Residue mutations of xylanase in Aspergillus kawachii alter its optimum pH

Accepted Manuscript Title: Residue mutations of xylanase in Aspergillus kawachii alter its optimum pH Author: Jin Qiu Hongjuan Han Baihui Sun Lei Chen Chengye Yu Rihe Peng Quanhong Yao PII: DOI: Reference:

S0944-5013(15)30006-9 http://dx.doi.org/doi:10.1016/j.micres.2015.09.002 MICRES 25819

To appear in: Received date: Revised date: Accepted date:

5-6-2015 17-8-2015 6-9-2015

Please cite this article as: Qiu Jin, Han Hongjuan, Sun Baihui, Chen Lei, Yu Chengye, Peng Rihe, Yao Quanhong.Residue mutations of xylanase in Aspergillus kawachii alter its optimum pH.Microbiological Research http://dx.doi.org/10.1016/j.micres.2015.09.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Residue mutations of xylanase in Aspergillus kawachii alter its optimum pH Jin Qiua,b, Hongjuan Hana, Baihui Suna,b, Lei Chena,b, Chengye Yua,b, Rihe Penga,b,*, Quanhong Yaoa,b,* [email protected] a

Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Institute,

Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai 201106, People’s Republic of China b

College of Food Science and Technology, Shanghai Ocean University, 999 Huchenghuan Rd, Shanghai 210306,

People’s Republic of China *

Corresponding authors. Shanghai Key Laboratory of Agricultural Genetics and Breeding Institute of

Agro-Biotechnology Research Shanghai Academy of Agricultural Sciences 2901 Beidi Road, Shanghai, People’s Republic of China. Tel.: +86-21-62203180; Fax: +86-21-62203180.

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Abstract Aspergillus kawachii and Aspergillus niger have been traditionally used as molds for commercial microbial fermentation because of their capability to grow in extremely acidic environments and produce acid-stable enzymes. Endo-1,4-β-xylanase cleaves the glycosidic bonds in the xylan backbone, consequently reducing the degree of polymerization of the substrate. The amino acid sequences of xylanases from A. kawachii and A. niger only differ in one amino acid residue. However, the xylanases from A. kawachii and A. niger show different optimum pH values of 2.0 and 3.0, respectively. In this study, we synthesized the A. kawachii xylanase gene (XynC) on the basis of the bias codon of yeast and mutated the gene in the dominating region related to optimum pH shifting during gene synthesis. After the overexpression of this gene in Pichia pastoris G115, the mutant (Thr64Ser) enzyme (XynC-C) showed an optimum pH of 3.8, which indicated partial alkalinity compared with the original xylanase from A. kawachii. Similar to that of the enzyme with one residue mutation (Asp48Asn), the optimum pH of the enzyme with two residue mutations (Thr64Ser and Asp48Asn) shifted to 5.0. The result indicated that mutation Asp48 was more important than mutation Thr64 in optimum pH shifting. We proposed a model that explains the lower optimum pH of XynC-C than other members of the xylanase family G. XynC-C showed similar proteolytic resistance and Km and Vmax values for beechwood xylan to other xylanases. Keywords: Aspergillus kawachii; xylanase; optimum pH; protein structure

