Improvement of the catalytic performance of a hyperthermostable GH10 xylanase from Talaromyces leycettanus JCM12802

Bioresource Technology 222 (2016) 277–284

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Improvement of the catalytic performance of a hyperthermostable GH10 xylanase from Talaromyces leycettanus JCM12802 Xiaoyu Wang a,b,1, Huoqing Huang a,1, Xiangming Xie b, Rui Ma a, Yingguo Bai a, Fei Zheng a,b, Shuai You a, Bingyu Zhang a, Huifang Xie a, Bin Yao a, Huiying Luo a,⇑ a b

Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, People’s Republic of China

h i g h l i g h t s
An acidic thermostable xylanase of GH10 (TlXyn10A) was identified in T.leycettanus.
Sequence analysis revealed seven residues probably involved in substrate contacting.
Mutant TlXyn10A_P with modifications at subsites +2 to +4 showed improved properties.
TlXyn10_P in combination with cellulase released the most reducing sugar form wheat straw.

a r t i c l e

i n f o

Article history: Received 2 August 2016 Received in revised form 30 September 2016 Accepted 1 October 2016 Available online 3 October 2016 Keywords: Talaromyces leycettanus Glycoside hydrolase family 10 (GH10) Xylanase Hyperthermostable Catalysis improvement

a b s t r a c t A xylanase gene of GH 10, Tlxyn10A, was cloned from Talaromyces leycettanus JCM12802 and expressed in Pichia pastoris. Purified recombinant TlXyn10A was acidic and hyperthermophilic, and retained stable over the pH range of 2.0–6.0 and at 90 °C. Sequence analysis of TlXyn10A identified seven residues probably involved in substrate contacting. Three mutants (TlXyn10A_P, _N and _C) were then constructed by substituting some or all of the residues with corresponding ones of hyperthermal Xyl10C from Bispora sp. MEY-1. TlXyn10A_P with mutations at subsites +2 to +4 exhibited improved specific activity (by 0.44fold) and pH stability (2.0–10.0). Molecular dynamics simulation analysis indicated that mutations E229I and F232E probably weaken the substrate affinity at subsites +3 to +4, and G149D may introduce a new hydrogen bond. These modifications altogether account for the improved performance of TlXyn10A_P. Moreover, TlXyn10A_P was able to hydrolyze wheat straw persistently, and has the application potentials in various industries. Ó 2016 Published by Elsevier Ltd.

1. Introduction Xylan is the second most abundant polysaccharide after cellulose in nature, accounting for approximately one-third of all renewable organic carbon on earth (Wong et al., 1988; Collins et al., 2005). As xylan exists ubiquitously, xylanases that randomly cleave the b-1,4-backbone of xylan can be applied in a large number of fields, such as brewing industry, food and feed production (Collins et al., 2005; Du et al., 2013; Passos et al., 2015; Rodríguez et al., 2012; Zhao et al., 2013), pharmacy (Christakopoulos et al., 2003), pulp bleaching (Techapun et al., 2003), waste treatment and bioconversion (Gupta et al., 2014;

⇑ Corresponding author. 1

E-mail address: [email protected] (H. Luo). Xiaoyu Wang and Huoqing Huang contributed equally to this work.

http://dx.doi.org/10.1016/j.biortech.2016.10.003 0960-8524/Ó 2016 Published by Elsevier Ltd.

Kaushik et al., 2014). Most xylanases are classified into glycoside hydrolase (GH; http://www.cazy.org) families 10 and 11, and other minorities belong to families 5, 8 and 30. Many kinds of microorganisms, including bacteria, yeasts, and fungi, are capable of producing xylanases. The enzyme fundamental properties, i.e., specific activity, thermal stability, and resistance to cations and chemicals, are vital factors that decide their potential applications. For example, alkaline xylanases are applied in the pulp bleaching industry (Ma and Yang, 2015; Techapun et al., 2003), acidic xylanases are supplemented in feed and food (Collins et al., 2005; Du et al., 2013; Zhao et al., 2013), and cold adapted xylanases (Chen et al., 2013; Wang et al., 2011, 2012a) are used in detergent and textile industries. Majority of fungal xylanases are acidic and have maximum activity at or near moderate temperatures (40–60 °C) (Collins et al., 2005), thus limiting their potential applications in some

