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

Bioresource Technology 104 (2012) 622–630

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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 Patricia Pavón-Orozco a,b, Alejandro Santiago-Hernández a, Anna Rosengren b, María Eugenia Hidalgo-Lara a, Henrik Stålbrand b,⇑ a b

Departamento de Biotecnología y Bioingeniería, CINVESTAV, Av. Instituto Politécnico Nacional No. 2508, CP 07360, México D.F., Mexico Department of Biochemistry, Chemical Center, Lund University, PO Box 124, S-221 00 Lund, Sweden

a r t i c l e

i n f o

Article history: Received 30 June 2011 Received in revised form 20 October 2011 Accepted 18 November 2011 Available online 25 November 2011 Keywords: Cellulase Xylanase Synergy Sugar cane bagasse

a b s t r a c t Synergy between Cellulomonas flavigena xylanase CflXyn11A and Trichoderma reesei endoglucanase TrCel7B was assessed during hydrolysis of alkaline pretreated sugar cane bagasse (SCB) after 12–48 h, applying the individual enzymes and mixtures of the enzymes. A high degree of synergy (6.3) between CflXyn11A and TrCel7B in hydrolysis of SCB was observed after 12 h in the equimolar mixture. A threefold decrease in the degree of synergy was observed with TrCel7B and the catalytic module of CflXyn11A; suggesting an important role played by the carbohydrate-binding module of CflXyn11A (CflXyn11A-CBM) in the observed synergy. Affinity electrophoresis and binding assays showed that CflXyn11A-CBM binds to xylans and to a lesser extent to cellulose. Our results suggest that synergy is more pronounced at early stages of hydrolysis. Furthermore, for the first time it is described that a CBM carried by a xylanase significantly enhances the synergy with a cellulase (threefold increase in synergy). Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Pretreatment of lignocellulosic biomass to facilitate hydrolysis of cellulose and hemicelluloses present in the complex network of the plant cell wall is an essential step towards economic production of biofuels and chemicals. The use of enzymes can increase yields, result in lower levels of by-products, and lower energy consumption (Kumar et al., 2009); however, the cost of the enzymes, their susceptibility to product inhibition and the need to develop optimized enzyme mixtures still has limited their application (Berlin et al., 2007). Microbial cellulases for lignocellulose saccharification have been studied extensively, both regarding cellulosomal enzyme complexes and free non-complexed enzymes (Lynd et al., 2002).

Abbreviations: GH, glycoside hydrolase; CMC, carboxymethylcellulose; SCB, sugar cane bagasse; HEC, hydroxyethylcellulose; CflXyn11A, full length CflXyn11A xylanase from Cellulomonas flavigena; core CflXyn11A, CflXyn11A catalytic domain; TrCel7B, the endoglucanase EGI from Trichoderma reesei; CBM, carbohydrate binding module; CflXyn11A-CBM, CBM of xylanase CflXyn11A; BSA, bovine serum albumin; MES, 2-(N-morpholino)-ethanesulfonic acid; BMCC, bacterial microcrystalline cellulose; PBMCC, prehydrolized BMCC; SSF, simultaneous saccharification and fermentation. ⇑ Corresponding author. Tel.: +46 222 8202; fax: +46 222 4116. E-mail address: [email protected] (H. Stålbrand). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.068

These studies have also investigated the well-known synergy between the major cellulases (endoglucanases and cellobiohydrolases) (Eriksson et al., 2002). Although, cellulase action benefits from accessory enzymes (Berlin et al., 2007; Várnai et al., 2010), the synergy between cellulases and hemicellulases (xylanase or mannanase) has been much less studied. Some studies have addressed the cellulase–hemicellulase synergy of cellulosomal enzyme systems of anaerobic bacteria such as Clostridium cellulovorans (Murashima et al., 2003; Beukes et al., 2008; Beukes and Pletschke, 2010). Only a few studies have addressed the potential synergy between free noncomplexed cellulases and hemicellulases of aerobic microbes during enzymatic hydrolysis of lignocellulosics (Öhgren et al., 2007; Kumar and Wyman, 2009). Cellulases and hemicellulases often carry a carbohydrate-binding module (CBM), i.e., an appended non-catalytic polysaccharide recognizing module (Boraston et al., 2004). These modules generally target the enzymes to the substrate and have enzyme–substrate proximity effects (Hervé et al., 2010). CBMs have been classified by amino acid sequence similarity in 64 families, many of which are further divided into subfamilies; see further ‘‘CAZy – the carbohydrate-active enzymes database’’ (http://www. cazy.org) (Cantarel et al., 2009). Members of CBM family I (CBMI) are known to bind to crystalline cellulose and/or chitin; CBMII subfamily a modules interact

