Protein Expression and Purification 94 (2014) 40–45

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Cellulose-inducible xylanase Xyl10A from Acremonium cellulolyticus: Purification, cloning and homologous expression Seiichiro Kishishita a, Miho Yoshimi a, Tatsuya Fujii a, Larry E. Taylor II b, Stephen R. Decker b, Kazuhiko Ishikawa a, Hiroyuki Inoue a,⇑ a b

Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA

a r t i c l e

i n f o

Article history: Received 8 August 2013 and in revised form 26 October 2013 Available online 7 November 2013 Keywords: Thermostable xylanase Hemicellulose Homologous expression Glycoside hydrolase family 10 Glycosylation

a b s t r a c t Cellulose-inducible endo-b-1,4-xylanase (Xyl10A) from the mesophilic fungus Acremonium cellulolyticus was purified, characterized, and expressed by a homologous expression system. A. cellulolyticus CF2612 produces a high level of xylanase upon induction by Solka-Floc cellulose. To identify this xylanase, the major fraction showing xylanase activity was purified from the CF-2612 culture supernatant, and its gene was identified from the genome sequence. Amino acid sequence homology of Xyl10A revealed that the purified xylanase, designated Xyl10A, exhibited significant homology to family 10 of the glycoside hydrolases (GH10), possessing a cellulose-binding module 1 in the C-terminal region. The xyl10A gene was cloned and expressed in A. cellulolyticus under the control of a glucoamylase promoter. Two recombinant Xyl10As (rXyl10A-I, 53 kDa, and rXyl10A-II, 51 kDa) were purified that have slightly different molecular weights based on SDS–PAGE. The rXyl10As had the same physicochemical and enzymatic properties as wtXyl10A: high thermostability (Tm 80.5 °C), optimum pH 5.0 and specific activity 232– 251 U/mg for birchwood xylan. The molecular weights of N-deglycosylated rXyl10As were consistent with that of wild-type Xyl10A (wtXyl10A, 51 kDa). Ó 2013 Elsevier Inc. All rights reserved.

Introduction Xylan is one of the major structural components of plant cell walls and the second most abundant renewable resource in nature. It consists of a backbone of b-1,4-D-xylose with short side chains of O-acetyl, a-L-arabinofuranosyl, D-a-glucuronic acid and phenolic acid [1]. Xylanases (endo-1,4-b-xylanases; EC 3.2.1.8), which catalyze the hydrolysis of b-1,4 bonds of xylan, are important enzymes for the degradation of hemicellulosic polysaccharides. Based on amino acid sequence similarities, most xylanases are classified into families 10 and 11 of the glycoside hydrolases (GH; http:// www.cazy.org/Glycoside-Hydrolases.html; [2]). GH10 xylanases generally have a molecular weight P30 kDa and a low pI. GH11 xylanases are generally smaller (approximately 20 kDa) and have a high pI [3]. The crystal structures of xylanases show that GH10 enzymes fold into a (b/a)8-barrel [4,5], whereas family 11 enzymes have a b-jelly roll structure [6]. Cellulose and xylan are linked together to form a lingocellulosic structure in plant cell walls [7]. Xylanase has been reported to act Abbreviations used: SF, Solka-Floc cellulose; CMC, carboxymethylcellulose; TSA, thermal shift assay; AA, amino acids. ⇑ Corresponding author. Fax: +81 82 423 7820. E-mail address: [email protected] (H. Inoue). 1046-5928/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2013.10.020