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high-temperature industries. During the bioconversion process, hot water, steam explosion, and acidic pretreatments are commonly used prior to or simultaneous to enzyme treatment (Mielenz, 2001; Saha, 2003). According to the congress mash protocol (EBC 4.5.1) (Kunze, 1999) in brewing industry, acidic thermostable xylanases are required for longer hydrolysis at 70 °C. In feed industry, xylanases must withstand the pelleting process temperature (typically 70–90 °C) and adapt to digestive tract (pH 4.8) (van Campenhout et al., 2003). Therefore thermo-acidic xylanases demonstrate great application potentials in these industries with advantages over their counterparts. To meet the demand of industrial requirements, gene mining and protein engineering are applied to develop hightemperature-active and thermostable xylanases. For example, GH10 xylanases XynA and XynB from Thermotoga maritima MSB8 (Winterhalter and Liebl, 1995) displayed maximum activity at 90–105 °C, while MpXyn10A from Malbranchea pulchella exhibited an optimum temperature of 80 °C (Ribeiro et al., 2014). By using protein engineering, GH10 xylanases were also modified for thermostability and catalysis improvement. The mutant of Xyn10A_ASPNG was more stable with the Tm increased by 17.4 °C (Song et al., 2015), and the Tm of XynAS9 was increased by 7 °C by introducing proline and glutamic acid residues (Wang et al., 2014). Except for thermostability, catalytic performance was undoubtedly another way of reducing associated costs in application. To our knowledge, most protein engineering for catalysis improvement is mainly carried on GH11 xylanases (Chen et al., 2012; Cheng et al., 2015; Hoffmam et al., 2016), and fewer were executed with GH10 xylanses. Xyn III fusing a xylan-binding domain with increased affinity to insoluble substrate showed improvement in both catalytic efficiency and thermostability (Matsuzawa et al., 2016), and Geobacillus stearothermophilus XynA with a single point mutation was found to have an increased catalytic efficiency by 3.46-fold (Wang et al., 2013b). Although hundreds of xylanases have been reported, robust enzymes are still needed for industrial applications. In this study, a highly thermostable xylanase, designated TlXyn10A, was identified in Talaromyces leycettanus JCM 12802 and successfully expressed in Pichia pastoris GS115. For commercial purpose, this thermostable xylanase was further modified by site-directed mutagenesis.

TaKaRa (Tsu, Japan). The total RNA isolation system kit was purchased from Promega (Madison, WI). Restriction endonucleases, T4 DNA ligase, and endo-b-N-acetylglucosaminidase H (Endo H) were purchased from New England Biolabs (Ipswich, MA). All other chemicals were of analytical grade and commercially available.

2. Methods

2.4. Heterologous expression in P. pastoris

2.1. Strains, media, vectors and chemicals

The cDNA fragment of mature TlXyn10A without the putative signal peptide was amplified with primer TlXyn10AEcorF and TlXyn10ANotR (Table S1). The PCR product was digested with EcoRI and NotI and then cloned into the pPIC9 vector in-frame fusion of the afactor signal peptide to construct the recombinant plasmid pPIC9Tlxyn10A. The recombinant plasmid was linearized using BglII and transformed into P. pastoris GS115 competent cells by electroporation according to the manufacturer’s instructions (Invitrogen). The positive transformants were screened via enzymatic activity measurement in shake tubes, and the transformant with the highest xylanase activity was cultivated for fermentation in a 1-L Erlenmayer flask as described by Luo et al. (2009).