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preferentially with crystalline cellulose while those belonging to CBMII subfamily b interact with xylan chains rather than cellulose (Bolam et al., 2001). Moraïs et al. (2010) reported that CBMs with affinity to xylan are important to efficient degradation of wheatstraw and that the proximity of CBMs to the xylanase catalytic module was essential. In the present study, enzymatic hydrolysis of sugar cane bagasse (SCB) was carried out. SCB is a xylan-rich lignocellulosic by-product of the sugar cane industry and a feedstock for the biotechnical production of fuels and sustainable products (Pandey et al., 2000). Since xylanase activity is beneficial to saccharification of the glucan-fraction in addition to the xylan fraction of pretreated SCB (Prior and Day, 2008), the current investigation studied the potential synergy between two individual non-complexed enzymes of two aerobes, CflXyn11A xylanase (endoxylanase, EC 3.2.1.8) from Cellulomomas flavigena (Amaya-Delgado et al., 2010) and TrCel7B cellulase (endoglucanase, EC 3.2.1.4) from Trichoderma reesei (Bailey et al., 1993), during hydrolysis of SCB. The potential role of CBMs in the synergy between cellulase and hemicellulase has not been extensively studied, and not at all regarding the degradation of SCB. Therefore, we chose to include this aspect in our study, with a focus on a CBM carried by a xylanase. CflXyn11A xylanase from C. flavigena carries a family II CBM (Amaya-Delgado et al., 2010) that has not been characterized, whereas TrCel7B cellulase from T. reesei carries a well characterized family I CBM with specific affinity to cellulose (Srisodsuk et al., 1997). Consequently, in order to evaluate the potential influence of the CflXyn11A-CBM on synergy, we created a truncated variant of this enzyme (core CflXyn11A), lacking the CBM and this variant was included in the study, along with the full length enzyme. 2. Methods 2.1. Substrates and polysaccharides Birchwood xylan (Roth 7500; MW 2500) was purchased from Carl Roth RG (Karlsruhe, Germany) for affinity electrophoresis assays and from Sigma (Sweden) for activity assays. Oatspelt xylan and Avicel (microcrystalline cellulose) were purchased from Fluka Biochemika (Germany) and Carboxymethylcellulose (CMC) from Sigma (Sweden). Hydroxyethylcellulose (HEC) 300 (Viscosity: 0.3 Pa. s 2% in water) was from SERVA (Heidelberg), Solka Floc BW 200 cellulose was from James River Corp. (Berlin, NH, USA). Oatspelt xylan was fractionated into water soluble and water insoluble fractions as previously described (Ghangas et al., 1989). D-Arabinose, D-galactose and D-xylose were purchased from Fluka Biochemika (Germany); D-glucose, D-mannose and p-nitrophenyl-glucopyranoside from Sigma (Sweden). Alkali-pretreated SCB was a kind gift by Dr. M.C. Montes Horcasitas (Departamento de Biotecnología y Bioingeniería, CINVESTAV, México). The alkali pretreatment was done according to De la Torre and Casas-Campillo (1984) by soaking 30 g of SCB in 2% sodium hydroxide, heating by direct steam until boiling (92 °C) for 15 min, cooling, filtration through cotton, washing with water until neutralized, and drying the water-insoluble material at 80 °C for 24 h. This method was chosen because Cellulomonas flavigena grows well on this type of SCB (De la Torre and Casas-Campillo, 1984) and increases the efficiency of enzymatic hydrolysis without significantly affecting the synergy between enzymes (xylanase, mannanase and arabinofuranosidase) (Beukes and Pletschke, 2010).

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et al., 2010) was used as a template for amplification of the coding region for both the full length CflXyn11A xylanase (CflXyn11A) and its catalytic domain (core CflXyn11A). pQE30 Xa expression vector (Qiagen, USA) was used to express the recombinant proteins, CflXyn11A and core CflXyn11A. Restriction enzymes were purchased from Fermentas (Sweden). E. coli M15 [pREP4] strain was grown on 2TY medium, supplemented with kanamycin (30 lg ml1) and ampicillin (100 lg ml1) for the expression of both full length CflXyn11A and core CflXyn11A. Recombinant T. reesei QM941 carrying the T. reesei gene encoding cellulase Cel7B (Collén et al., 2001) was used for the expression of TrCel7B. 2.3. Cloning and expression of recombinant xylanase enzymes Standard molecular biology techniques were performed as described (Sambrook and Russell, 2001). The coding region of the Cflxyn11A gene was amplified by PCR using forward primer DXQE (50 -CGGGATCCATGACCGCAAGTTTCGCAACCC-30 ) and reverse primer RXQE (50 -CCCAAGCTTTCAGCAGAACGCGCTGGGCGT-30 ) with restriction sites BamHI and HindIII (underlined), respectively. The coding region for the catalytic module of the Cflxyn11A gene, core CflXyn11A, was amplified by using the forward primer LSCD (50 CGGATCCGCGGTGACGTCCAACCAGACC30 ) and reverse primer LSCR (50 -CAAAGCTTCGAGCCCTCGGACACCGT-30 ) with restriction sites BamHI and HindIII (underlined). PCR was performed with Pfu DNA Polymerase (Fermentas, Sweden) under the following conditions: one cycle of 10 min at 95 °C, followed by 35 cycles of 1 min at 94 °C, 45 s at 56 °C, 2.5 or 1 min at 72 °C; one cycle of 7 min at 72 °C in a Peltier Thermal Cycler PTC-200 (MJ Research DNA Engine). PCR products were purified from 1% agarose gel by using the Sigma Gel Extraction Kit (Sigma, Sweden). For the expression of the full length CflXyn11A xylanase, the coding region of Cflxyn11A gene (1 kb) was directionally cloned into pQE-30 Xa vector, in order to yield the construct Cflxyn11A pQE-30 Xa. For the expression of the catalytic domain of the CflXyn11A xylanase (core CflXyn11A), the corresponding coding region of Cflxyn11A gene (0.75 kb) was directionally cloned into pQE-30 Xa vector to yield the plasmid core Cflxyn11A pQE-30 Xa. Plasmids were analyzed by digestion with restriction enzymes and DNA sequencing at Labmet (Lund, Sweden). The DNA sequence was analyzed and compared to other sequence databases, using available online tools (http://www.expasy.ch/, http://www.ncbi.nlm.nih.gov/). Subsequently, Cflxyn11A pQE-30 Xa and core Cflxyn11A pQE-30 Xa plasmids were transformed into E. coli M15 [pREP4] for protein expression. Recombinant enzymes were expressed in E. coli M15 [pREP4] cells harboring the Cflxyn11A/pQE-30 Xa or pQE-30 core Cflxyn11A Xa construct. E. coli cultures, grown overnight in 2TY medium, were diluted 100-fold in 2TY medium and incubated at 37 °C, 200 rpm, until reaching a cell density of OD600 nm = 0.6. IPTG was added to 1 mM final concentration and cultures were incubated at 30 °C, 200 rpm, for 5 h. Cells were harvested by centrifugation (11,900g at 4 °C for 20 min), the pellet was recovered and cell lysis was performed by incubating with 0.1 mg/ml lysozyme in lysis buffer (100 mM NaCl, 2 mM EDTA, 50 mM Tris HCl, pH 8) at 4 °C for 1 h. The lysate was centrifuged at 11,900g, 4 °C 10 min. Both soluble and insoluble fractions were recovered and further analyzed by xylanase activity assays and SDS–PAGE, for the presence of recombinant proteins, full length CflXyn11A and core CflXyn11A. 2.4. Purification