coordinately with cellulase in enzymatic degradation of lignocellulosic biomass such as a pretreated plant residue. Addition of xylanase activity to cellulose cocktails has been shown to synergistically improve cellulose digestion in saccharifications of lingocellulosic biomass [8]. Xylanase from Cellulomonas flavigena has showed a high degree of synergy (6.3-fold) with endoglucanases from Hypocrea jecorina during hydrolysis of alkaline pretreated sugarcane bagasse [9]. Furthermore, it has been reported that the expression of fungal cellulolytic and xylanolytic genes is related at the transcriptional level. A transcriptional activator, XlnR from Aspergillus niger, mediates the xylan-dependent expression of the xylanolytic genes as well as the cellulolytic genes [10]. Xyn III (a GH10 xylanase) from Trichoderma reesei PC-3–7 is induced by Avicel, L-sorbose and sophorose, but not by xylose, xylooligosaccharides or birchwood xylan [11]. These studies strongly suggest that xylanase plays important roles in the degradation of lingocellulosic biomass by fungal cellulase system. Acremonium cellulolyticus, isolated by Yamanobe et al. in 1982, is one of the high cellulolytic enzyme-producing fungi [12]. Fujii et al. reported that the culture supernatant from A. cellulolyticus has higher cellulase specific activity and yields more glucose from lignocellulosic materials than the culture supernatant from T. reesei [13]. The mutant strain A. cellulolyticus CF-2612, which is the most

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efficient producer of cellulolytic enzymes within this species, was isolated from the mutant strain C1 by random mutagenesis [14]. The culture supernatant of CF-2612 grown in the presence of Solka-Floc (SF) cellulose has showed four times higher specific activity of xylanase than wild type, concomitant with improvement in glucose production yield from lignocellulosic materials [13,15]. This cellulose-induced xylanase may play an important role in cellulolytic biomass degradation of A. cellulolyticus. We have now purified and characterized cellulose-inducible GH10 xylanase (Xyl10A) from A. cellulolyticus, and herein report a comparison of the physicochemical and enzymatic properties between wild-type Xyl10A (wtXyl10A) and recombinant Xyl10A (rXyl10A). Both purified enzymes showed high thermostability (Tm 80.5 °C). Materials and methods Materials Birchwood xylan, carboxymethylcellulose (CMC) and Avicel PH101 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Xyloglucan (from tamarind) and low-viscosity wheat arabinoxylan were purchased from Megazyme (Wicklow, Ireland). All other chemicals were of the highest grade commercially available. Strain and culture conditions A. cellulolyticus CF-2612 was maintained on potato dextrose agar plates [14]. The A. cellulolyticus YP-4 uracil autotroph was maintained on potato dextrose agar plates containing uracil and uridine at final concentrations of 1 g/L each [16]. Transformants of A. cellulolyticus YP-4 were maintained on MM agar plates [17]. Purification and identification of wtXyl10A A. cellulolyticus CF-2612 was grown at 30 °C on medium (pH 4.0) containing, per liter: 50 g Solka Floc (SF; Fiber Sales & Development, Urbana, OH, USA), 24 g KH2PO4, 1 g Tween 80, 5 g (NH4)2SO4, 1.2 g MgSO47H2O, and 4.0 g urea. The culture supernatant was clarified by filtration (using a 0.22 lm filter) and then desalted on an AKTA purifier (GE Healthcare, Buckinghamshire, UK) using a HiPrep 26/10 Desalting column (GE Healthcare) equilibrated with 20 mM 2-(N-morpholino)ethanesulfonic acid buffer, pH 6.5, containing 0.01% NaN3. The desalted sample was applied to a Resource Q column (6 ml, GE Healthcare) equilibrated with the same buffer. Xylanase activity was observed in the flow-through fractions. The active fractions were pooled and concentrated by a Vivaspin 20 concentrator (10,000 MWCO, Sartorius AG, Goettingen, Germany). An equal volume of 2.0 M ammonium sulfate was added to pooled fractions and applied to a Resource ISO hydrophobic interaction column (10 ml, GE Healthcare) equilibrated with 1.0 M ammonium sulfate in 20 mM sodium acetate (pH 5.5). The bound protein was eluted with a 300 ml linear gradient of 1.0–0.2 M ammonium sulfate. The peak of xylanase activity was at 0.70 M ammonium sulfate. The purified enzyme was concentrated, dialyzed against 20 mM sodium acetate buffer (pH 5.0), and stored at 4 °C until use. The purity and size of the protein was analyzed by SDS–PAGE using precast NuPAGE 4–12% polyacrylamide Bis–Tris gels (Life Technologies, Carlsbad, CA, USA). Xyl10A was identified by Stanford Mass Spectrometry Services (Stanford University, Stanford, CA, USA). Trypsin-digested peptide fragments of the purified enzyme were analyzed using an electrospray ionization-quadrupole time-of-flight (Micromass Q-Tof) mass spectrometer (Micromass UK, Manchester, UK). Scaffold 3 software (Proteome Software, Portland, OR, USA) was used to validate protein identification. A draft

