Expression and Characterization of Glycosyl Hydrolase Family 115 α-Glucuronidase fromScheffersomyces stipitis

Original Research Expression and Characterization of Glycosyl Hydrolase Family 115 a-Glucuronidase from Scheffersomyces stipitis Paramjit K. Bajwa, Sean Harrington, Mehdi Dashtban, and Hung Lee School of Environmental Sciences, University of Guelph, Guelph, Canada Abstract The gene encoding glycosyl hydrolase family 115 aglucuronidase from Scheffersomyces stipitis (ssagu115) was cloned and expressed in Pichia pastoris strain X33. The recombinant enzyme was purified to homogeneity by Ni-chelation affinity chromatography. The apparent molecular weight of the secreted recombinant enzyme was about 150 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recombinant a-glucuronidase was able to remove 4-Omethylglucuronic acid groups from several polymeric xylans (beech, birch, and oat spelt xylan) as well as xylooligosaccharides (aldotetraouronic acid and aldopentaouronic acid). The enzyme was more active towards xylooligosaccharides than xylans. Substrate inhibition was observed with aldouronic acids at concentrations above 23 mM. The Km values of the enzyme towards aldotetraouronic and aldopentaouronic acids were 8.2 mM (5 mg/mL) and 9 mM (6.5 mg/mL), respectively. For beech and birch xylan, the Km values were 9.5 mg/mL and 35 mg/mL, respectively. The enzyme was active over the pH range of 3.0–8.5, with maximal activity at pH 4.0. The optimum temperature for activity of the enzyme was 50C. The enzyme exhibited synergy with endoxylanases from the GH10 and GH11 families on hydrolysis of beech xylan, birch xylan, and oat spelt xylan, with the greatest synergistic effect being shown with the GH10 endoxylanase. Treatment of polymeric xylan with SsAgu115 led to reduced solubility and increased precipitation from solution. Insoluble xylans have the potential to form hydrogels, which may have pharmaceutical and biomedical applications. Key words: a-glucuronidase, glycosyl hydrolase, 4-Omethylglucuronic acid, pentose-fermenting, Pichia pastoris, Scheffersomyces stipitis, xylan, yeast a Introduction -glucuronidase catalyzes the hydrolysis of a-(1-2)linked 4-O-methylglucuronic acid (MeGlcA) residues from xylooligosaccharides or xylan.1,2 To date, only two glycosyl hydrolase (GH) enzyme families, 98 INDUSTRIAL BIOTECHNOLOGY A P R I L 2 0 1 6 namely GH67 and GH115, have been identified that exhibit aglucuronidase activity on xylooligosaccharides or xylan.1,2 Most of the known a-glucuronidases that remove MeGlcA residues from xylooligosaccharides belong to the GH67 family. Members of this family remove MeGlcA residues only from the terminal non-reducing end of the xylooligosaccharides but not for polymeric xylan. In 2009, Ryebova et al. reported the existence of an extracellular a-glucuronidase from Scheffersomyces stipitis that could remove MeGlcA residues from the internal chain of xylan. This activity is not exhibited by members of the GH 67 family. The discovery of the S. stipitis enzyme led to the classification of a new GH115 family. Prior to this discovery, another a-glucuronidase from Schizophyllum commune was shown to exhibit activity towards polymeric xylan.3 At that time, the GH115 family was not known and the authors reported a lack of sequence homology of this enzyme with other known GH67 enzymes. Subsequently, a-glucuronidase from another S. commune strain was characterized and confirmed to be a member of the GH115 family.4 About 265 members of the GH115 family have been identified by sequence analysis.5 The majority are from bacterial sources. Among these, three prokaryotic enzymes from Bacteroides ovatus, Bacteroides thetaiotaomicron, and Streptomyces