The donor strain T. leycettanus JCM 12802 was purchased from Japan Collection of Microorganisms RIKEN BioResource Center (Tsukuba, Japan). It was cultured at 42 °C in potato dextrose broth (PDB) for genomic DNA extraction, or inducing medium containing 5.0 g/L (NH4)2SO4, 1.0 g/L KH2PO4, 0.5 g/L MgSO47H2O, 0.2 g/L CaCl2, 10.0 mg/L FeSO47H2O, 30.0 g/L wheat bran, 30.0 g/L soybean meal, and 30 g/L corncob for complementary DNA preparation. The DNA isolation kit and DNA Pfu polymerase were purchased from Tiangen (Beijing, China). The plasmids pEASy-T3 (TransGen, Beijing, China) and pPIC9 (Invitrogen, Carlsbad, CA) were used as cloning and expression vectors, respectively. Escherichia coli Trans1-T1 (TransGen) was grown in Luria-Bertani (LB) medium at 37 °C for gene cloning and sequencing. P. pastoris GS115 (Invitrogen) cultivated in yeast peptone dextrose (YPD) medium at 30 °C was used for gene expression. The media for heterologous gene expression were prepared according to the manual of the Pichia Expression kit (Invitrogen). Birchwood xylan from Sigma-Aldrich (St. Louis, MO) was used as the substrate. The DNA purification kit, Genome Walking kit, and LA Taq DNA polymerase were purchased from

2.2. Cloning of the DNA and cDNA of xylanase-encoding gene Tlxyn10A The genomic DNA of T. leycettanus JCM12802 was extracted as described by Cubero et al. (1999) and used as the PCR template. The core region of the xylanase-encoding gene Tlxyn10A was amplified by a degenerate primer set specific for fungal GH10 endo-xylanases (Luo et al., 2009). PCR products (500 bp) were purified, ligated with pEASY-T3 vector, and transformed into E. coli Trans1-T1 for sequencing. Thermal asymmetric interlaced (TAIL)-PCR was then conducted to obtain the 50 and 30 flanking regions of the core region with the TaKaRa genome walking kit and a few specific primers (Table S1). The integrated gene fragment was recovered by assembling the 50 and 30 flanking and core regions. Three-day-old mycelia of T. leycettanus JCM12802 were harvested and ground to a fine powder in liquid nitrogen. The total RNA was extracted and purified using the SV Total RNA Isolation System (Promega) according to the manufacturer’s instructions. The total RNA was then used as a template for cDNA synthesis with the ReverTra Ace-a-TM kit (TOYOBO, Osaka, Japan). The cDNA fragment coding for the mature protein was amplified using the specific primers Tlxyn10AF and Tlxyn10AR as shown in Table S1. The PCR products were purified and ligated into the pEASy-T3 vector for sequencing. 2.3. Sequence analysis The sequence assembly was performed using the Vector NTI (Invitrogen). The nucleotide sequence was translated into amino acids using the ExPASy Translate tool (http://web.expasy.org/tanslate/). The nucleotide and protein sequences were aligned using the BLASTx and BLASTp programs (http://www.ncbi.nlm.nih.gov/ BLAST/), respectively. The signal peptide was predicted using the SignalP (http://www.cbs.dtu.dk/services/SignalP/). Multiple sequence alignments were performed with ClustalW (http:// www.clustal.org/).

2.5. Purification of recombinant TlXyn10A The induced culture was centrifuged at 12,000g for 10 min at 4 °C to remove cell debris, followed by concentration through a Viva flow 200 ultrafiltration membrane (cutoff 10 kDa; Vivascience, Hannova, Germany) and dialysis in 20 mM citric acidNa2HPO4 (buffer A; pH 6.6) at 4 °C overnight. The crude enzyme was loaded onto a HiTrap Q Sepharose XL 5 mL FPLC column (GE