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1. Introduction The common hemicellulose xylan accounts for up to one-third of the total dry weight of plants. Enzymes such as endoxylanase, β-xylosidase, acetyl xylan esterase, α-glucuronidase, α-arabinofuranosidase, feroryl, and p-coumaroyl esterases are involved in xylan degradation. Endo-β-1,4-xylanase catalyzes the hydrolysis of the main backbone of xylan. On the basis of amino acid sequence similarities, the majority of endoxylanases fall into families 10 and 11 of glycosyl hydrolase. Xylanases are found in bacteria, actinomycetes, fungi, and protozoa. Fungal xylanases have attracted considerable attention because of their special properties, such as broad pH adaptability, good thermostability, strong proteolytic resistance, and high specific activity. Xylanases are mainly applied as bleaching agents replacing toxic chlorine-containing chemicals in the paper and pulp industries. Moreover, incorporating xylanase into lignocellulosic feeds for animal nutrition reduces intestinal viscosity and enhances feed conversion efficiency. Several acidophilic xylanases from fungi have been reported to date. Acidophilic and acidic-stable xylanases benefit processes that require low pH condition to avoid microbial contamination. Certain Aspergillus strains can grow in extremely acidic environments and produce acid-stable enzymes that are peculiar to them. These enzymes display properties appropriate for potential industrial applications. Aspergillus kawachii, used in the shochu industry in Japan, produces a large quantity of citric acid and several interesting acid-stable enzymes, such as α-amylases, glucoamylases, xylanases, and acidophilic proteinases. A. kawachii is phylogenetically close to Aspergillus niger, which is also used industrially to produce citric acid. However, the former is distinct from the latter because the endo-β-1,4-xylanase from A. kawachii shows more acidophilicity than the homo-enzyme from A. niger. A. niger Xylanase I, whose optimum pH is 3.0, has one residue difference from A. kawachii XynC; however, the optimum pH of XynC is 2.0. To elucidate the mechanism underlying the optimum pH determination of fungal family 11 xylanase, we studied two mutations in A. kawachii xylanase. One mutation was near the region that has one residue difference from A. niger, and the other mutation was near the catalytic residues. We then compared the optimum pH and other characteristics, such as substrate specificity, temperature stability, and proteolytic resistance, of the mutant enzyme (XynC-C) with those of its origin. We elucidated the catalytic mechanism under low pH conditions on the basis of the 3D structure of the enzyme.

2. Materials and methods 3

2.1 Microorganism and chemicals All chemicals used were of analytical grade. Pichia pastoris strain G115 (His-Mut+), which was used as a host for heterologous expression of xylanase, was purchased from Invitrogen. (Carlsbad, CA, USA). Vector pYPX88 (GenBank Accession No: AY178045) which was used as an expression vector was prepared in our laboratory. Genomic DNA and plasmid isolation and purification kits were purchased from TIANGEN (Beijing, China). Restriction endonucleases, T4 DNA ligase, DNA polymerase, dNTPs and GC buffer I were purchased from TaKaRa (Otsu, Japan). Beechwood xylan (X4252-25G) and proteases including pepsin and trypsin, were purchased from Sigma (St. Louis, MO).

2.2 Gene synthesis and sequence analysis In consideration of the amino acid sequence, the mutational xylanase gene XynC-C with an additional C-terminal 6× histidine tag sequence at the 3′ end was optimized using codon usage bias and was synthesized via successive polymerase chain reaction (Xiong et al., 2004). The amplification reaction mixture (25 µL) was composed of 2.5 µl of 10 × PCR buffer, 2.5 µl of 25 mM Mg2+, 2 µl of 10 mM dNTPs, 1 µL of each primer (10 mM), 10 ng of each inner primers, 100 ng of outer primers, and 0.5 U of Pyrobest™ DNA polymerase. The PCR cycling parameters were an initial denaturation step at 94 ℃ for 5 min, 25 cycles of amplification (denaturation at 94 ℃ for 20 s, annealing at 58 ℃ for 20 s, extension at 72 ℃ for 30 s), and a final elongation step at 72 ℃ for 10 min. The resulting PCR product was separated by electrophoresis in a 1% (w/v) agarose gel and recovered. The amplified fragment was digested with Bam HI and Sac I and then transformed into Bam HI/Sac I site of pYPX88 vector to obtain a genomic library. Errors in the synthetic gene were corrected by the overlap extension PCR method (Xiong et al., 2006). The vector pYPX88 contains a 357-bp fragment of α-factor prepro-leader MF4I (GenBank accession No: AY145833) with P. pastoris preferred codon usage, which substituted for the wild-type α-signal sequence to enhance the expression level (Xiong et al., 2003). Routine DNA manipulations were performed by standard recombinant method (Sambrook et al., 1989).