pristinaespiralis, and two eukaryotic enzymes from S. stipitis and S. commune have been shown to display a-glucuronidase activity.2–4,6–9 Members of the GH115 family share 31–45% primary sequence identity and the conserved residues are distributed throughout the protein sequences without clear regions of homology. The native GH115 enzyme was purified from S. stipitis, which is a naturally occurring xylose-fermenting yeast with the rare ability to hydrolyze xylan and ferment the resultant xylose directly to ethanol.10 Sequence analysis of the S. stipitis GH115 (SsAgu115) confirmed its unique identity, distinct from GH67 as well as other previously characterized GH family enzymes.2 Although SsAgu115 is active against both xylooligosaccharides and polymeric xylan, its activity was higher against the former.8 This suggests that synergistic activity of GH115 with endoxylanases is required for appreciable MeGlcA liberation. However, no information is available on the kinetic parameters for a eukaryotic GH115 enzyme. In this study, the gene encoding a-glucuronidase from S. stipitis was cloned and expressed in Pichia pastoris. The recombinant enzyme was subjected to kinetic and functional characterization using several xylo-oligosaccharides and xylans. This is the first report on kinetic characterization of a eukaryotic GH115 a-glucuronidase. We also determined the synergistic action of SsAgu115 and endo-1,4-b-xylanases belonging to the GH10 and GH11 families on hydrolysis of beech xylan, birch xylan, and oat DOI: 10.1089/ind.2015.0031 GH115 a-GLUCURONIDASE FROM S. STIPITIS spelt xylan. In addition, the ability of SsAgu115 to reduce the solubility of polymeric xylan was assessed. Selective removal of MeGlcA reduces the solubility of xylan. Insoluble xylans may form hydrogels that have biotechnological applications in the pharmaceutical and biomedical fields.11,12 Materials and Methods CHEMICALS, MICROBIAL STRAINS, VECTORS, CULTURE MAINTENANCE All chemicals were purchased from Sigma–Aldrich (Oakville, Canada). A genomic DNA isolation kit was purchased from Qiagen (Toronto, Canada). Ni Sepharose excel affinity resin was purchased from GE Healthcare Life Sciences (Mississauga, Canada). EKMax enterokinase and NuPAGE Novex 10% Bris-Tris gel were purchased from Life Technologies (Burlington, Canada). GH10 xylanase from Bacillus stearothermophilus T6 and GH11 xylanase from Thermomyces lanuginosus were purchased from Megazyme (Wicklow, Ireland) and Sigma-Aldrich, respectively. a-glucuronidase assay and D-glucuronic/D-galacturonic acid assay kits were purchased from Megazyme. Beech xylan, birch xylan, and oat spelt xylan were purchased from Sigma-Aldrich. S. stipitis NRRL Y-7124 (NRC 2548) was obtained from the National Research Council Canada Culture Collection (Ottawa). P. pastoris X-33 and yeast expression vector pPICZaC were obtained from Life Technologies (Norwalk, CT). pPICZaC has an a-factor secretion signal for directing expression of the recombinant protein and a zeocin antibiotic resistance gene for selection in both Escherichia coli and P. pastoris. S. stipitis and P. pastoris cultures were individually maintained on YPD agar (dextrose 2%, peptone 2%, yeast extract 1%, and agar 1.5%) plates at 4C and were subcultured at monthly intervals. All the strains were stored in 25% (w/v) glycerol for long-term preservation. CLONING, EXPRESSION VECTOR CONSTRUCTION, AND MAINTENANCE IN E. COLI S. stipitis genomic DNA was purified using a DNeasy minikit from Qiagen (Hilden, Germany) according to the manufacturer’s directions. The ssagu115 gene (GenBank accession number NC_009046) was amplified from S. stipitis genomic DNA using forward (5¢-GTGCGGTACCCATATGTTGTTTCATACTTCC AGCGT-3¢) and