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Healthcare, Uppsala, Sweden) equilibrated with buffer A. A linear gradient of NaCl (0–1.0 M) was used to elute the proteins. Fractions bearing xylanolytic activity were pooled, dialyzed in buffer A, and concentrated by ultrafiltration at 4000g for 40 min at 4 °C using an Amicon Ultra Centrifugal Filter Device PL-10 (Millipore, Billerica, MA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with the 5% stacking gel and 12% separation gel. Protein concentration was determined using the Bradford method with bovine serum albumin as the standard. 2.6. Biochemical characterization of purified recombinant TlXyn10A The standard assay for xylanase activity was performed at 80 °C for 10 min in citric acid-Na2HPO4 (pH 4.5) containing 1.0% (w/v) beechwood xylan. The reducing sugar released was determined using 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). For assays of pH adaptability and stability, glycine-HCl (pH 1.0–2.5), citric acid-Na2HPO4 (pH 2.5–9.0) and glycine-NaOH (pH 9.0–10.5) were used. The optimal pH for TlXyn10A activity was determined at 70 °C and pH 2.0–7.0. The optimal temperature for TlXyn10A activity was determined by performing the activity assay at 40– 90 °C at the optimal pH. The pH stability of TlXyn10A was determined by pre-incubating the enzyme at 37 °C for 1 h in buffers of pH 1.0–10.5 and measuring the residual activities under 80 °C and pH 4.5 for 10 min. Thermal stability was monitored by assessing the residual xylanase activities under standard conditions after incubating the enzyme at 85 °C, 90 °C or 95 °C for 1 h at the concentration of approximately 50 lg/mL without the presence of substrate. The effect of different metal ions and chemical reagents on the TlXyn10A activity was determined in the presence of 5 mM of NaCl, KCl, LiCl, MgSO4, CaCl2, HgSO4, AgNO3, ZnSO4, FeCl3, NiSO4, CuSO4, SDS, EDTA, and b-mercaptoethanol. The reaction systems without any chemical addition were treated as the control. 2.7. Homology modeling and structure analysis Modeller 9.13 was used to build the homology model of TlXyn10A. Pymol 0.99rc was employed to represent the homology model and align structures. The catalytic amino acid residues and substrate binding sites were predicted by Hotspot Wizard (Pavelka et al., 2009) (http://loschmidt.chemi.muni.cz/hotspotwizard/). The conserved substrate contacting residues were also identified by Hotspot Wizard. 2.8. Site-directed mutagenesis Three mutants (TlXyn10A_P, TlXyn10A_N and TlXyn10A_C) harboring different mutation combinations were then generated by site-directed mutagenesis using overlap PCR with Pfu polymerase (Tiangen) with primers in Table S1. Heterologous expression and biochemical characterization of mutant enzymes followed the procedures as described above.

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heated over the temperature range of 30–100 °C at a rate of 1 °C/ min in Nano-DSC (TA Instruments, New Castle, DE). The experiment was repeated at least twice. 2.11. Hydrolysis of wheat straw TlXyn10A_P and a commercial cellulase (Shengdong, Hunan, China) were used to degrade the steam explosion pretreated wheat straw. The experiment groups were designed as follows: CK, no enzyme added; Cellulase, 100 U of cellulase; TlXyn10A_P, 100 U of TlXyn10A_P; TlXyn10A_P + Cellulase, 50 U of each enzyme. The 50-mL reaction systems containing 1 g wheat straw in 100 mM citric acid-Na2HPO4 buffer (pH 4.5) with/without enzyme(s) were incubated at 70 °C for 8 h. Aliquots (1 mL) were removed at 1-h intervals, and the amounts of reducing sugar were determined by the DNS method (Miller, 1959). After 8-h hydrolysis, the enzymes were removed from the reaction system through the Nanosep centrifugal 3 K device (Pall, New York, NJ). The end products were analyzed by high-performance anion-exchange chromatography equipped with a pulsed amperometric detector ICS-5000 (HPAEC-PAD) (Dionex, Sunnyvale, CA). Aliquots (500 ll) of diluted hydrolysate samples were eluted by 100 mM NaOH at the rate of 1 mL/min and at the temperature of 22 °C. Glucose, xylose, xylooligosaccharides and cellooligosaccharides with the polymerization degree of 66 were used as the standards. Each experiment was repeated three times. 3. Results and discussion 3.1. Gene cloning and sequence analysis The full-length cDNA of Tlxyn10A (GenBank accession No. KX228205) was successfully amplified by specific primers TlXyn10AF and TlXyn10AR (Table S1). TlXyn10A contains 1095 bp that encodes a 364-residue polypeptide. The first 17 amino acid residues were predicted to be a putative signal peptide, and the mature protein was predicted to have a calculated molecular mass of 38.7 kDa. The deduced amino acid sequence of TlXyn10A is most similar to a structure-resolved GH10 endo-1,4-b-xylanase 4F8X of Penicillium canescens (76% identity). 3.2. Expression and purification of the recombinant protein The gene fragment coding for the mature TlXyn10A without signal peptide was successfully obtained from cDNA by PCR with primers TlXyn10AEcoF and TlXyn10ANotR (Table S1) and expressed in P. pastoris GS115. Substantial xylanase activity (180 U/mL) was detected in the cultures of positive transformants. Recombinant TlXyn10A was then purified to electrophoretic homogeneity and detected by SDS-PAGE. As shown in Fig. 1, recombinant TlXyn10A was highly glycosylated; after Endo H treatment, the enzyme migrated a single band with apparent molecular weight of 39 kDa, which is in agreement with its calculated value (38.7 kDa).