2.3 Electroporation and screening transformants The synthetic plasmid was linearized with Bgl II and transformed into P. pastoris strain G115 cells by electroporation method. P. pastoris strains were grown in YPD broth (1% tryptone, 0.5% yeast extract and 0.5% NaCl) at 30 ℃ with vigorous aeration to an OD600 of 1.5-2.0. Cells were harvested and washed three times in 4

sterile cold de-ionised water by centrifugation at 3000 g for 10 min and finally resuspended in 0.6mol/l sorbitol. Electroporation was performed using a Bio-Rad Gene Pulser and Pulse Controller as described by Dower et al (Dower et al., 1988). Cells to be transformed were first thawed on ice, 500 ng DNA in 10 µ1 of water was added, gently mixed and transferred to a pre-chilled 0.2 cm gap electroporation cuvette. Cells were then plated out onto SD-his medium and grown at 28 ℃ for 2 days. The transformants were inoculated from a single colony into a broth for overnight culture. The transformants were screened for their ability to grow on histidine deficient medium. Small-scale expression experiments were performed to detect expression of the recombinant protein. The his transformants were streak cultivated on BMGY (2% peptone, 1% yeast extract, 1.34% YNB, 0.4 µg/ml biotin, ＋

1% glycerol) at 28 ℃ with constant shaking at 225 rpm for 24 h. Cells grown in BMGY were inoculated into 96-well plates containing 50 µl BMMY medium and 1% methanol respectively and incubated for 10 h at 28 ℃, 225 rpm. Subsequently, the positive colonies were selected by enzymatic activity determination. One strain showing the highest xylanase activity was selected for further analysis. The strain was cultivated in 50 ml BMGY until the OD600 reached 3.0. The cells were harvested and resuspended in equal volume of BMMY. To maintain induction, methanol was added to the culture to a final concentration of 1% every 24 h. After 3 days of cultivation, the xylanase activity of the cultures reached their highest level. All purification procedures were performed at 4 ℃. Aliquots of culture supernatant were taken daily and examined for protein production by SDS-PAGE, and the xylanase activity was assayed at the same time.

2.4 Protein purification and enzyme activity assay The culture was induced with methanol for 72 h (OD600 = 5–6) and then centrifuged at 3000 g for 10 min at 4 °C to remove cell debris and purify the enzyme. The His-tagged protein was purified using Ni2+-NTA Agarose (Qiagen, Valencia, CA). After purification, the production was analyzed using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and visualized with Coomassie Brilliant Blue R-250 staining. The concentrations of soluble proteins in the filtered fermentation culture samples were determined using a Bradford assay kit. Xylanase activity was determined by measuring the release of reducing sugar from beechwood xylan using the 3, 5-dinitrosalicylic acid (DNS) reagent (Miller, 1959). The standard assay for xylanase activity was performed at 45 ℃ for 10 min in citrate buffer (pH 3.8). The reaction mixture (200µl) contained 50 µl of the diluted enzyme solution and 100µl of 1% beechwood xylan. The reaction was stopped by the addition of 150 µl of DNS reagent. 5

Absorbance of samples was measured at 540 nm against the substrate blank. One unit of xylanase activity was defined as the amount of enzyme required to liberate 1 µmol of xylose from xylan in 1 min under its optimal assay conditions.

2.5 Optimal pH and stability of xylanase The optimal pH of the purified synthetic enzyme was determined at 45 ℃ in buffers with pH ranging from 2.6 to 8.0. The pH stability was estimated by measuring the residual enzyme activity after pre-incubating the enzyme in different buffers of pH 2.6-8.0 at 37 ℃ for 3 h; The buffers used were: 0.2 M glycine-HCl (pH 2.6-3.0), and 0.2 M Na2HPO4-citric acid buffer (pH 3.0-8.0). The xylanase activity was assayed under standard conditions.

2.6 Other properties of XynC-C The optimal temperature of xylanase was determined over the range of 20-90 ℃. The thermostability of the enzyme was determined by pre-incubating the enzyme in citrate buffer (pH 3.8) at 20-100 ℃ without substrate for 30 min and measuring the residual enzyme activity under the standard conditions. According to the Lineweaver-Burk method (Lineweaver and Burk, 1934), the Km and Vmax values for the xylanase were determined at 45 ℃ using 1.0-18.0 mg/ml beechwood xylan as the substrate. The reaction mixture was incubated at 45 ℃ for 10 min and the enzyme activity was measured as above. The effects of different metal ions and chemical reagents on the purified recombinant enzyme activity were evaluated by individually adding 10 mM (final concentration) of NaCl, KCl, CaCl2, NiSO4, CuSO4, MgSO4, FeSO4, FeCl3, MnSO4, ZnSO4, NH4Cl, AlCl3, CrCl3, CoCl2, DTT, SDS, pepsin or trypsin to the purified recombinant enzyme after 30min of incubation at 37 ℃. The system without any additive was used as a control (CK). Substrate specificity of the purified recombinant enzyme was investigated in the standard assay system containing the following substrates (1%; w/v): beechwood xylan, gather wood glucan and microcrystalli.