2.2. Strains and plasmids Plasmid DNA of Escherichia coli XL1-Blue MRF’, carrying the C. flavigena gene encoding xylanase CflXyn11A (Amaya-Delgado

Crude extract of soluble CflXyn11A was desalted by ultrafiltration using a Stirred Cell System (Amicon). The sample was equilibrated in buffer A (5 mM NaCl, 20 mM Tris–HCl buffer, pH 7.5)

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and loaded into a 50 ml Source Q column (Pharmacia Biotech), equilibrated in same buffer. Proteins were eluted from the column with a linear gradient of NaCl (5–500 mM) in buffer A. Fractions and the flow through were collected and analyzed for xylanase activity. Samples were concentrated and equilibrated by ultrafiltration in 20 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer, pH 6.5 and loaded onto a 10 ml Resource S column (Pharmacia Biotech), previously equilibrated in buffer B (5 mM NaCl, 20 mM MES buffer, pH 6.5). Proteins were eluted from the column with a linear gradient of NaCl (5–500 mM) in buffer B. Fractions and the flow through were collected and analyzed for xylanase activity. Fractions with xylanase activity were collected, concentrated and buffer exchanged against 50 mM citrate–phosphate buffer, pH 6.5. A final purification process was carried out, as previously described (Santiago-Hernández et al., 2007), which utilized the affinity of xylanase toward SCB. In brief, samples were incubated with 10 mg/ml SCB in 50 mM citrate–phosphate buffer, pH 6.5 at 4 °C for 16 h. SCB was recovered by centrifugation (11,900g, 4 °C, 30 min), and the pellet was resuspended and washed in 1 M NaCl. SCB was again recovered by centrifugation (11,900g, 4 °C, 30 min) and the pellet was resuspended in 3 M guanidine hydrochloride in 50 mM citrate–phosphate buffer, pH 6.5, and then incubated at 4 °C for 18 h in a tube inverter (VWR), at a rate of 20 inversions per min. The supernatant was recovered by centrifugation and dialyzed against 15 mM citrate–phosphate buffer, pH 6.5 at 4 °C and concentrated by ultrafiltration. The pellet was resuspended in 50 mM citrate–phosphate buffer, pH 6.5, the suspension was centrifuged (11,900g, 4 °C, 30 min) and the pellet was resuspended in 6 M urea in 15 mM citrate–phosphate buffer, pH 6.5 and incubated at 4 °C for 18 h in a tube inverter, at a rate of 20 inversions per min. The supernatant was recovered by centrifugation (11,900g, 4 °C, and 30 min) and dialyzed against 50 mM citrate–phosphate buffer, pH 6.5 and concentrated by ultrafiltration. For the core CflXyn11A, inclusion bodies were recovered as described by Sambrook and Russell (2001). Supernatant was collected and dialyzed against 20 mM Tris–HCl buffer, pH 8.5. Cellulase TrCel7B from T. reesei was expressed from genetically modified T. reesei QM9414 under control of the gpdA promoter from Aspergillus nidulans and purified as previously described (Collén et al., 2001). The purity of all enzymes was verified by SDS–PAGE. The gel was scanned using an Image Scanner III with software LabScan 6.0 and purity estimated using ImageQuant TL 7.0 (GE Health Care Bioscience AB, Uppsala, Sweden). 2.5. Activity assays Enzymatic activities were determined by quantifying reducing sugars using the DNS method as described previously (SantiagoHernández et al., 2007). Cellulase activity was measured by incubating enzyme at 50 °C for 20 min in 50 mM sodium acetate buffer, pH 5, containing 1% CMC (Jeoh et al., 2002). Xylanase activity was assayed by incubating enzyme with 0.25% birchwood xylan in 50 mM citrate–phosphate buffer, pH 6.5. Glucosidase activity was determined with p-nitrophenyl-glucopyranoside (Sigma) as described previously (Plant et al., 1988). The molar amount of each recombinant enzyme was determined from the amount of protein in each enzyme preparation. Units of activity were defined as lmol of reducing sugar per minute. Specific activity was expressed in terms of terms U/lmol of protein (values of U/mg protein are also given for each enzyme). Molecular weight was estimated by SDS–PAGE and used to determine moles of enzyme.