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genome sequence of A. cellulolyticus (unpublished data) was searched for the xyl10A coding region using In silico Molecular Cloning gene analysis software (in silico biology Inc., Yokohama, Japan) based on the internal amino acid sequence. The sequence of the genomic region encoding xyl10A was deposited to the GenBank/EMBL/DDBJ database under accession number AB796434. For phylogenetic analysis, amino acid sequences of 55 fungal GH10 xylanases were aligned and a maximum likelihood tree was calculated by program MAFFT [18]. Cloning of xyl10A and construction of expression vector Construction of a xyl10A expression vector for A. cellulolyticus was performed as described previously [16]. The genomic region encoding xyl10A was amplified from CF-2612 chromosomal DNA using the forward primer 50 -ATTGTTAACAAGATGACTCTAGT AAAGGCTATTC (with an HpaI site underlined) and the reverse primer 50 -AATCCTGCAGGTTACAAACATTGGGAGTAGTATGG (with an SbfI site underlined). The expression plasmid pANC208 was constructed by introducing the xyl10A fragment digested with HpaI/ SbfI into the EcoRV/SbfI site of pANC202 containing a glucoamylase (glaA) promoter and terminator. All ligated gene fragments and their ligation sites were verified by sequencing. Homologous expression and purification of rXyl10A Protoplasts of A. cellulolyticus YP-4 were transformed with pANC208 by nonhomologous integration into the host chromosomal DNA [17]. Gene integration into prototrophic transformants was verified by genomic PCR. Chromosomal DNA of the transformants was purified using the Gentra Puregene Yeast/Bact. Kit (Qiagen). rXyl10A-I and rXyl10A-II were purified from culture supernatant of Y208 (YP-4 transformed with pANC208) grown in medium containing 20 g/L soluble starch (Wako Pure Chemical Industries, Osaka, Japan) and 2.0 g/L urea. rXyl10As were purified and stored using the same procedure used for wtXyl10A. Enzyme and protein assay Xylanase activity was measured at 45 °C in 50 mM sodium acetate buffer (pH 5.0) containing 1% (w/v) birchwood xylan (Sigma– Aldrich) as the standard assay conditions. The substrate specificity of Xyl10A was examined using 1% (w/v) CMC, 1% (w/v) Avicel, or 1% (w/v) xyloglucan. The reducing sugars produced in the reaction mixture were measured by the dinitrosalicylic acid assay [14]. Protein concentration was determined by the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA) using bovine serum albumin as the standard. One unit of enzyme activity was defined as the amount producing 1 lmol reducing sugar per min. Effects of pH and temperature The optimal pH for Xyl10A activity was measured using a dinitrosalicylic acid assay with 1% birchwood xylan in McIlvaine’s buffer in a pH range of 2.0–8.0 [19]. In order to determine the stability with respect to pH, purified xylanases were incubated in McIlvaine’s buffer in a pH range of 2.0–8.0 at 4 °C for 24 h, and then the residual activities of treated enzymes were measured by the standard assay procedure. The optimal temperature for Xyl10A activity was determined by the standard assay procedure at temperatures ranging from 40 to 90 °C. For the temperature stability assay, the assay conditions were essentially the same as described above except that the enzyme was exposed to temperatures ranging from 30 to 90 °C without the substrate. The remaining activity was then assayed after a 10 min exposure. A protein thermal shift assay (TSA) [20] was performed using a CFX Connect real-time PCR

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detection system (Bio-Rad, Hercules, CA, USA) as described previously [16].