2.7 Sequence analysis and three-dimensional structure model Analysis of the deduced amino acid sequence was done using the NCBI BLAST tool. Alignment of protein sequences between XynC-C and other published family 11 xylanase was performed with DNAMAN. Swiss-Model (http://swissmodel.expasy.org/) was utilized to model the three-dimensional structure of XynC-C (Beg et al., 6

2001).The template structure was 1T6G (PDB code), which shared the highest sequence identity (99%) with XynC-C among the proteins in the Protein Data Bank (PDB) (http://www.rcsb.org/). The comparison was achieved by DALI server (http://www.ebi.ac.uk/dali/Interactive.html). A single model was selected for further analysis. The three dimensional structures of modeled proteins were analyzed using the DSViewerPro60.

2.8 Site-directed mutagenesis of XynC The substitutions were introduced in XynC by PCR technology (Peng et al., 2006). The used primers in Thr64Ser

were

5’-

AATGCCATCTCCTACTCTGCCGAG-3’

GGCAGAGTAGGAGATGGCATTGGA-3’.

The

used

primers

in

Asp48Asn

and were

as

follows:

5’5’-

GTCTCTTCCGACTTCGTCGTTGGT-3’ and 5’- AACGACGAAGTCGGAAGAGACACC-3’. The xylanase activity was assayed under standard conditions.

3. Results 3.1 Gene sequence analysis of XynC-C The entire open reading frame of XynC-C generated a single band of 636 bp encoding a protein of 195 amino acids. We synthesized the current sites in consideration of the yeast bias codon. The G + C content of the synthetic XynC-C was approximately 52.2%. To facilitate protein purification, additional histidine tag sequences were attached at the 3′ end.

3.2 Enzyme expression and purification The structural gene (without the signal peptide coding sequence) coding for XynC-C was expressed in Pichia pastoris G115 and induced with 1% methanol at 28 °C for 3 days. The His6-tagged xylanase was secreted into the culture supernatant and showed a xylanase activity of 600.5 U/mg. The xylanase in the culture supernatant was concentrated and further purified to electrophoretic homogeneity via Ni2+-NTA metal chelating affinity chromatography. The purified enzyme preparation contained a single band of approximately 23 kDa on SDS–PAGE, which approached the calculated molecular weight (http://web.expasy.org/compute_pi/) of 23.3 kDa, as shown in Fig. 1.

3.3 Effects of pH on XynC-C activity and stability 7

The optimal pH for the mutant xylanase XynC-C was pH 3.8, and more than 40% of the maximal activity was retained at pH 2.0 (Fig. 2a). This finding is similar to the xylanase from A. niger (3.0). The profiles showed that XynC-C was highly stable (50% activity remaining) under an extremely acidic pH ranging from 2.6 to 6.0 (Fig. 2a). When the Asp48 of XynC-C was replaced with Asn through site-directed mutagenesis, the optimal pH for the xylanase activity of purified D48N shifted to pH 5.0, and nearly 60% of the maximal activity was retained at pH 3.5 (Fig. 2a).