2.9. Molecular dynamics (MD) simulation 3.3. Biochemical properties of TlXyn10A Amber 14 package was used to carry out MD simulation at a temperature of 353 K for a 20 ns process with force fields ff99SB, gaff and GLYCAM_06j-1. Trajectory data were analyzed with CPPTRAJ software (Roe and Cheatham, 2013). 2.10. Differential scanning calorimetry (DSC) analysis The melting temperatures (Tm) of TlXyn10A and TlXyn10A_P were analyzed by using DSC. The proteins were diluted in 10 mM citric acid-Na2HPO4 (pH 6.0) to approximately 0.25 mg/mL, and

By using beechwood xylan as the substrate, TlXyn10A exhibited maximum activities at pH 4.5 (Fig. 2a) and 80 °C (Fig. 2b), and remained more than 70% activity over the acidic (pH 4.0–5.0) and thermophilic (70–85 °C) ranges. TlXyn10A showed stability over a broad pH range of 2.0–8.0, retaining more than 80% of the initial activity after incubation at 37 °C for 1 h (Fig. 2d). Moreover, the enzyme was stable at high temperatures, retaining 81.7%, 74.8% and 58.2% of its initial activity after incubation for 1 h at 85 °C, 90 °C and 95 °C, respectively (Fig. 2c).

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Fig. 1. SDS-PAGE analysis of TlXyn10A and its mutants. Lanes: M, the molecular mass standards; 1, 4, 7 and 10, the crude enzymes; 2, 5, 8, and 11, the purified recombinant enzymes; 3, 6, 9, and 12, the deglycosylated enzymes with Endo H treatment.

Fig. 2. Characterization of the purified recombinant TlXyn10A and its mutants with and without N-deglycosylation. (a) Effect of pH on the xylanase activities. (b) Effect of temperature on the xylanase activities. (c) Thermostability of the xylanase activities. (d) pH stability of TlXyn10A and TlXyn10A_P.

Thermostable xylanases are applied in broad fields including brewing industry, food and feed production (Collins et al., 2005; Du et al., 2013; Passos et al., 2015; Rodríguez et al., 2012; Zhao et al., 2013), pharmacy manufacture (Christakopoulos et al., 2003), pulp bleaching, waste treatment and bioconversion (Gupta et al., 2014; Kaushik et al., 2014). Disulfide bonds are considerate to be vitally important in maintaining protein thermostability (Hattori et al., 2015; Wang et al., 2012b; Yin et al., 2015). The three putative disulfide bonds of TlXyn10A may contribute to its stability (Fig. 3b). To obtain xylanases with excellent thermostability, two common strategies, i.e. gene mining and protein engineering, are commonly used. The pH and temperature optima of this enzyme are similar to that of xylanases from Malbranchea pulchella (Ribeiro et al., 2014), Thermoascus aurantiacus (Gomes et al.,

1994), and Penicillium funiculosum (Lafond et al., 2011). However, TlXyn10A is more thermostable than most known thermophilic xylanases, including MpXyn10A (Ribeiro et al., 2014), GtXyn10 (Wang et al., 2015) and CbXyn10C (Xue et al., 2015). The specific activity, Vmax, Km, and kcat/Km values of TlXyn10A were also determined to be 2240 ± 34 U/mg, 2542 ± 22 U/mg, 1.01 ± 0.05 mg/mL and 1626 ± 81 mL/s/mg, respectively (Table 1). TlXyn10A displayed strong resistance to all tested metal ions and chemical reagents b-mercaptoethanol and EDTA, and the enzymatic activity was promoted by b-mercaptoethanol (1.64-fold) and Na+ (1.22-fold), respectively. However, it was completely inhibited by SDS (Table 2). The relationship between thermostability and SDS resistance is not validated yet (Zheng et al., 2015).

