrained by the high histidine concentration.

To further improve the xylanase activity, the transformant with the highest activity was subjected to high-cell-density fermentation in a 5-L fermenter. The end of the glycerol batch phase was indicated by a spike in the DO caused by the exhaustion of glycerol. The cell-wet weight reached about 52 g/L, corresponding to an OD600 value of around 35 in the phase. During the glycerol-fed-batch, the biomass increased exponentially to 277 g/L with an OD600 value of around 242 when all the glycerol had been consumed and the dissolved oxygen level approached 100% of saturation. After 2 hours, the induction phase began with methanol added at a gradually varying flow until the rate of 10.9 mL/h/L fermentation volume was reached, which lasted about 72 hours. The xylanase expression level of codons optimized gene was increased to 13-fold higher than that of the shake flask culture with the maximum activity of 20424.2 U/mL and a total protein of 584.4 mg/L. The cell wet weight increased up to 310 g/L, and the OD600 value reached 320 at the end of the induction phase (Fig. 3).

SDS-PAGE analysis of the fermentation supernatant sample taken after a different induction time point was performed. The recombinant xylanase showed three apparent molecular sizes of about 21, 22.5, and 24 kDa (Fig. 4), which was the same as the native recombinant xylanase. The different bands appearing on the electrophoresis are due to a difference in the degree of glycosylation of the polypeptide chains (Deng et al. 2006).

Biochemical Characterization of the Recombinant Syn-xylanase

The biochemical properties of a xylanase will impact its commercial effective-ness. Effects of temperature on activity and stability of the recombinant enzyme are shown in Fig. 5. The optimal temperature was 50oC. The enzyme displayed about 90% of its peak activity in the temperature range 37 to 40oC, i.e. the body temperature of animals that might receive the enzyme in their diet. However, enzyme activity declined rapidly at temperatures in excess of 50oC, indicating potential problems if the enzyme was used at high temperatures, such as those used for pelleting or extruding diets (Lawrence 1970).

Fig. 3. Growth process and secretion curve of recombinant syn-xylanase in high-cell-density fermentation: (a) Growth process of the recombinant yeast cells (b) Expression of syn-xylanase and secretion of total protein in a 5-L fermenter

Fig. 4. SDS-PAGE analysis of recombinant syn-xylanase in P. pastoris: The fermentation supernatant was collected at different induction times. Lane M: Protein molecular weight standards. The lane heading 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h indicated the induction time.

The optimal pH for the recombinant xylanase was about 5.0. The enzyme retained about 90% of its activity after being incubated at pH 3.0 to 8.0 for 1 hour at room temperature. Enzymes used in animal feeds must survive transit through the stomach, and the pH in the animal’s stomach on full feed rarely rises above pH 3.0 (Lawrence 1970).

Fig. 5. Characterization of the recombinant syn-xylanase: (a) Effect of pH on xylanase activity. The enzyme activity was determined under different pH values at 50oC. The highest xylanase activity was taken as 100% in an assay of pH optimum. (b) Effect of temperature on xylanase activity. The highest activity was taken as 100% in an assay of temperature optimum. All these assays were performed as described above using 1% birchwood xylan as the substrate.

Fig. 6. HPLC analysis of the hydrolysis product: The birchwood xylan was incubated in pH 5.0, 50 mM sodium citrate buffer with syn-xylanase at 45oC for 8 hours (a) and 24 hours (b) 9.869 min implied the retention time of standard xylose.

The kinetic parameters km and Vmax of recombinant xylanase for birch xylan were 16.7 mg/mL and 188.7 mg/mL min, respectively. The enzyme activity was not or only a little affected by 1 mM Fe3+, Ca2+, K+, Mg2+, Zn2+, Ba2+, Co2+, Al3+, and Fe2+, in which the enzyme was activated by Cu2+ and inhibited by Mn2+. The addition of excess EDTA (10 mM) did not affect the activity, suggesting the cation cofactors were not required for the enzymatic reaction.

The hydrolysis product of birchwood xylan by syn-xylanase was analyzed by HPLC, as shown in Fig. 6. As the reaction time increased, birchwood xylan was degraded into xylooligosaccharide and xylose. Xylooligosaccharide was the major hydrolytic product relative to the xylose. After 24 hours of incubation, the yield of xylooligosaccharide increased, but the concentration of xylose remained constant at 0.388 mg/mL. The results showed that the syn-xylanase has great potential in the bioconversion of lignocellulosic waste to xylooligosaccharide. Xylooligosaccharides have attracted more and more interest because of their beneficial effects as bifidobacterium growth-promoting factors (Vasquez et al. 2000).


The xynB gene from A. niger was optimized, synthesized, and expressed successfully in P. pastoris GS115, as shown in this paper. The culture and induction conditions for the recombinant expression of xylanase were optimized. The xylanase activity (20424.2 U/mL) was significantly enhanced by high-cell-density fermentation in a 5-L fermenter; the activity was about 13-fold and 39-fold higher than that of the syn-xynB and initial recombinant strain in a shake flask, respectively. Compared to the non-optimized control, the enzymatic properties were not changed. The enzyme showed great potential in the natural biodegradation process of hemicellulose. The present results will also promote the industrial application of the enzymatic process.


This work was supported by the National Natural Science Foundation of China (Grant No. 31070515) and the Priority Academic Program Development of Jiangsu Higher Education Institutions funded the project


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Article submitted: December 20, 2011; Peer review completed: March 25, 2012; Revised version received and accepted: March 30, 2012; Published: April 16, 2012.