3.4 Other characteristics of the mutant xylanase XynC-C The optimal temperature for enzyme activity was 45 °C (Fig. 2b). Xylanase XynC-C was stable at 40 °C for 30 min but became unstable at 40 °C (Fig. 2b). Xylanase XynC-C only retained approximately 77% maximal activity after incubation for 30 min at 40 °C without a substrate. The effects of various metal ions and chemical reagents on XynC-C activity were evaluated, and the results are shown in Table 1. Compared with the activity of the standard xylanase (CK), the activity of XynC-C slightly increased in the presence of a few metal ions (DTT, Zn2+, K+, Mg2+, Co2+, Ca2+,Al3+, NH4+, Fe3+, Fe2+, Ni2+, Na+, and Cr3+) at low concentrations. The activity of XynC-C was inhibited by Cu2+, SDS, and Mn2+; strongly inhibited (>50%) by SDS and Mn2+; and partially inhibited by Cu2+. XynC-C was strongly resistant to protease digestion. After treatment at 37 °C for 30 min with pepsin or trypsin, the enzyme retained more than 90% of the maximum xylanase activity (Table 1). The activity of XynC-C was measured under standard conditions in the presence of different substrates. The Km and Vmax values of XynC-C for beechwood xylan were 10 mg/mL and 1250 µmol/min/mg, respectively. These values were in agreement with those of other fungal xylanases that range from 0.09 mg/mL to 40.9 mg/mL for Km and from 0.106 µmol/min/mg to 6300 µmol/min/mg for Vmax (Beg et al., 2001). The Km value of XynC-C toward beechwood xylan was 10 mg/mL, which indicated the high affinity of XynC-C to xylan. XynC-C showed specificity to polymeric xylan sources but not to other substrates such as Gather wood glucan and microcrystalli.

3.5 Amino acid homology alignment The amino acid homology alignment of XynC-C with 13 other xylanases from different sources was conducted (Supplemental Fig. 1). Fourteen of the xylanases used in the comparison are summarized in Table 2. Twenty-eight 8

of the XynC-C residues were strictly conserved in the 14 aligned proteins (Supplemental Fig. 1). XynC-C belongs to family 11 of glycohydrolases and lacks xylane binding, cellulose binding, and thermostabilizing domains (Gilbert et al., 1999). A few structural differences from other family 11 xylanases are concentrated in the coiled regions. However, certain differences are also present in the protein core. For example, the missing N-terminal extension limits the large β-sheet B to eight instead of nine strands. Two putative catalytic glutamate residues in XynC-C, namely, Glu90 and Glu181, are highly conserved among family 11 members (as shown in black frame in Supplemental Fig. 1). As in other acidophilic xylanases, an Asp residue (Asp48) is present in the conserved domain of xylanases (as shown in the black frame in Supplemental Fig. 1). This residue is replaced by Asn in other xylanases. In fact, only one different residue is found between xylanases from A. kawachii and A. niger. In particular, Asn61 in A. kawachii is Lys61 in A. niger. The optimum pH values of the xylanases from A. kawachii and A. niger are 2.0 and 3.0. However, the optimum pH of XynC-C, in which threonine is replaced with serine, shifted to 3.8. After further mutation of Asp48Asn, the optimum pH shifted to 5.0. This phenomenon indicates that Thr64 and Asp48 are both related to the shifting of optimum pH.

3.6 Structural analysis of XynC-C The overall structure of XynC-C is dominated by one α-helix and two strongly twisted β-sheets that are packed against each other. The shape of the molecule resembles a right hand: the two β-sheets and the α-helix form fingers and a palm, a long loop between the B7 and B8 strands forms a thumb, and a loop between the B6 and B9 strands forms a cord (Fig. 3a). The larger, eight-stranded β-sheet (sheet B) is highly twisted around a deep, long cleft that is large enough to accommodate several xylose residues. The only α-helix is embedded on the rear side of this sheet. This structure is stabilized mostly by hydrophobic interactions with the β-sheet and by several hydrogen bonds and electrostatic interactions between highly conserved amino acid residues. This type is the protein fold of family 11 xylanases. The backbone structure of XynC-C is highly similar to that of Xylanase I from A. niger. Thus, this structure was used as a model for molecular analysis. The amino acid sequences of Xylanase I and XynC-C differ in only two residues (as shown in Fig. 4b). In particular, Asn61 and Ser64 in XynC-C are Lys61 and Thr64 in Xylanase I. The region corresponds to the back of the XynC-C. The large cleft, which is created by the highly twisted β-sheet B, is ideally suited to serve as a substrate-binding 9