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Fig. 3. Structure analysis of modeled TlXyn10A. (a) The catalytic cleft of TlXyn10A binding a xylopentaose at subsites 1 to +4. The putative catalytic residues and mutated sites are indicated. (b) The opposite side of modeled TlXyn10A with three disulfide bonds indicated. (c) Xylopentaose binding in the catalytic cleft by forming hydrophobic interaction with F232 and a hydrogen bond with E229. (d) Xylopentaose forming a hydrogen bond with substituted D149 via its thermal motion and the movement of loop 4. Table 1 The specific activities and kinetic values of purified TlXyn10A and its mutants.a

a

Sample

Specific activity (U/mg)

Vmax (U/mg)

Km (mg/mL)

kcat/Km (mL/s/mg)

TlXyn10A TlXyn10A_P TlXyn10A_N TlXyn10A_C

2240 ± 34 3232 ± 76 1327 ± 32 1769 ± 16

2542 ± 22 3852 ± 55 1520 ± 24 2015 ± 24

1.01 ± 0.05 1.49 ± 0.06 1.09 ± 0.06 1.11 ± 0.04

1626 ± 81 1665 ± 61 901 ± 50 1175 ± 42

Data are shown as mean ± standard deviation (n = 3).

Table 2 Effect of metal ions and chemical reagents (5 mM) on the activity of purified recombinant TlXyn10A. Chemicals

Relative activity (%)a

Control Na+ Mg2+ Mn2+ K+ Ni2+

100.0 ± 1.6 122.3 ± 1.9 115.5 ± 4.5 115.0 ± 1.6 111.3 ± 5.1 110.6 ± 9.4

Cr3+ Zn2+

107.0 ± 5.9 104.0 ± 1.0

Chemicals 2+

Ca Ag+ Fe3+ Cu2+ Pb2+ bMercaptoethanol EDTA SDS

Relative activity (%) 102.8 ± 7.6 98.5 ± 3.2 95.8 ± 1.0 95.1 ± 6.3 93.2 ± 1.2 164.1 ± 4.6 102.2 ± 1.7 0.9 ± 0.4

a Values represent the mean ± standard deviation (n = 3) relative to the untreated control samples.

rel (TIM barrel) of typical GH10 members. Based on the Hotspot Wizard analysis, two conserved catalytic glutamates (Glu145 and Glu257) were found on strands b-4 (acid/base) and b-7 (nucleophile), respectively (Fig. 3a), and nine conserved residues Asn60, Lys63, His96, Trp100, Asn144, His227, Glu225, Trp300, and Trp308 probably involved in catalysis and substrate binding were identified. Deduced TlXyn10A contains three disulfide bonds (Fig. 3b). Disulfide bonds are considerate to be vitally important in maintaining protein thermostability (Hattori et al., 2015; Wang et al., 2012b; Yin et al., 2015). The three putative disulfide bonds of TlXyn10A may contribute to its stability. 3.5. Identification of non-conserved xylan contacting residues and mutant designing

3.4. Homology modeling and modeled structure analysis The modeled structure of TlXyn10A was built with 4F8X and 1E5N as the templates. Modeled TlXyn10A folded into a (a/b)8 bar-

To identify non-conserved xylan contacting residues, six GH10 xylanase-xylooligosaccharide complexes (PDB IDs: 1E5N, 1US2, 3MMD) were aligned with the modeled structure using pymol

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Fig. 4. The distances between the Cb of key residues and the O atom of ligand calculated by the molecular dynamics simulation. (a) N148 (the wild type)/D149 (the mutant) and ligand. (b) N197 (the wild type)/Y197 (the mutant) and ligand. (c) E229 (the wild type)/I229 (the mutant) and ligand. (d) F232 (the wild type)/E232 (the mutant) and ligand.

3.6. Comparison of the enzymatic properties of wild type and mutant enzymes

Fig. 5. The reducing sugar yields of different experiment groups over 8 h.