reverse primers (5¢-GTGCGCATGCGCTCT TCCGCACTTTTTGATGTAAGTTTCTGGTGGTCC-3¢). Restriction sites used for directional cloning are underlined in the above sequences. Amplification was carried out using Phusion high-fidelity DNA polymerase under the following conditions: denaturation at 98C for 3 min, followed by 25 cycles of amplification at 98C for 30 s, 50C for 50 s, and 72C for 2 min. The part of the gene coding for the first 18 amino acids was omitted from the sequence during polymerase chain reaction (PCR) cloning. This sequence corresponds to the enzyme secretion signal sequence and is not preserved in the mature extracellular protein of 979 amino acids.2 The ssagu115 gene was cloned in pUC19 previously digested with KpnI and SphI and maintained in E. coli TOP10 cells. The ssagu115 gene was also cloned into pTYB1 previously digested with Nde1 and Sap1 and maintained in T7 Express Competent E. coli cells. ssagu115 was subcloned into pPICZaC downstream of the AOX1 promoter and the a-factor signal sequence using the In-Fusion EcoDry Cloning kit (Clontech Laboratories, Mountain View, CA) following the manufacturer’s directions. The sequences for the forward primer and reverse primers were: 5¢-TCTCTCGAGAAGAGACATCA TCATCATCATCATgacgacgacgacaagTTGGGTGGGTTGCAAA ATATT-3¢ and 5¢-TCTAAGGCTACAAACCTATTTGATGT AAGTTTCTGGTGGTG-3¢, respectively (sequences specific to ssagu115 are shown in normal upper case letters while vectorspecific sequences are shown in bold upper case letters). Sequences for N-terminal 6·His-tag (upper case italics) and enterokinase cleavage site (lower case) were incorporated in the forward primer. The recombinant plasmid (pPICZaC-SsAgu115) was transformed into HST08 E. coli stellar competent cells, and positive clones were selected on low salt Luria-Bertani (LB) agar (1% tryptone, 0.5% yeast extract, 0.5% NaCl, and 1.5% agar, pH 7.5) plates supplemented with 25 lg/mL zeocin. The correct insert was confirmed by DNA sequencing in both directions. TRANSFORMATION OF P. PASTORIS CELLS P. pastoris X-33 cells were transformed with 5–10 lg of PmeI-linearized recombinant plasmid (pPICZaC-SsAgu115) by electroporation using the Gene Pulser (Bio-Rad; Hercules, CA) following the Invitrogen transformation protocol described in the manual for Pichia expression vectors. The electroporated cells were spread on YPDS (2% peptone, 2% dextrose, and 1 M sorbitol) agar plates supplemented with 100 lg/mL zeocin and incubated at 30C for 3–5 days until colonies formed. About 30 randomly selected colonies were individually streaked on 100 lg/mL zeocincontaining YPDS agar plates. The presence of the ssagu115 gene in the transformants was confirmed by PCR using insert- and vector-specific primers with yeast genomic DNA as the template. EXPRESSION OF SSAGU115 IN P. PASTORIS Expression studies were done following the Invitrogen protocol from the Pichia expression kit. Ten positive transformants were grown individually in 5 mL of YPD medium in 50-mL sterile tubes at 30C for 24 h with shaking at 200 rpm. Each culture was then inoculated into 25 mL of BMGY medium (2% w/v peptone, 1.34% w/v yeast nitrogen base, 4 · 10-5% w/v biotin, 1% v/v glycerol, and 100 mM potassium phosphate, pH 6.0) in a 250-mL Erlenmeyer flask and shaken (200 rpm) overnight at 30C. The cells were centrifuged at 10,000·g for 10 min at 4C and resuspended in 25 mL of BMMY induction medium (1% w/v yeast extract, 2% w/v peptone, 1.34% w/v yeast nitrogen base, 4 · 10-5 % w/v biotin, 1% v/v methanol, and 100 mM potassium phosphate, pH 6.0). To maintain induction, methanol was added every 24 h to a final concentration of 1.0% (v/v) for 5 days. Samples were withdrawn every 24 h and extracellular protein expression was determined by SDS-PAGE (NuPAGE Novex 10% Bris-Tris gel). After 5 days, a-glucuronidase activity was measured and the clone with the highest activity was selected for scale-up production. Large-scale (2-L) protein expression was carried out following the Invitrogen protocol described in the Pichia expression kit. His-tagged SsAgu115 was purified from the supernatant by immobilized metal ion affinity chromatography (IMAC), using ª M A R Y A N N L I E B E R T , I N C .  