pocket. This pocket is lined with several aromatic residues (Tyr141, Tyr91, Tyr92, Tyr97, Tyr86, Trp55, Trp83, Tyr81, Tyr175, Trp183, Tyr119, Tyr113, Tyr100, Tyr77, Tyr17, Tyr21, Phe138, Phe142, and Phe49) (Fig. 3c) that are perfectly positioned to stabilize xylose residues (Fushinobu et al., 1998). Among these aromatic residues, the side chains of Tyr91, Tyr141, Trp55, Tyr119, Tyr113, and Phe49 are stretched into the other side of the cleft; Tyr86, which is on a β-turn, is highly solvent exposed. Hence, these residues probably exert no influence on substrate binding. Tyr77 on the β-strand B5, Tyr81 on the β-strand B5 in the middle of the cleft, Tyr92 on the β-strand B6, and Trp183 are located on the β-strand B4 that is near the active site Glu181. These residues are believed located near the active site in the cleft (Fig. 3d). Therefore, these four residues might be involved in substrate binding. Tyr100 is located on the “cord,” quite near Tyr97, which is on β-strand B5 (Fig. 3d). The two residues probably affect the entrance of xylan. Two conserved glutamate residues, namely, Glu90 and Glu181 (Fig. 3a), could be identified as the catalytic residues (Ko et al., 1992; Wakarchuk et al., 1994; Bray and Clarke, 1994). Glu90 is shown on the upper side and Glu181 on the lower side. Both residues extend their side chains to the bottom of the cleft from opposite sides. These residues correspond to Glu170 as the acid/base catalyst and to Glu79 as the nucleophile of A. niger Xylanase I. An Asp residue (Asp48) is present in the conserved domain of our xylanase and is near the two catalytic residues (Glu90 and Glu181), as in A. niger Xylanase I. This residue may be replaced by Asn in other fungal xylanases. The Asp residue is believed to be a hydrogen-bond linked to the glutamic acid residues that can serve as general acid/base catalysts for hydrolase activity (Fushinobu et al., 1998).

4. Discussion Recent studies have discovered immense biotechnological applications of xylanases in the food, feed, and paper-pulp industries. Enzyme activity is a principal factor determining the economics of any process. The methylotrophic yeast P. pastoris has been recognized recently as an efficient host for high levels of heterologous expression. The following assumptions were based on analyses of the conserved residues in the cleft. Interestingly, aromatic residues have the hydrophobic face of the side chain on the cleft surface. These residues can realize hydrophobic stacking interactions with the sugar residues, and their hydroxy groups can form hydrogen bonds (Cregg et al., 2000). We proposed that the structural biology community uses the system in which sites are labeled from +n to −n, with +n at the non-reducing end and −n at the reducing end. Cleavage occurs between the +1 and −1 sites (Davies 10