Diego, CA, USA). Protein molecular weight standards were used to estimate the molecular weight of proteins (MultimarkÒ Invitrogen, Sweden). Affinity gel electrophoresis was performed in nonreducing 7.5% polyacrylamide gels polymerized in the absence or presence of soluble substrates 0.1% (w/v): CMC, birchwood xylan, oatspelt xylan and HEC. Bovine serum albumin (BSA) was included as a reference protein. Electrophoresis was performed at 4 °C for 2 h, in glycine buffer pH 10, using a Mini PROTEAN II system (BioRad) at 150 V. Proteins were visualized by Coomassie blue R-250 (BioRad) staining. Binding was visually assessed according to the retention or migration of proteins. 2.7. Carbohydrate and lignin analysis Sugar composition and lignin content of pretreated SCB were determined according to the standardized methods of NREL (Sluiter et al., 2005). The total amount of sugars in the supernatant of hydrolysates was determined by acid hydrolysis (Öhgren et al., 2007). Briefly, samples were autoclaved at 121 °C for 1 h with 4% H2SO4 and subsequently neutralized. Monomeric sugars were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection HPAEC-PAD (Dionex), employing an ED40 electrochemical detector and equipped with CarboPac PA10 analytical and guard columns (Dionex). Injection volume was 20 ll and 0.1 mM NaOH was used as eluent at a flow rate of 1 ml/min. D-arabinose, D-galactose, D-glucose, D-mannose and D-xylose were used as standards. Three samples of each of the analyzed materials were hydrolyzed with three injections from each. 2.8. Hydrolysis experiments Enzymatic hydrolysis of SCB (10 mg/ml) was performed in 50 mM sodium acetate buffer, pH 5. The reactions were initiated by mixing the enzyme solution with the substrate in a 1.8 ml screw-cap tube. The working volume was 1.5 ml. Incubations were performed at 37 °C in an incubator equipped with a tube inverter, at 20 inversions per min. Supernatants were withdrawn at 0, 12, 24 and 48 h. Hydrolysis was stopped by boiling samples for 5 min, followed by centrifugation (11,900g at room temperature for 5 min). Samples were frozen for further analysis. Reaction mixtures with different molar ratios, from 0% to 100% of cellulase and xylanase were used. In all assays, the final molar concentration of the enzymes was kept constant at 0.35 mM. Commercial b-glucosidase from Sigma (Sweden) was present at 0.01 mM in all reactions. 2.9. Binding assays Enzyme binding to water-insoluble substrates (Avicel, Solka Floc BW 200 cellulose, insoluble fractions of oatspelt xylan and SCB) was determined by measuring the residual activity in the supernatant, after incubation at 4 °C for 1 h. Enzyme (0.5–5 lM) was incubated with 20 mg/ml substrate in hydrolysis buffer (50 mM sodium acetate buffer, pH 5), at 4 °C for 1 h, and supplemented with 0.1 mg/ ml BSA in a tube inverter, at 20 inversions per min. Percentage of bound protein is presented as the percentage of residual activity found in supernatant after incubation, in comparison to initial activity (Hägglund et al., 2003). Binding during hydrolysis conditions was assayed by incubating each enzyme mixture with 10 mg/ml substrate in hydrolysis buffer supplemented with 0.1 mg/ml BSA in a tube inverter, at 20 inversions per min at 37 °C for 30 min.

2.6. Gel electrophoresis

2.10. Synergism

SDS–PAGE electrophoresis was performed using polyacrylamide precast gels (Nu-PAGE 4–12% Bis-Tris) from Novex (San

The degree of synergy is given as the ratio of the specific activities of the enzymes in the hydrolysis of SCB when the enzymes are

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1

2

3

4

5

6

KDa

60

36 31 22

E D C

B

A

Fig. 1. Purified recombinant and commercial enzymes. 4–12% SDS–PAGE analysis of recombinant enzyme preparations stained with Coomassie blue. Lane 1, molecular marker; lane 2 (A), core CflXyn11A (20.6 kDa); lane 3 (B), full length CflXyn11A (36 kDa); lane 4 (C), commercial b-glucosidase (56 kDa); lane 5 (D), TrCel7B (60.7 kDa); lane 6 (E), BSA (58.3 kDa).

together divided by the sum of the specific activities for the enzymes alone (Beukes et al., 2008). Synergy was determined by quantification of reducing sugar release (with glucose as standard) or glucose release, both expressed in units of enzymatic activity per lmol of protein. One unit of enzymatic activity was defined as the lmol of sugar released per minute. 3. Results and discussion The focus of this study was to investigate the synergy between xylanase CflXyn11A from C. flavigena and cellulase TrCel7B from T. reesei during the hydrolysis of SCB. The analysis of the sugar composition of the SCB showed a high content of xylan (20.3% ± 0.5) and a lower amount of polymeric arabinose (2.9% ± 0.7). This is in accordance with investigations showing that the predominant xylans in SCB are arabinoxylans (Peng et al., 2009). SCB from various sources contains cellulose (Pandey et al., 2000) so there is also some polymeric glucose present in pretreated SCB (6.8% ± 0.2). This glucan value is lower than that of other alkaline-treated SCB (Beukes and Pletschke, 2010; Pandey et al., 2000). Other sugars present in polymeric form were galactose (0.7% ± 0.2) and mannose (0.6% ± 0.5). The levels of acid and water soluble lignin were 20.3% ± 6.9 and 0.12% ± 0.09, respectively.