VOL. 12 NO. 2  APRIL 2016 INDUSTRIAL BIOTECHNOLOGY 99 BAJWA ET AL. Ni Sepharose excel affinity resin according to the manufacturer’s instructions. His-tagged protein was eluted using an elution buffer comprised of 50 mM NaH2PO4 (pH 7.0), 0.5 M NaCl, 250 mM imidazole, and 10% (v/v) glycerol. Finally, the His-tag was cleaved using enterokinase. Ten milligrams of the purified protein dissolved in 50 mM NaH2PO4 (pH 7.0), 50 mM NaCl, and 1 mM CaCl2 was mixed with enterokinase (100 U), and the reaction mixture was incubated at 23C for 16–18 h and loaded onto a Ni Sepharose excel affinity resin column to remove any uncleaved protein. The uncleaved protein was bound to the resin while the cleaved protein along with enterokinase was eluted in the flow through. Enterokinase was removed by passing the flow through containing the cleaved protein through Amicon ultra-15 centrifugal filter units with a 50-kDa cut-off. The protein was finally exchanged into the storage buffer containing 50 mM NaH2PO4 (pH 7.0), 0.5 M NaCl, and 10% glycerol and stored at -20C. ENZYME SUBSTRATES AND ASSAY Aldotetraouronic acid (UXX) was prepared by hydrolyzing beech xylan with GH10 endo-1,4-b-xylanase from B. stearothermophilus T6. GH10 xylanase generates predominantly UXX along with small amounts of xylobiose and xylotriose from xylan hydrolysis. In aldotetraouronic acid generated by a GH10 xylanase, the non-reducing xylose residue of xylotriose is substituted with MeGlcA.13 The reaction mixture containing 50 mM potassium phosphate buffer (pH 6.0), 4% (w/v) beech xylan, and 100 U of GH10 xylanase was incubated for 18–24 h at 40C. Aldopentaouronic acid (XUXX) was prepared by hydrolyzing beech xylan with recombinant GH11 endo-1,4-bxylanase from T. lanuginosus. GH11 xylanase generates predominantly XUXX along with small amounts of xylobiose and xylotriose from xylan hydrolysis. In XUXX generated by a GH11 xylanase, the xylose residue immediately following the non-reducing residue of xylotetrose is substituted with MeGlcA.13 The reaction mixture containing 50-mM potassium phosphate buffer (pH 6.0), 4% (w/v) beech xylan, and 2,500 U of GH11 xylanase was incubated for 18–24 h at 40C. Acidic xylooligosaccharides were purified from the digestion mixture by passing through a column of AG1–X8 anion exchange resin (acetate form). After eluting the neutral sugars by water, acidic oligosaccharides were eluted by 3 M acetic acid. Acetic acid was removed by evaporation. SsAgu115 activity was measured using the a-glucuronidase kit from Megazyme. Hydrolysis of xylan or acidic xyloologosaccharides by GH115 a-glucuronidase released 4-Omethylglucuronic acid, which was in turn oxidized by nicotinamide adenine dinucleotide (NAD)-dependent uronate dehydrogenase to D-glucarate. The coupled reaction was followed by measuring the formation of NADHat 340 nm. One unit of a-glucuronidase activity was the amount of enzyme required to liberate one lmole of MeGlcA per min. INITIAL CHARACTERIZATION OF SSAGU115 To compare the ability of the recombinant SsAgu115 to hydrolyze substrates with different degrees of polymerization, the enzyme reactions containing 2.8 lg of purified enzyme and 10 mg/mL of the individual substrates (XUXX, UXX, beech, 100 INDUSTRIAL BIOTECHNOLOGY A