et al., 1997). The location of the aromatic residues that are near the active site in the cleft was analyzed; on the basis of this analysis, Tyr92, Tyr81, Tyr21, and Trp183 were thought to be located near the active site in the cleft (Fig. 3d). Therefore, these four residues might be involved in substrate binding. Using the molecular model of the xylo-oligomer, Törrönen et al. (1994) estimated that a space is available for at least four xylose units in the cleft (the residues of which are Tyr96, Tyrl79, Trpl8, and Phe180); the cleavage site is located between sites Trpl8 and Phe180. Thus, we deduced that Tyr100 (corresponding to Tyr96), Tyr183 (corresponding to Tyrl79), and Tyr21 (corresponding to Trpl8) are the substrate binding sites. However, no aromatic residue corresponded to Phe180; hence, the cleavage site was undetermined. Xylanase activity was also reported to be inhibited by N-Bromosuccinimide (NBS) (Nakamura, 2003; Yewang et al., 2007). This finding suggests that tryptophan and tyrosine are involved in catalysis. These conclusions are consistent with Nakamura, who suggested that tryptophan and tyrosine are involved in catalysis (Törrönen and Rouvinen, 1995). Trichoderma reesei Xylanase II (Sansen et al., 2004), Xylanase J (Nakamura, 2003), and three sites (Trp, Tyr, and Tyr) were predicted to bind xylose units in the sites −2, −1, and +1, respectively, in B. circulans Xylanase A (Karshikoff and Ladenstein, 1998). The three amino acids corresponded to Tyr77, Tyr81, and Tyr92 in XynC-C. A pairwise alignment between XynC-C and B. circulans Xylanase A, T. reesei Xylanase II, and Xylanase J showed 93%, 87%, and 33% structural similarities, respectively. The low percentage difference between Xylanase J and XynC-C can be attributed to the xylan-binding domain of Xylanase J on the C-terminal. Therefore, Tyr77, Tyr81, and Tyr92 were most probably involved in substrate binding, and the cleavage site may be located between sites Tyr81 and Tyr92. Tyr100 and Tyr97 are on the surface of the cleft and may have influenced the entrance of xylan. The mechanism underlying the substrate binding of XynC-C requires further investigation in terms of the crystal structure of the enzyme–substrate complex. Structural analysis shows that XynC-C has a highly similar 3D structure to A. niger Xylanase I. Hence, the properties of XynC-C may be the same as those of A. niger Xylanase I. Xylanase from A. kawachii (XynC) has an optimum pH of 2.0 (Fushinobu et al., 1998), XynC-C has an optimum pH of 3.8, and Xylanase I from A. niger has an almost similar optimum pH of 3.0 (Krengel and Dijkstra, 1996). Xylanase I has one residue difference from XynC, which is equal to N61K mutant on XynC. However, Xylanase I has an optimum pH similar to that of XynC-C, which is equal to the S64T mutant on XynC. The Ser64 in XynC-C and Lys61 in Xylanase I are in the same region on the top of β-strand A5. Hence, this region may be related to optimum pH shifting. In XynC-C, T64S is near Asn61 (Lys61 in Xylanase I as shown in Fig. 3b). 11

As expected, the optimum pH shifted from 3.8 to 5.0 when the Asp48 of XynC-C was replaced with Asn. Fushinobu et al. (1998) investigated the Asp37 (Asp48 in XynC-C and Xylanase I) of xylanase from A. kawachii and found that it was replaced with Asn; hence, the optimum pH shifted to 5.0. Although the region on the top of β-strand A5 can also shift the optimum pH from 2.0 (xylanase from A. kawachii) to 3.8 (XynC-C), D48N had more influence on optimum pH shifting. Therefore, this residue is expected to critically influence the pH dependence of xylanase activity. The Asp or Asn residue is believed to be a hydrogen bond linked to the glutamic acid residues that can serve as general acid/base catalysts for hydrolase activity (Luo et al., 2009a). Thus, Asp48 is mostly involved in the optimal pH of xylanase. This finding is consistent with the conclusion that the optimum pH of family 11 xylanases is influenced by the raspartic acid in acidic xylanases and by asparagines in alkaline xylanases (Joshi et al., 2000). Several structures of the thermophile xylanase revealed that the enzyme has a single N-glycosylation site on which N-glycans are known to have a stabilizing effect, which may prevent the aggregation of unfolded protein molecules (Wang et al., 1996). Therefore, in the region of the top of β-strand A5, Lys may contribute in increasing the positive charges of xylanase and in shifting the optimum pH from 2.0 to 3.0. However, Thr64 may increase any hydrogen bonds with the rest of the molecule. At present, the contribution of the region on the top of β-strand A5 to the optimum pH shifting of xylanase is unknown.

5. Conclusions Tyr77, Tyr81, and Tyr92 were most probably involved in substrate binding. The cleavage site may be located between sites Tyr81 and Tyr92. This study is the first to demonstrate that the region on the top of β-strand A5 is involved in optimum pH shifting. However, Asp48 was suggested to be a more indispensible site for optimum pH shifting than the D48N mutant.

Conflict of interest statement The authors declare that they have no conflict of interest.