0.99rc (Fig. S1a), and their ligands were used as probes to pick up xylan contacting residues. Residues of TlXyn10A near these ligands within 6 Å or on the flexible loops were selected as potential substrate contacting sites (Fig. S1b). After excluding highly conserved ones, seven residues (A101, Q103, N148, G149, N197, E229 and F232) were selected to design the mutants. Commonly, it is difficult to decide which amino acid to replace the original one when the mutable sites were determined. The BLASTp program was carried out to search for reference protein sequence. Of all sequences with >50% similarity, Xyl10C demonstrated the most thermostable and with great catalytic performance (Luo et al., 2009). Sequence alignment (Fig. S2) of TlXyn10A to the Xyl10C (indicated the potential substituents of these residues, and three mutants of different mutation combinations, i.e., TlXyn10A_P (N148S, G149D, N197Y, E229I and F232E from +2 to +4 site), TlXyn10A_N (A101Q and Q103E from 1 to 2 site on the loop 3), and TlXyn10A_C (A101Q, Q103E, N148S, G149D, N197Y, E229I and F232E) (Fig. 3a) were designed, and generated by overlap PCR.

The recombinant mutant enzymes were purified and characterized as described above. All mutants exhibited maximum activities at pH 4.5 and 80 °C as the wild type (Fig. 2a, b). TlXyn10A_P showed greater adaptability to higher temperature. The mutants were stable at 75 °C (Fig. 2c). Of them, TlXyn10A_P showed similar thermostability to TlXyn10A. DSC analysis over the temperature range of 30–100 °C indicated that the Tm values of TlXyn10A and TlXyn10A_P were 74.11 °C and 72.93 °C (Fig. S3), respectively. TlXyn10A_P also had a widened pH stability profile (Fig. 2d). When compared with the wild type, TlXyn10A_P retained 15% and 30% more activities with the respective treatment of citric acidNa2PO4 (pH 2.5) and Gly-NaOH (pH 10.0). Moreover, TlXyn10A_P shower greater stability than GH10 xylanases from Rhizopus oryzae (Xiao et al., 2014) and Sphingobacterium sp. TN19 (Luc et al., 2009) at pH lower than pH 4.0 and higher than pH 8.0. The specific activities and kinetic values of TlXyn10A mutants towards birchwood xylan were also compared with that of the wild type (Table 1). All mutants showed decreased substrate affinity, and mutant TlXyn10A_P had improvement in specific activity, and velocity. Catalytic performance is generally elevated by decreasing the Km values or synchronously decreasing Km and increasing Vmax (Choi et al., 2016; Hoffmam et al., 2016; Xu et al., 2016). However, in this study, TlXyn10A_P is low in substrate affinity but high in reaction velocity. 3.7. MD simulation Modeled TlXyn10A and TlXyn10A_P were subjected to the 20 ns MD simulation using the Amber 14 package. As shown in Fig. 4, mutations N148S/G149D caused loop 4 much closer to the ligand, while E229I and F232E demonstrated impaired binding ability to

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Fig. 6. HPAEC analysis of the hydrolysis products of wheat straw. 1, the xylooligosaccharide and cellooligosaccharide standards; 2, the control without enzyme addition; 3–5, the hydrolysis products released from cellulase group, TlXyn10A_P group, and TlXyn10A_P and cellulase group, respectively. G1, glucose; G2, cellobiose; X1, xylose; X2; xylobiose; and X3, xylotriose.