P R I L 2 0 1 6 birch, and oat spelt xylan) suspended in 50 mM sodium acetate buffer (pH 5.0) were incubated for 15 min at 23C. The effect of substrate concentration on a-glucuronidase activity was evaluated using varying concentrations of XUXX (0.78-35 mM) and UXX (0.94-42 mM). For beech and birch xylan, enzyme activity was measured at substrate concentrations ranging from 2.85-60 mg/mL. The xylan substrates were added to water and dissolved by heating. Due to the lower solubility of xylans, concentrations higher than 60 mg/mL could not be tested. The assay was performed at 23C and pH 5.0. The Km and Vmax values were estimated from the substrate dependence data using the nonlinear regression function of Graph Pad Prism version 6.04 for Windows (GraphPad Software, La Jolla, CA). The Kcat values were calculated based on a molecular weight (MW) of 150 kDa determined by SDS-PAGE. For determining the optimal temperature and pH, the assays were performed using 2.8 lg of the purified enzyme and XUXX as the substrate. For optimal temperature determination, the reaction mixtures were incubated for 15 min in 50 mM sodium acetate buffer (pH 5.0) at 23, 35, 45, 50, and 60C. Optimal pH was determined at 23C using buffers adjusted to pH values ranging from 3.0 to 8.5 (sodium acetate, pH 3.0–5.5; potassium phosphate, pH 6.0–8.5). ENZYME SYNERGY STUDIES Potential synergy between a-glucuronidase and xylanases was evaluated by hydrolyzing individual substrates (beech, birch, and oat spelt xylans) using 2.8 lg of purified SsAgu115— either alone or in combination with 10 U of a GH10 or GH11 xylanase and 10 mg of the substrate in a standard assay done for 15 min at 50C. EFFECT OF SSAGU115 ON XYLAN SOLUBILITY The ability of SsAgu115 to reduce the solubility of polymeric xylan was assessed qualitatively by incubating 25 mg/mL beech xylan with 5 and 10 lg of purified SsAgu115 in 50 mM acetate buffer pH 5.0 in a total volume of 1 mL. The reaction mixture was incubated at 22C and 40C for 24 h, and the solubility of xylan was assessed by measuring the turbidity of the reaction mixture at 600 nm. Heat-killed enzyme was used in the control samples. The amount of glucuronic acid released was estimated using the a-glucuronidase kit from Megazyme. To estimate the total glucuronic acid content of xylans, approximately 100 mg of each polysaccharide was digested with 5 mL of 2 M H2SO4 in screw cap glass tubes and incubated at 100C for 6 h. The tubes were cooled to room temperature, and 7 mL of 2 M NaOH were added. The contents of the tube were transferred to a 100 mL volumetric flask, and the volume was adjusted to 100 mL with distilled water. The solution was centrifuged at 1,500 g for 10 min prior to measuring the amount of glucuronic acid, as described above. A control sample with glucuronic acid was treated the same way to determine the extent of its degradation during acid hydrolysis, and this was taken into account when calculating the total amount of glucuronic acid in xylan. ADDITIONAL PROCEDURES Protein concentration was measured by the Bradford assay using bovine serum albumin as the standard.14 The MW of the GH115 a-GLUCURONIDASE FROM S. STIPITIS enzyme was determined by SDS-PAGE and NativePAGE (NativePAGE Novex 4–16% Bis-Tris Gels). EXPERIMENTAL REPLICATION AND DATA ANALYSIS All enzyme assays were conducted 3 times and the values shown in the figures are the averages – standard deviations from 3 independent sets of experiments. Results and Discussion CLONING OF SSAGU115 GENE AND ITS EXPRESSION IN P. PASTORIS The 2,940-bp ssagu115 gene was cloned by PCR from the S. stipitis genomic DNA into pPICZaC downstream of the AOX1 promoter and the a-factor signal sequence to yield pPICZaC-SsAgu115. The