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CflXyn11A have already been described (Amaya-Delgado et al., 2010). Hitherto, xylanases from this family appear to have only demonstrated activity on xylan (Collins et al., 2005); however, CflXyn11A presented a specific activity of 14,068 U/lmol (390.8 U/mg), on birchwood xylan and interestingly an activity of 252.7 U/lmol (7.0 U/mg) using CMC as a substrate, which is indicative of endoglucanase activity. Xylanases with endoglucanase side-activity have previously been found in family 10, but not in family 11 (Collins et al., 2005). Prior to SCB hydrolysis assays, the pH and temperature stability of the variants of xylanase CflXyn11A were determined. The fulllength and the core enzyme exhibited pH stability at pH 4–7 (retaining >90% activity following incubation at 37 °C for 24 h) and with thermal stability at 37 °C (retaining 100% activity after incubation at pH 6.5, for 48 h). At pH 5, the CflXyn11A xylanase activity decreased to 95% during the first 3 h at 37 °C; subsequently activity remained constant for the next 24 h. Theoretical pIs of 9.5 and 8.7 for CflXyn11A and core CflXyn11A, respectively, were estimated by applying informatics tools (http://www.expasy.org). TrCel7B cellulase activity has been well characterized (Bailey et al., 1993; Eriksson et al., 2002), its xylanase activity has been reported (Bailey et al., 1993) and the X-ray structure of its catalytic core module has been determined (Protein Data Bank Accession No. 1EG1). Under the hydrolysis conditions utilized in the present study, TrCel7B retained 100% of its initial activity after 120 h incubation at 37 °C in sodium-acetate buffer pH 5. TrCel7B presented a specific activity of 14,985 U/lmol (249.8 U/mg) on CMC which concurred with previous studies (Eriksson et al., 2002). TrCel7B showed a specific activity of 351.52 U/lmol (5.9 U/mg) using birchwood xylan as the substrate. 3.3. Sequence similarity analysis of CflXyn11A-CBM Previously it was shown that CflXyn11A is a modular enzyme with a C-terminal CBM from family II (Amaya-Delgado et al., 2010). Sequence similarity analysis of the CBM of CflXyn11A xylanase (CflXyn11A-CBM), applying the BLAST algorithm (Expasy Proteomic Service) showed highest sequence identity (up to 88.6%) with subfamily IIb of CBMs. Sequence alignment clearly showed that CflXyn11A-CBM has the characteristics of a subfamily IIb CBM (Fig. 2). It lacks a stretch of eight residues (including an exposed Trp) which is present in subfamily IIa CBMs (Bolam et al., 2001). Two other tryptophans are conserved in family II (Fig. 2) and are involved in xylan binding for the two subfamily IIb CBMs of GH11 xylanase XynD (Bolam et al., 2001). Although many subfamily IIb CBMs are specific for xylan, an Arg of the XynD CBMs appears to be important for cellulose binding (Bolam et al., 2001). This Arg also appears to be present in CflXyn11A-CBM as judged from the alignment (Fig. 2). 3.4. Affinity characterization of CflXyn11A-CBM

3.1. Cloning, expression and purification of enzymes CflXyn11A and core CflXyn11A, expressed in E. coli, were observed as 36 kDa and 22 kDa bands, respectively (Fig. 1A and B). The purity of CfXyn11A and core CfXyn11A was at least 95%; only a minor contaminant was evident, manifesting as a faint band of approximately 45 kDa. Recombinant TrCel7B appeared as a single band of approximately 60 kDa (Fig. 1C). SDS–PAGE analysis of the commercial b-glucosidase also revealed a single band (Fig. 1D). 3.2. Enzymatic activity of the recombinant CflXyn11A xylanase and TrCel7B cellulase CflXyn11A was previously identified as a member of family 11 of GHs and likewise the biochemical properties of recombinant

To determine the binding specificity of CflXyn11A-CBM, the binding of recombinant CflXyn11A xylanase from C. flavigena was characterized by nondenaturing affinity gel electrophoresis using soluble substrates. Results indicated that full length CflXyn11A is retarded when Oatspelt or birchwood xylan were included in the gel, whereas no retardation was observed when cellulose derivatives (CMC or HEC) were assayed (Fig. 3). A control protein (BSA) and core CflXyn11A were not retarded (Fig. 3), confirming that the retardation of CflXyn11A was caused by the presence of the CflXyn11A-CBM. In the case of insoluble substrates, full length and core CflXyn11A were incubated in the presence of either SCB, the water insoluble fraction of oatspelt xylan or cellulose (Avicel or Solka floc). The percentage of bound enzyme was calculated as the percentage