the ligand when compared to the wild type. Because the distances between Cb at position 229 or 232 and xylopentaose were quite stable in wild type TlXyn10A while those of TlXyn10A_P were expanded and more fluctuant. TlXyn10A is more likely to trap substrate in the catalytic cleft. The MD simulation results are consistent with the experimental kinetics. It revealed that TlXyn10A_P harboring mutations E229I and F232E might lose substrate binding at subsites +3 to +4 (Fig. 4c, d). Moreover, TlXyn10A_P lost a hydrogen bond between E229 and substrate and hydrophobic interaction of F232 (Fig. 3c); instead, G149D on the loop 4 is able to form a hydrogen bond with substrate (Fig. 3d). Therefore the movement of loop 4 containing N148S and G149D may play a role in the efficient products release (Fig. 3d), making contribution to the great increase in reaction velocity. Of all mutations, E229I and F232E showed the most significant distance curves between the wild type and mutant enzymes, and probably play a major role in the improvement of catalytic performance. Namely, the improvement of Vmax might be mainly caused by the weakened affinity of mutant at subsites +3 to +4. Of the seven selected residues for mutagenesis, Q103 are conserved of GH10 xylanases, while others are not. Mutations A101Q and Q103E showed negative effects on enzyme catalysis of TlXyn10A_N. Multiple sequence alignment of 50 protein sequences with Hotspot Wizard indicated that the residues corresponding to the non-conserved N148, G149, E229 and F232 of TlXyn10A are Asn with the frequency of Asn (72.0%), Asp or Glu (91.0%), Ile (67.5%) and Glu (30.5%). The four corresponding residues in highly active AuXyn10A from Aspergillus usamii (Wang et al., 2013a) are Asn, Glu, Ser and Gly, respectively, while CbXyn10C from Caldicellulosiruptor bescii (Xue et al., 2015) with high Vmax and low Km values have Asp, Glu, Asn and Trp, respectively. The big variations of these non-conserved residues suggest that the catalytic behavior of GH10 xylanases is far more complicated. It inferred that Asp and Glu at positions 148 and 149 might be involved in product release, while Asn and Trp at positions 229 and 232 are related to ligand binding.

239.3 lg/g reducing sugar after 8 h reaction. The highest reducing sugar yield (518.9 lg/g biomass) was achieved by the enzyme combination of TlXyn10A_P and cellulase at 8 h, which is higher than the cellulase alone. The hydrolysis products of wheat straw by commercial cellulase, TlXyn10A_P and their combination were also analyzed by HPAEC-PAD. Glucose (232.8 lg/g), xylose (43.9 lg/g) and cellobiose (38.2 lg/g) were the main products of cellulase group. In contrast, the xylanase group mainly produced xylose (48.8 lg/g) and xybiose (125.5 lg/g). When combined both enzymes at half dosage, more reducing sugars were released, including 251.0 lg/g glucose, 43.4 lg/g cellobiose, 106.9 lg/g xylose, and 94.2 lg/g xylobiose (Fig. 6). TlXyn10A_P yielded reducing sugar at 70 °C and pH 4.5, meeting the demand of hemicellulose decomposition (high temperature and diluted-acid), and enzymatic activity could reduce the consumption of H2SO4 (Talebnia et al., 2010). Most of the biomass degradation were carried out at a lower temperature (Gonçalves et al., 2015; Sharma et al., 2015), but TlXyn10A_P hydrolyzed the wheat straw for eight hours at 70 °C, for the higher reaction temperature would lead to lower related cost after elevated temperature pretreatment.

3.8. Synergetic action towards wheat straw hydrolysis

Acknowledgements

In order to study the cooperative effects of TlXyn10A_P and cellulase, a simulated biomass degradation experiment was carried out. As shown in Fig. 5, the commercial cellulase showed great capacity to degrade wheat straw, releasing 335.1 lg/g of reducing sugar within 5 h. After that, no more reducing sugar was released. TlXyn10A_P was able to hydrolyze the wheat straw, releasing

This work was supported by the National Natural Science Foundation of China (no. 31472127) and the National High-Tech Research and Development Program of China (863 Program, no. 2013AA102803) and the National Science Fund for Distinguished Young Scholars of China (no. 31225026) and the China Modern Agriculture Research System (no. CARS-42).

4. Conclusion A highly thermostable xylanase, TlXyn10A, was identified in T. leycettanus JCM12802 and successfully produced in P. pastoris. Recombinant TlXyn10A demonstrated excellent thermostability and resistance to all tested metal ions. Mutation of some potential ligand contacting residues, i.e., E229I and F232E, caused weakened affinity at subsites +3 to +4, promoted reaction velocity, and increased high specific activity. Besides the improved catalytic performance, the pH stability of mutant TlXyn10A_P was also enhanced, and demonstrated persistent hydrolysis of wheat straw for up to 8 h. All these properties make TlXyn10A_P an excellent candidate to be potentially applied in various industrial fields.

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