recombinant plasmid was transformed into E. coli HST08 cells for maintenance and into P. pastoris X33 cells for expression. SsAgu115 was expressed as a secreted protein by P. pastoris cells under the control of AOX1 promoter. Ten zeocin-resistant clones were randomly selected, cultured independently in BMMY medium for 72 h, and assayed for extracellular a-glucuronidase activity. All 10 clones showed good and comparable enzyme activity; the clone with the highest activity was selected for protein purification. No activity was detected in the culture supernatant of a clone transformed with the insert-free pPICZaC vector. About 5 mg of His-tagged recombinant enzyme was purified per liter of culture supernatant. Both the native S. stipitis a-glucuronidase and the recombinant form expressed in Saccharomyces cerevisiae have a MW of 120–125 kDa.2,15 However, the MW of the recombinant enzyme secreted in the P. pastoris cultures was about 150 kDa, as determined by SDS-PAGE. The higher MW of the recombinant enzyme secreted by P. pastoris was likely due to greater glycosylation, as reported by some researchers for other recombinant enzymes.16–18 The MW of the recombinant S. stipitis a-glucuronidase on non-denaturing native gel electrophoresis was about 380 kDa, suggesting the presence of more than one subunit. The optimal pH (pH 4.0) and temperature (50C) of the recombinant a-glucuronidase closely resembled those reported for the native enzyme (pH 4.4, 60C).2 BIOCHEMICAL PROPERTIES OF RECOMBINANT S. STIPITIS a-GLUCURONIDASE Substrate preference. To examine the substrate preference of the recombinant a-glucuronidase, its specific activity was measured against short and longer chain substrates, including terminally or internally substituted aldouronic acids (UXX and XUXX) and several xylans. Both terminally (UXX) and internally substituted (XUXX) aldouronic acids were hydrolyzed by the a-glucuronidase, with 1.7-fold higher specific activity against UXX (24 U/mg) than XUXX (14 U/mg) (Fig. 1). Interestingly, the specific activities of the recombinant enzyme towards aldouronic acids were about 3-fold higher compared to those reported for the native enzyme under similar assay conditions.8 The reason for this difference is not known. While the recombinant a-glucuronidase was able to release MeGlcA from xylan, it was more active against xylooligosaccharides, as has been reported for the native enzyme.8 Fig. 1. Specific activities of SsAgu115 on aldotetraouronic acid (UXX), aldopentaouronic acid (XUXX), beech, birch, and oat spelt xylan (10 mg/mL each) at 23C in 50 mM sodium acetate buffer (pH 5.0). Among the xylans tested, the enzyme was most active against beech xylan (specific activity of 4 U/mg), followed by birch xylan (1 U/mg). Activity against oat spelt xylan was negligible. Differences in enzyme activity against different xylans could be attributed to differences in the degree of MeGlcA substitution and the presence of other side groups between the xylans. Beech and birch are angiosperms, in which the xylan is highly substituted with MeGlcA residues (20–35%).19–21 Oat spelt xylan is an arabinoglucuronoxylan.19–21 It is possible that SsAgu115 was hindered by the presence of L-arabinofuranosyl substituents, and that the activity of a-glucuronidase on oat spelt xylan might be enhanced by a-L-arabinofuranosidase. Substrate dependence and kinetic parameters. To our knowledge, no information is available on the kinetic parameters for a eukaryotic GH115 enzyme. In this study, kinetic and functional characterization was done on SsAgu115. The kinetic parameters were determined using UXX, XUXX, beech, and birch xylan as substrates. SsAgu115 exhibited higher maximum activity (Vmax) and affinity (lower Km) for xylooligosaccharides compared to polymeric xylans (Table 1). Substrate inhibition was observed at high concentrations of aldouronic acids, whereas normal Michaelis–Menten kinetics were observed with the polymeric substrates within the concentration ranges tested. The low solubility of xylan prevented higher concentrations from being Table 1. Kinetic parameters for SsAgu115 Vmax (lmole/ min/mg) Km (mg/mL) Kcat (min-1) Kcat/Km (min-1/ mg/mL) UXX 24.9 5.1 4,150 813.7 XUXX 18.4 6.5 3,066 471.6 Beech xylan 8.7 9.5 1,450 152.6 Birch xylan 7 35 1,166 33.3 SUBSTRATE ª M A R Y A N N L I E B E R T , I N C .  VOL. 12 NO. 2  APRIL 2016 INDUSTRIAL BIOTECHNOLOGY 101 BAJWA ET AL. Fig. 2. Effect of substrate concentration on SsAgu115 activity towards aldouronic acids. Inset: Lineweaver–Burke plot. Symbols: UXX and A XUXX.  tested. a-glucuronidase activity increased with increasing aldouronic acid concentrations to a maximum, beyond which a further increase in substrate concentration (above 14 and 17 mg/ mL [23 mM] for UXX and XUXX, respectively) resulted in decreased enzyme activity (Fig. 2). Substrate inhibition (above 6 mM UXX and 25 mM UXXX) with aldouronic acids has also been reported for the Aureobasidium pullulans GH67 a-glucuronidase.22 The Kcat of a-glucuronidase against beech xylan was about 3- and 2-fold lower than UXX and XUXX, respectively (Table 1). Against birch xylan, the Kcat was about 3.5- and 2.6-fold lower than UXX and XUXX, respectively. The enzyme showed a decreasing reaction rate and turnover number with substrates of longer chain length. The substrate affinity and catalytic efficiency also declined with increasing chain length. Against birch xylan, the apparent Km was 3.6-fold higher and catalytic efficiency was 4-fold lower compared to beech xylan (Table 1). SYNERGY BETWEEN a-GLUCURONIDASE AND ENDOXYLANASES Most of the a-glucuronidases characterized to date belong to the GH67 family. Since these enzymes remove MeGlcA residues only from the terminal nonreducing end of xylooligosaccharides, but not from polymeric xylan, they require the synergistic action of endoxylanase and b-xylosidase for efficient liberation of MeGlcA from polymeric substrates.3,23,24 GH115 enzymes, unlike GH67 enzymes, can remove terminal as well internal MeGlcA residues from polymeric xylan as well as xylooligosaccharides.2 Therefore, it was of interest to assess if and to what extent synergies exist between the endoxylanases and GH115 enzyme. In this study, the potential synergistic influence of GH10 and GH11 endoxylanase on SsAgu115 was examined by incubating 102 INDUSTRIAL BIOTECHNOLOGY A P R I L 2 0 1 6 Fig. 3. Synergistic effect of GH10 and GH11 endoxylanases on SsAgu115 activity towards beech, birch, and oat spelt xylan. Purified SsAgu115 (2.8 lg) was tested either alone or in combination with 10 U of a GH10 or GH11 xylanase for 15 min at 50C in a standard assay mixture that contained 10 mg of the substrate. Symbols: beech xylan, birch xylan, and , oat spelt xylan. SsAgu115 and one of the endoxylanases with a xylan substrate. The activity of a-glucuronidase was clearly enhanced in the presence of GH10 or GH11 xylanase (Fig. 3). On beech xylan, 1.3- to 2-fold more MeGlcA was released when SsAgu115 was tested together with a GH10 or GH11 xylanase. On birch xylan, 4-fold more MeGlcA was released by the combined action of Fig. 4. Effect of SsAgu115 on solubility of beech xylan (25 mg/ml) incubated at (A) 22C and (B) 40C for 24 h. Symbols: 5 lg enzyme, 5 lg heat-killed enzyme, 10 lg enzyme, and 10 lg heat-killed enzyme. GH115 a-GLUCURONIDASE FROM S. STIPITIS a-glucuronidase and endoxylanases. The extent of MeGlcA removal from oat spelt xylan was slightly