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GGSCTVSATKTQEWGDRFNVTYTVSGSN-----SWTVTVYPSNGQSIQNHWNANRNGN----TFTPN--------GSGNTFGVTYYKGPNNVNWTPSASTGCSVTATRAEEWSDRFNVTYSVSGSS-----AWTVNLALNGSQTIQASWNANVTGSGSTRTVTPN--------GSGNTFGVTVMKNGSSTTPAATCA TGSCSVSAVRGEEWADRFNVTYSVSGSS-----SWVVTLGLNGGQSVQSSWNAALTGSSGTVTARPN--------GSGNSFGVTFYK—-NGSSATPGAPAGCQVLWG-VNQWNTGFTANVTVKNTSSAPVDGWTLTFSFPSGQQVTQAWSSTVTQSGSAVTVRNAPWNGSIPAGGTAQFGFNGSHTGTNAAPTAF--

CflXyn11A CBM CfXynD CBMIIb-1 CfXynD CBMIIb-2 CfCex CBMIIa

Fig. 2. Amino acid sequence alignment of CflXyn11A-CBM (Swiss-Prot Accession No. A2AWV8) with both CBMIIb-1(N-terminal) (46% identity) and CBMIIb-2 (C-terminal) (48% identity) of CfXynD from C. fimi (Swiss-Prot Accession No. P54865) and CBMIIa of CfCex from C. fimi (22% identity) (Swiss-Prot accession number P07986) using the ClustalW algorithm. h Conserved tryptophans in type II CBMs. The third Trp is present in subfamily IIa but not in family IIb due to a eight residue deletion. The Arg referred to in the text is present three residues down-stream from the first Trp.

(A) 1 3

2

3 (B) 1 3

2

3 (C) 1 3

2

3

(D) 1 3

2

3

Fig. 3. Affinity gel electrophoresis of CflXyn11A and core CflXyn11A against several soluble polysaccharides. Lane 1, control BSA; lane 2, core CflXyn11A, lane 3 CflXyn11A in 7.5% nondenaturing polyacrylamide gels containing no polysaccharide (A) or 0.1% Oatspelt xylan (B), birchwood xylan (C) or CMC (D).

Table 1 Binding of xylanase CflXyn11A to insoluble substrates. %a

Avicel cellulose Solka cellulose SCB Oatspelt xylan

48.2 ± 3.7 32.3 ± 3.4 70.9 ± 2.6 79 ± 6.4

±, Standard deviations. a Percentage of enzyme bound to the substrate.

8

6

mM

Substrate

10

4

of activity which remained in the supernatant after 1 h incubation. The highest percentage (71–79%) of bound CflXyn11A was found in the insoluble oatspelt xylan and SCB incubations (Table 1). The truncated enzyme, core CflXyn11A bound significantly less to the polysaccharides (3–5%). The results suggest that CflXyn11A binds with between 1.3- and 3-times higher degree to xylan (oatspelt xylan) than to cellulose (Avicel and Solka floc). The binding to both soluble and insoluble xylan and weaker binding to cellulose appear to be features similar to those found in the two well-characterized subfamily IIb CBMs of XynD (Bolam et al., 2001). A contributing factor to the observed greater binding (71%) to SCB than to cellulose (Table 1) is likely the fact that xylan is the predominant polysaccharide in the SCB substrate used. Future studies may reveal if CflXyn11A also binds to lignin in analogy with several cellulases for which CBMs appear to be important for unspecific lignin binding (Palonen et al., 2004). 3.5. Synergy between CflXyn11A and TrCel7B The synergistic action between xylanase CflXyn11A and cellulase TrCel7B was determined by the independent quantification of reducing sugars and glucose released during the hydrolysis of SCB. To avoid potential inhibition of TrCel7B by cellobiose (Claeyssens et al., 1990), b-glucosidase was supplemented in all incubations. In control experiments with all substrates used, b-glucosidase alone did not produce any detectable reducing sugars or glucose. For synergy assays, the recombinant enzyme combinations were set up in a number of reactions containing different mo-

2

0 0

12h

24h

48h

t(h) Fig. 4. Reducing sugar release during the enzymatic hydrolysis of SCB at different molar ratios of CflXyn11A and TrCel7B. (d) TrCel7B (C), (s) CflXyn11A (X), (.) 25C:75X, (4) 50C:50X, (j) 75C:25X.

lar ratios for each enzyme, where the total molar protein concentration was kept constant. Previous synergy studies with cellulases, using cellulose and modified cellulose (bacterial microcrystalline cellulose, BMCC and prehydrolized BMCC) as a substrate have revealed that the ratio of the concentrations of the total enzyme to that of the substrate is an important variable affecting synergy (Jeoh et al., 2002). Furthermore, competition and also synergy in binding are sensitive to loading molar ratio (Jeoh et al., 2006). Hydrolysis conditions were decided by evaluating pH stability and thermostability for each enzyme. At 37 °C, both CflXyn11A and TrCel7B maintained 100% of their initial activities for at least 120 h in 50 mM Na-acetate buffer, pH 5.0. Individual activity against SCB was evaluated by an analysis of reducing sugar at the highest individual molar concentration used in experiments (0.35 mM). CflXyn11A and TrCel7B manifested an enzymatic

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Reducing sugars 12h hydrolysis