Table 1 Physicochemical and enzymatic properties of Xyl10As from A. cellulolyticus. Properties

wtXyl10A

rXyl10A-I

rXyl10A-II

Molecular weight (kDa) Thermostabilityb Optimum pH

51 80.5 5

53 80.5 5

51 80.5 5

Substrate specificity (U/mg) Xylan (birchwood) CMC Avicel Xyloglucan

232 n.d.c n.d. n.d.

251 n.d. n.d. n.d.

250 n.d. n.d. n.d.

a

Deglycosylation assay Purified Xyl10A was N-deglycosylated using Endo Hf (70 kDa, New England BioLabs Inc., Ipswich, MA, USA) following the manufacturer’s instructions. Protein samples were denatured by heating at 94 °C for 10 min with 0.5% SDS and 40 mM dithiothreitol. Xyl10A (3 lg) was treated with Endo Hf (2 U) in 50 mM sodium citrate buffer (pH 5.5) for 4 h at 37 °C. BSA (68 kDa, 10 lg) was added for Endo Hf stabilization. Deglycosylated and untreated Xyl10A were analyzed by SDS–PAGE. Results and discussion Purification and characterization of wtXyl10A in A. cellulolyticus To identify a cellulose-induced xylanase from A. cellulolyticus, a major fraction showing xylanase activity was purified to electrophoretic homogeneity from A. cellulolyticus CF-2612 cultures grown with SF. The molecular weight of purified wtXyl10A was estimated at 51 kDa (wtXyl10A) by SDS–PAGE (Fig. 1). Table 1 summarizes the properties of wtXyl10A. The specific activity of the purified protein was 232 U/mg for birchwood xylan. There was no activity toward CMC, Avicel or xyloglucan, implying that the cellulose-induced Xyl10A possesses high substrate specificity for xylan. The Tm of wtXyl10A was 80.5 °C based on TSA results. This high thermostability is beneficial for biomass saccharification at high temperature. Several xylanases have been purified and characterized from A. cellulolyticus in previous studies using both culture supernatant of Y-94 wild-type and commercial enzymes [21,15]. Nihira et al. reported finding three endo-xylanases (Xylanase I, Xylanase II and Xylanase III) from A. cellulolyticus [21]. The molecular weights of these xylanases ranged from 25.5 to 33.5 kDa and optimum temperatures were 50–55 °C. Mitsuishi et al. also reported finding three thermostable xylanases (Xn-A, Xn-B and Xn-C) from A. cellulolyticus [15]. Xn-A, B and C have molecular weights ranging from 35 to 51 kDa, with highest activity

M

1

2

3

M

a

The apparent molecular weight of the protein was determined by SDS–PAGE. Thermostability was determined by a thermal shift assay as described in ‘‘Materials and methods’’. c Not detected. b