increased in the presence of a combination of enzymes. Since the activity of SsAgu115 is lower on polymeric substrates compared to xylooligosaccharides, the presence of endoxylanases increased the efficiency of side chain removal by hydrolyzing the polymer into shorter chain substrates against which the a-glucuronidase exhibited higher activity. Since the enzyme was more active on terminally substituted aldouronic acids compared to internally substituted aldouronic acids, the synergistic action was higher in the presence of GH10 (which generated UXX) compared to GH11 endoxylanase (which generated XUXX). It is not currently known how the other side chains in polymeric xylan affected SsAgu115 activity. A better picture would be available when SsAgu115 is used in combination with endoxylanases and other debranching enzymes such as acetylxylan esterase and a-Larabinofuranosidase. When GH115 a-glucuronidase from S. commune was used in combination with acetylxylan esterase, xylanase, and b-xylosidase, more MeGlcA was released from acetylated glucuronoxylan from birch.3 However, no synergistic action was seen between a-glucuronidase and a-L-arabinofuranosidase in removing MeGlcA groups from arabinoglucuronoxylan from spruce pulp, although a-L-arabinofuranosidase efficiently removed arabinose from spruce pulp.3 EFFECT OF SSAGU115 ON XYLAN SOLUBILITY The presence of the various side groups on the xylan backbone renders xylan more soluble in water.3 Therefore, removal of side chains is expected to change the solubility, rheological property, and possibly the potential biotechnological applications of xylans. In this study, we assessed the ability of GH115 a-glucuronidase to reduce the solubility of beech xylan by incubating the xylan suspensions with GH115 enzyme for 24 h. Treatment with the recombinant SsAgu115 resulted in increased viscosity visible by eye and precipitation of the xylan as indicated by increased optical density (OD)600 (Fig. 4). At 22C, OD600 increased from 0.52 to 1.47 and from 0.53 to 1.63 over 24 h when beech xylan was incubated with 5 lg and 10 lg of enzyme, respectively. This corresponded to removal of about 48% of MeGlcA side groups from xylan. The increase in OD600 (0.5 to 1.79 and 0.51 to 1.90 with 5 lg and 10 lg of enzyme, respectively) and percent removal of MeGlcA side groups (56%) over 24 h were higher when the incubation temperature was raised to 40C. OD600 did not increase over 24 h in the control samples, to which heat-killed enzyme was added. Conclusions SsAgu115 was subjected to kinetic and functional characterization. The enzyme was more active against xylooligosaccharides than polymeric xylan. SsAgu115 acts in synergy with GH10 and GH11 endoxylanases. Removel of MeGlcA side groups by SsAgu115 reduces xylan solubility. Insoluble xylans are potential raw materials for production of hydrogels.11 A bioactive substance can be encapsulated within the hydrogel and used for sustained slow-release or targeted drug delivery.12 The utilization of SsAgu115 for reducing xylan solubility is a new area of research with novel applications that hold considerable promise and warrant further investigation. Acknowledgments This research was funded by the Ontario Ministry of Research & Innovation. We thank Emma Master, Weijun Wang, and Thu Vuong from University of Toronto for providing valuable suggestions. Author Disclosure Statement No competing financial interests exist. REFERENCES 1. Nurizzo D, Nagy T, Gilbert HJ, Davies, GJ. The structural basis for catalysis and specificity of the Pseudomonas cellulosa a-glucuronidase, GlcA67A. Structure 2002;10:547–556. 2. Ryabova O, Vrsanska M, Kaneko S, et al. 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