Acknowledgements The research was supported by the Key Project Fund of the Shanghai Municipal Committee of Agriculture (zhongzi2013-8, zhongzi2014-2), International Scientific and Technological Cooperation (13440701700), 12

Agriculture science technology achievement transformation fund (133919N1300, 143919N0300), National Natural Science Foundation (31071486, 31200212, 31200075, 31200076). Basic research was done in the field of the science and technology project of the Science and Technology Commission of Shanghai Municipality (14JC1403602). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Figure Captions Fig. 1 SDS-PAGE analysis of the purified recombinant XynC-C. Lane 1 is the protein molecular mass markers; lane 2 is the purified XynC-C. Fig. 2 Effects of pH and temperature on the activity and stability of XynC-C and D48N mutant of XynC-C. a Effect of pH on the activity of XynC-C and D48N mutant of XynC-C. The optimal pH values of XynC-C (squares) and D48N mutant of XynC-C (circles) were assayed with pH varying from 2.6 to 8.0. The pH stability of XynC-C (triangles) was obtained after incubation with pH varying from 2.6 to 8.0 at 37 °C for 3 h, and the residual activities were measured at 45 °C for 10 min. b Effect of temperature on the activity of XynC-C and D48N mutant of XynC-C. The optimum temperature of XynC-C (squares) was assayed with temperature varying from 20 °C to 90 °C. Thermal stability of XynC-C (triangles) was obtained after pre-incubation of the enzyme solution for 30 min at 20 °C to 100 °C. Residual xylanase activities were measured at 45 °C for 10 min. Fig. 3 The three-dimensional structure of XynC-C. a The location of residues in 3D structure of XynC-C. The two β-sheets are shown in blue (sheet A and sheet B), and the helices are shown in red. Catalytic residues Glu90 and Glu181 are shown in scaled ball and stick. Asp48 is shown near the Glu181. b The location of the different amino acids from A. niger xylanase. Two residues are shown in scaled ball and stick. c The location of the aromatic residues in cleft. 19 residues are shown in scaled ball and stick. d The location of the residues which would be involved in the binding of the substrate and the entrance of xylan . The catalytic residues Glu90 and Glu181 are shown in yellow.

18

Fig. 1

19

Fig. 2 a

b

20

Fig. 3 a

b

c

d

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Tables Table 1 Effects of different metal ions and other reagents on XynC-C activity. Effectors

Specific activity (U/mg)

Effectors

Specific activity (U/mg)

CK

350.67±3.61

NH4+

382.89±4.01

DTT

687.82±4.03

Fe3+

453.50±1.01

Zn2+

446.10±2.02

Fe2+

483.17±2.34

k+

417.44±4.01

Ni2+

606.50±1.34

Mg2+

497.20±3.02

Na+

403.99±2.19

Co2+

337.64±1.05

Cu2+

323.09±03.01

SDS

193.70±1.02

Cr3+

1014.71±2.31

Mn2+

259.29±0.98

pepsin

363.96±1.08

Ca2+

603.16±1.02

trypsin

339.94±2.06

Al3+

585.02±3.00

*Activities were investigated after incubation of the enzyme with the reagents for 30 min at 37 ℃. In the control, no reagent was added to the reaction system. Activity under standard reaction conditions (45 ℃, pH 3.8). Values are the means of three replications ± SD.

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Table 2 Optimum pH of xylanase from different stains.

Optimum pH

Organism

Code

Reference

pH 2 - 3

Aspergillus kawachii

XynC

Fushinobu et al., 1998

Aspergillus niger

Xylanas I

Krengel and Dijkstra, 1996

Trichodrema Reesei

TAX

Sansen et al., 2004

Talaromyces versatilis

xynB

Lafond et al., 2014

Aspergillus kawachii

XynC-C

This paper

Talaromyces versatilis

xynF

Lafond et al., 2014

Penicillium oxalicum

XYN11A

Liao et al., 2014

Trichodrema Reesei

XYNII

Törrönen et al., 1994

pH 5 - 6

Aspergillus awamori

AWX

Umsza-Guez et al., 2011

pH above 6

synthetic construct

xyn11Ts

Törrönen and Rouvinen, 1995

Bacillus sp. 41M-1

xylanase J

Bai et al. 2014

Bacillus agaradhaerens

BAX

Sabini et al. 1999

Bacillus sp. SN5

Xyn11A-LC

Bai et al. 2014

Streptomyces sp.TN119

XynB119

Junpei et al., 2011

pH 3 - 4

pH 4 - 5

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