at 80 °C and pH 4.9. Although wtXyl10A has similar properties to these enzymes, the amino acid sequences of Xn-A, B and C have not yet been reported. Identification of xyl10A Peptide fragment masses from purified wtXyl10A were assigned to the deduced amino acid sequence of a hypothetical protein in the A. cellulolyticus genomic sequence (Fig. 2), corresponding to 21% coverage of the sequence. The Xyl10A coding region in the chromosome included three introns (AB796434). xyl10A was 1221 bp, encoding 407 amino acids (AA) including a deduced signal peptide (19 AA), catalytic domain (315 AA), a serine/ threonine-rich linker region (38 AA), and a carbohydrate binding module 1 (CBM1, 35 AA) (Fig. 2). N118 was deduced as an N-glycosylation site by the NetNGlyc 1.0 server. Two catalytic glutamate residues (E148 and E255) were conserved in Xyl10A [22]. Based on a BLAST search, Xyl10A had significant identity to the following GH10 xylanases from fungi: 96% to Talaromyces funiculosus (CAG25554); 86% to Talaromyces stipitatus (XP_002484128); 85% to Talaromyces marneffei (XP_002150978); 64% to Penicillium digitatum (AFS18477.1); 63% to Aspergillus clavatus (XP_001268415); and 63% to Penicillium chrysogenum (XP_002564127). Thus, physiological properties and amino acid sequence homology indicate that Xyl10A belongs to GH10 family. Xyl10A from A. cellulolyticus showed highest homology to the xylanases from Talaromyces, suggesting that A. cellulolyticus is closely related to the Talaromyces genus. Phylogenetic analysis of GH10 xylanases Sato et al. reported that GH10 xylanases can be divided into two groups based on phylogenetic analysis of introns: group I contains

Fig. 1. SDS–PAGE of Xyl10A. Lanes: M, molecular weight marker; 1, wtXyl10A; 2, rXyl10A-I; 3, rXyl10A-II.

Fig. 2. Amino acid sequence of Xyl10A. Box, putative N-glycosylation site; dotted box, conserved catalytic glutamate residues; underlines, peptide identified by on mass spectrometry; dotted lines, putative signal peptide; italics, linker region; boldface, CBM1 region.

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enzymes from both bacteria and fungi, and group II contains only fungal enzymes [23]. The same analysis for Xyl10A from A. cellulolyticus indicated that Xyl10A belonged to group I of this classification. We constructed a cladogram from an alignment of the full-length amino acid sequences of 55 fungal GH10 xylanases including A. cellulolyticus Xyl10A (Fig. 3). Fungal GH10s were divided in three groups: group I (27 sequences) and group III (11 sequences) from the Ascomycota and group II from the Basidiomycota. Many fungal GH10 xylanases contain CBM1, and significant correlation was observed between this grouping of xylanases and the position of CBM1. GH10 proteins belonging to group I contained CBM1 at the C-terminus, whereas GH10 proteins belonging to group III had no CBM1. On the other hand, GH10 proteins belonging to group II contained CBM1 at the N-terminus. These results indicate that acquisition of CBM happened before speciation of the two phyla. Expression and characterization of rXyl10A A starch-inducible homologous expression system was used to produce rXyl10A. This expression system has the advantage of producing a recombinant protein without contamination of celluloseinducible cellulases and hemicellulases [16]. The genomic region including xyl10A was cloned downstream of the glaA promoter to construct the starch-inducible expression plasmid pANC208, and

rXyl10A was expressed in the culture supernatant of an A. cellulolyticus transformant by soluble starch induction. Following hydrophobic chromatography, two rXyl10A peaks (rXyl10A-I and rXyl10A-II) were observed and purified individually, although a single peak was observed for wtXyl10A. The molecular weights of rXyl10A-I and rXyl10A-II were estimated to be about 53 and 51 kDa, respectively, by comparison of their relative mobility in SDS–PAGE (Fig. 1). wtXyl10A and rXyl10A-II had similar apparent molecular weights while that of rXyl10A-I appeared slightly higher. This difference in size is due to differences in glycosylation, as discussed later. The enzymatic properties of rXyl10A-I and rXyl10A-II are summarized in Table 1. The optimum pH values and temperatures for wtXyl10A, rXyl10A-I and II showed a similar trend (Fig. 4). Interestingly, these xylanases retained more than 80% of their activity after incubation for 24 h at different pH values ranging from 2.0 to 9.0 (data not shown). The residual activities of wtXyl10A, rXyl10A-I and rXyl10A -II were, respectively 84.4%, 84.0% and 84.7% at 75 °C, and 10.0%, 9.3% and 10.0% at 80 °C (Fig. 5). These results correlated well with the Tm results (80.5 °C) of Xyl10As from a protein thermal shift assay (Table 1). Depending on optimal temperature, xylanases can be classified as mesophilic (40–60 °C), thermophilic (50–80 °C) or hyperthermophilic (>80 °C) [24]. Most xylanases from fungi are mesophilic with optimal temperatures typically between 50 and 60 °C [25], but several hyperthermophilic 1_AB796434_Acremonium_cellulolycus N