(A) 0.016

7

0.014

6

0.012 0.010 4 0.008 3 0.006

Degree of synergy

U/micromol

5

2 0.004 1

0.002 0.000

0 100c

25c 75x

50c 50x

75c25x

100x

molar ratio U/micromol degree of synergy

(B) 0.008

3.0

U/micromol

2.0 0.004 1.5 0.002 1.0

0.000

Degree of synergy

2.5

0.006

0.5

-0.002

0.0 100c

25c75x

50c50x

75c25x

100x

molar ratio

(C) 0.018 0.016 0.014

100C 100X 25C 75X 50C 50X 75C 25X

U/micromol

0.012 0.010 0.008 0.006 0.004 0.002 0.000 Ara

Glc

Xyl

Man

Saccharides Fig. 5. Synergy between CflXyn11A and TrCel7B at 12 h hydrolysis at different molar ratios (given as molar %) of CflXyn11A and TrCel7B. (s) U/lmol, (d) Degree of synergy. (A) Synergy quantified by reducing sugars release, (B) synergy quantified by glucose release, (C) total monomers of solubilized saccharides after acid hydrolysis. Explanation: 100C, 100% TrCel7B; 75C:25X, 75% TrCel7B:25% CflXyn11A; 50C:50X, 50% TrCel7B:50% CflXyn11A; 25C:75X, 25% TrCel7B:75% CflXyn11A; 100X, 100% CflXyn11A.

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Table 2 Degree of synergy between CflXyn11A and TrCel7B during SCB hydrolysis. Time (h)

12 24 48

Degree of synergya 100Cb

25C:75X

50C:50X

75C:25X

100Xc

1 1 1

4.64 0.95 1.62

6.26 1.57 1.5

3.85 1.43 1.57

1 1 1

a Degree of synergy was calculated as the ratio of the specific activities of the enzymes in the hydrolysis of SCB when they are together divided by the sum of the individual specific activities at the concentration used. b Percentage of cellulase TrCel7B used in the mixture of hydrolysis. c Percentage of xylanase CfXyn11A used in the mixture of hydrolysis.

specific activity of 0.002 U/lmol protein and 0.0025 U/lmol protein, respectively, on SCB. Binding assays (30 min), under hydrolysis conditions, showed that CflXyn11A and TrCel7B bound to approximately 13% (CflXyn11A) and 17% (TrCel7B), with remaining activity in solution of 87% and 83%, respectively. This indicates that both enzymes were exposed to both insoluble and soluble fractions of the substrate. Independent analysis of the reducing sugar and glucose released, clearly indicated that during the hydrolysis of SCB, synergy was occurring between CflXyn11A and TrCel7B (Figs. 4 and 5). The rate of reducing sugars released during the first 12 h of SCB incubation with either CflXyn11A or TrCel7B was 0.02 mM/h; whereas mixtures of both enzymes showed an increase in the rate of hydrolysis of up to approximately 0.2 mM/h (Fig. 4). This indicates a remarkable increase of up to 10-fold in the rate of hydrolysis, evaluated after 12 h when both enzymes were present, as compared to when enzymes were acting individually. After 12 h, the rate of hydrolysis increased for the enzymes acting alone, whereas the rate of hydrolysis stayed relatively unchanged for the mixed enzymes (Fig. 4). The degree of synergy was evaluated after 12, 24 and 48 h, at different molar ratios (TrCel7B: CflXyn11A) of 25C:75X, 50C:50X and 75C:25X. Synergy was observed for all the molar ratios and for all the samples assayed, according to the rate of release of reducing sugars (Fig. 5A) or glucose (Fig. 5B). The highest degrees of synergy observed during the hydrolysis of SCB for the molar ra-

tios assayed in terms of the release of reducing sugars are summarized in Table 2. The highest degree of synergy was observed after 12 h for all the different molar ratio mixtures. Also, the highest rate based on release of reducing sugars (0.015 U/lmol) was evident after 12 h in the mixture 50C:50X, concurring with the highest degree of synergy (6.26) (Fig. 5A, Table 2). The degree of synergy observed after 12 h (3.8–6.3) (Table 2) was two to threefold higher than that previously reported for synergy studies on xylanase and cellulase from Clostridium, carried out for 24 h or longer (Beukes and Pletschke, 2010) which in turn was similar to the synergy observed after 24 and 48 h in this work (Table 2). Thus findings here suggest a remarkable synergy between xylanase CflXyn11A and cellulase TrCel7B, most pronounced at the early stages of SCB degradation. The highest degree of synergy in terms of the release of glucose was reached in the mixture 25C:75X, synergy 2.7 with an activity of 0.006 U/lmol (Fig. 5B). The supernatants from the enzyme and SCB incubations were subject to acid hydrolysis in order to analyze the monomeric composition of solubilized saccharides. As expected, saccharides contained glucose and xylose units and also arabinose units (Fig. 5C). Minor amounts of mannose were detectable only with 100% of TrCel7B.

3.6. Synergy between core CflXyn11A and TrCel7B The binding of enzymes to cellulose via CBMs has been proposed to be one factor which can affect the synergy between cellulases (Jeoh et al., 2002). To investigate whether the CflXyn11ACBM affects the xylanase–cellulase synergy observed in the current work, we also evaluated synergy between core CflXyn11A and TrCel7B, applying the same conditions as above. A synergistic effect among the enzymes was apparent in reducing sugar analysis after 12 h of SCB hydrolysis with the highest degree of synergy also here in the 50C:50X mixture (Fig. 6B). However the degree of synergy was threefold lower than that observed in the case of the fulllength enzyme, as presented in Fig. 6. After 24 and 48 h of SCB hydrolysis, the degree of synergy was similar to that observed in the case of the full-length enzyme (data not shown).