N N N N N N N N N N

N N N N N N N N N N N N C C C C C C C

Group I (Ascomycota)

Group II (Basidiomycota)

C

Group III (Ascomycota)

0.08 Fig. 3. Phylogenetic tree of GH10 family xylanases. A set of 55 GH10 family xylanases from the Ascomycota, Basidiomycota and bacteria were aligned using the MAFFT program [18]. Xylanase with C-terminal CBM1 and xylanase with N-terminal CBM1 are marked C and N, respectively.

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Fig. 4. Optimal temperature and pH of wtXyl10A and rXyl10As. (A) Effect of temperature on activity. Enzyme activity of wtXyl10A (circles), rXyl10A-I (squares), and rXyl10AII (triangles) were measured at various temperatures at pH 5.0. (B) Effect of pH on activity. Enzyme activity of wtXyl10A (circles), rXyl10A-I (squares), and rXyl10A-II (triangles) were measured across a pH range of 2.0–9.0.

Fig. 6. SDS–PAGE of deglycosylated Xyl10A. Lanes: M, molecular weight marker; 1, wtXyl10A; 2, deglycosylated wtXyl10A; 3, rXyl10A-I; 4, deglycosylated rXyl10A-I; 5, rXyl10A-II; 6, deglycosylated rXyl10A-II.

Fig. 5. Thermostability of Xyl10A. The residual activities of wtXyl10A (circles), rXyl10A-I (squares), and rXyl10A-II (triangles) were measured at 50 °C after a 10 min heat treatment.

fungal GH10 xylanases have been reported. GH10 xylanase from Aspergillus fumigatus showed an optimum temperature of 90 °C, the highest reported optimum temperature of a fungal GH10 xylanase [26]. GH10 xylanase from Bispora sp. showed an optimum temperature of 85 °C and GH10 xylanase from T. funiculosus, which has significant homology with Xyl10A, showed an optimal temperature of 80 °C [27,28]. Xyl10A from A. cellulolyticus is thus on the high end of thermophilic group of xylanases.

Deglycosylation of wtXyl10A and rXyl10As Molecular weights of wtXyl10A (51 kDa) and rXyl10As (rXyl10A-I, 53 kDa; rXyl10A-II, 51 kDa) were higher than the calculated value (42 Da), suggesting that Xyl10As are glycosylated. In fact, one N-glycosylation site (N118) was predicted in Xyl10A. In addition, the linker region of Xyl10A contains many serine/threonine residues, which are often O-glycosylated at the linker region of fungal glycosidases [29]. To investigate the glycosylation of Xyl10A, both

wtXyl10A and rXyl10As were enzymatically N-deglycosylated. After removal of N-glycosylated sugar, only rXyl10A-I showed a smaller molecular weight (Fig. 6). N-deglycosylated rXyl10A-I had the same apparent size as wtXyl10A and rXyl0A-II. This result suggests that rXyl10A-I is a higher glycosylated isoform of rXyl10A, probably due to incomplete processing of N-deglycosylation during secretion under starch induction. wtXyl10A was not sensitive to Endo Hf, but the deglycosylated wtXyl10A band in SDS–PAGE seemed to be broader than that of rXyl0A-I and rXyl0A-II, suggesting that there may be some differences in O-glycosylation patterns between cellulose and starch induction. This difference may be due to differential expression of glycosylation and deglycosylation enzymes under different induction conditions. Heterologous expression systems have been widely applied to many proteins for overexpression and protein engineering. However, in the case of a GH10 family xylanase (XynD) from T. funiculosus, the expressed xylanase was heavily glycosylated during heterologous expression in yeast [27]. Although wtXyl10A and XynD share 96% amino acid sequence homology, wtXyl10A showed 4–5 times higher specific activity for birchwood xylan than XynD. This suggests that hyperglycolylation has a negative effect on xylanase activity of XynD. In the case of the rXyl10As, there were no significant differences in xylanase properties compared with wtXyl10A, so this homologous expression system is suitable for development of Xyl10A for industrial applications.