0.0035

2.5

0.0030

2.0

0.0025

1.5

0.0020

1.0

0.0015

0.5

0.0010

Degree of synergy

U/micromol

molar ratio vs U/micromol molar ratio vs Degree of synergy

0.0 100c

25c75x

50c50x

75c25x

100x

molar ratio Fig. 6. Synergy between core CflXyn11A and TrCel7B at 12 h hydrolysis at different molar ratios (given as molar%) of core CflXyn11A and TrCel7B. Synergy quantified by reducing sugars release. (s) U/lmol, (d) Degree of synergy. Explanation: 100C, 100% TrCel7B; 75C:25X, 75% TrCel7B:25% core CflXyn11A; 50C:50X, 50% TrCel7B:50% core CflXyn11A; 25C:75X, 25% TrCel7B:75% core CflXyn11A; 100X, 100% core CflXyn11A.

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3.7. General discussion on xylanase–cellulase synergy Since xylan interacts with cellulose (Teleman et al., 2001) and is believed to be intertwined among the cellulose fibers in plant cell walls, it needs to be made partially soluble in order to expose the cellulose fibers to cellulolytic enzymes (Murashima et al., 2003; Kumar and Wyman, 2009). Several authors have shown how xylan can be made soluble from pretreated lignocellulosics with the aid of xylanases (see e.g., Suurnäkki et al., 1997; Teleman et al., 2001) facilitating the exposure of more cellulose fibers (Shoham et al., 1992). However, as previously mentioned, synergy with xylanase–cellulase has only been described in a few cases, although certain studies have included SCB (Beukes et al., 2008; Beukes and Pletschke, 2010). Once the hemicellulose component of the plant biomass is made partially soluble by the hydrolysis of xylan, the exposure of the cellulose fibers to cellulase may increase, thus permitting the degradation of the cellulose fibers. Thus, the synergy observed in the current work may be explained by the concerted action of both the xylanase and the cellulase on SCB. Interestingly, we observed the highest xylanase–cellulase synergy (6.3) achieved so far using SCB as a substrate (Fig. 5A) and this value was obtained after 12 h of incubation. At prolonged incubations (24 and 48 h) synergy decreased to values reaching up to 1.6 (Table 2), and these values are similar to the xylanase–cellulase synergy observed in previous studies on SCB after 24 and 48 h incubations (1.7–2.3) using cellulosomal enzymes (Beukes and Pletschke, 2010). Thus, the enhanced and high synergy at early stages of incubation evident in this study represents a new interesting finding. (Table 2). It should be emphasized that this result was obtained when the full-length xylanase CflXyn11A was used. When using the truncated xylanase (core CflXyn11A) lacking the CBM, the synergy after 12 h of incubation was approx. threefold lower compared to the full-length enzyme, with a top value of 2.0 (Fig. 6). The synergy then decreased slightly reaching similar values to those of the full-length enzyme after 24 and 48 h incubation (1.5–1.6 for the equimolar mixes) (Table 2). Our findings suggest that the CBM of CflXyn11A plays an important role in synergy, especially during the initial stage of SCB hydrolysis, increasing the degree of synergy during the first 12 h of SCB hydrolysis. The importance of a xylan-binding CBM for the efficient degradation of wheat-straw by a T. fusca xylanase has previously been described (Moraïs et al., 2010). Also, in our case the degradation was greater when the xylanase carried a CBM: compared to core CflXyn11A, the full-length enzyme released 1.8-fold higher values for reducing sugar when incubated for 12 h with SCB (Figs. 5A and 6). Also, cellulose-binding CBMs naturally fused to hemicellulases can markedly enhance hemicellulose hydrolysis in complex substrates containing cellulose (Black et al., 1997; Hägglund et al., 2003) and the global importance of CBMs that target different components for cell-wall degradation has recently been high-lighted (Hervé et al., 2010). The binding of the fulllength xylanase not only to xylan but also to cellulose (Table 1) may be an advantage for the binding and removal of xylan found in the close vicinity of cellulose, making it more accessible to the cellulase, and thus possibly contributing to the enhanced synergy when the CBM is present, especially at the early stages of hydrolysis.

4. Conclusions Synergy studies between CflXyn11A and TrCel7B showed the highest degree of synergy (6.3) in an equimolar mixture of the enzymes at early stages of SCB hydrolysis (12 h); where a threefold decrease in the degree of synergy was observed with core CflXy-

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n11A. These data suggest an important role for the CflXyn11ACBM in the synergy inherent in SCB hydrolysis. It would be an interesting future topic to evaluate if the family I CBM carried by TrCel7B also influences the cellulase–xylanase synergy observed. Our results can potentially contribute to the formulation of optimized enzyme mixtures for the saccharification of plant biomass for further fermentation, e.g., to liquid fuel. The discovery that the synergy is highest during early stages (12 h) of hydrolysis may be particularly important for the development of fed-batch simultaneous saccharification and fermentation (SSF) strategies (Öhgren et al., 2007).

Acknowledgements This work was supported by grants from the Swedish Agency for Innovation (VINNOVA), the Crafoord Foundation (Sweden) and the Departamento de Biotecnología y Bioingeniería, CINVESTAV, México. P. Pavón-Orozco received a scholarship from CONACYT, México.

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