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Conclusion A thermostable xylanase of A. cellulolyticus, Xyl10A, was purified, cloned and expressed homologously. Purified wtXyl10A showed high thermostability (Tm 80.5 °C) with a broad pH optimum (pH 4–8). These properties are suitable for many industrial applications such as saccharification of cellulolytic biomass. The sequence of the cloned Xyl10A gene indicated that Xyl10A belongs to the GH10 family. rXyl10As showed two distinct molecular weights due to differences in N-type glycosylation, but showed no significant differences in enzymatic properties. The favorable pH and temperature characteristics of Xyl10A and benefits of the homologous expression system suggest that Xyl10A is a promising enzyme for further development for industrial applications. Acknowledgment This work was supported by the Japan–U.S. Cooperation Project for Research and Standardization of Clean Energy Technologies. References [1] M.P. Coughlan, G.P. Hazlewood, Beta-1,4-D-xylan-degrading enzyme systems: biochemistry, molecular biology and applications, Biotechnol. Appl. Biochem. 17 (1993) 259–289. [2] P.M. Coutinho, B. Henrissat, Carbohydrate-active enzymes: an integrated database approach, in: H.J. Gilbert, G.J. Davies, B. Henrissat, B. Svensson (Eds.), Recent Advances in Carbohydrate Bioengineering, The Royal Society of Chemistry, Cambridge, UK, 1999. pp. 3–12. [3] J. Beaugrand, G. Chambat, V.W.K. Wong, F. Goubet, C. Remond, G. Paes, S. Benamrouche, P. Debeire, M. O’Donohue, B. Chabbert, Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkaliextractable arabinoxylans, Carbohydr. Res. 339 (2004) 2529–2540. [4] G.W. Harris, J.A. Jenkins, I. Connerton, N. Cummings, L. Loleggio, M. Scott, G.P. Hazlewood, J.I. Laurie, H.J. Gilbert, R.W. Pickersgill, Structure of the catalytic core of the family F-xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites, Structure 2 (1994) 1107–1116. [5] L. Lo Leggio, S. Kalogiannis, M.K. Bhat, R.W. Pickersgill, High resolution structure and sequence of T. aurantiacus xylanase. I. Implications for the evolution of thermostability in family 10 xylanases and enzymes with (beta)alpha-barrel architecture, Proteins 36 (1999) 295–306. [6] G. Sidhu, S.G. Withers, N.T. Nguyen, L.P. McIntosh, L. Ziser, G.D. Brayer, Sugar ring distortion in the glycosyl-enzyme intermediate of a family G/11 xylanase, Biochemistry (Mosc.) 38 (1999) 5346–5354. [7] N.C. Carpita, D.M. Gibeaut, Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth, Plant J. 3 (1993) 1–30. [8] M.J. Selig, E.P. Knoshaug, W.S. Adney, M.E. Himmel, S.R. Decker, Synergistic enhancement of cellobiohydrolase performance on pretreated corn stover by addition of xylanase and esterase activities, Bioresour. Technol. 99 (2008) 4997–5005. [9] P. Pavon-Orozco, A. Santiago-Hernandez, A. Rosengren, M.E. Hidalgo-Lara, H. Stalbrand, The family II carbohydrate-binding module of xylanase CflXyn11A from Cellulomonas flavigena increases the synergy with cellulase TrCel7B from Trichoderma reesei during the hydrolysis of sugar cane bagasse, Bioresour. Technol. 104 (2012) 622–630.

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