Figures
Abstract
A novel β-xylosidase gene of glycosyl hydrolase (GH) family 3, xyl3A, was identified from the thermophilic fungus Humicola insolens Y1, which is an innocuous and non-toxic fungus that produces a wide variety of GHs. The cDNA of xyl3A, 2334 bp in length, encodes a 777-residue polypeptide containing a putative signal peptide of 19 residues. The gene fragment without the signal peptide-coding sequence was cloned and overexpressed in Pichia pastoris GS115 at a high level of 100 mg/L in 1-L Erlenmeyer flasks without fermentation optimization. Recombinant Xyl3A showed both β-xylosidase and α-arabinfuranosidase activities, but had no hydrolysis capacity towards polysaccharides. It was optimally active at pH 6.0 and 60°C with a specific activity of 11.6 U/mg. It exhibited good stability over pH 4.0–9.0 (incubated at 37°C for 1 h) and at temperatures of 60°C and below, retaining over 80% maximum activity. The enzyme had stronger tolerance to xylose than most fungal GH3 β-xylosidases with a high Ki value of 29 mM, which makes Xyl3A more efficient to produce xylose in fermentation process. Sequential combination of Xyl3A following endoxylanase Xyn11A of the same microbial source showed significant synergistic effects on the degradation of various xylans and deconstructed xylo-oligosaccharides to xylose with high efficiency. Moreover, using pNPX as both the donor and acceptor, Xyl3A exhibited a transxylosylation activity to synthesize pNPX2. All these favorable properties suggest that Xyl3A has good potential applications in the bioconversion of hemicelluloses to biofuels.
Citation: Xia W, Shi P, Xu X, Qian L, Cui Y, Xia M, et al. (2015) High Level Expression of a Novel Family 3 Neutral β-Xylosidase from Humicola insolens Y1 with High Tolerance to D-Xylose. PLoS ONE 10(2): e0117578. https://doi.org/10.1371/journal.pone.0117578
Academic Editor: Linsheng Song, Institute of Oceanology, Chinese Academy of Sciences, CHINA
Received: September 28, 2014; Accepted: December 28, 2014; Published: February 6, 2015
Copyright: © 2015 Xia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: This work was supported by the National High-Tech Research and Development Program of China (863 Program, 2012AA022105) and the National Science Foundation for Distinguished Young Scholars of China (31225026) and the 948 program of the Ministry of Agriculture (2014-S1) and the China Modern Agriculture Research System (CARS-42). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Hemicelluloses, mainly composed of hetero-1,4-β-d-xylans and hetero-1,4-β-d-mannans, are the second most abundant renewable polysaccharides in nature [1]. As the major component of hemicellulose, xylan is composed of a backbone of β-1,4-linked d-xylopyranosyl residues and side chains of different substituents. Thus, the degradation of xylan requires action of a battery of debranching and depolymerizing activities, which is achieved by the synergic hydrolysis of multiple enzymes. Specifically, xylanase attacks internal xylosidic linkages on the backbone, β-xylosidase releases xylosyl residues by endwise attack of xylooligosaccharides, and other debranching enzymes including α-l-arabinofuranosidase, α-glucuronidase and esterases remove branched chains [2]. Single xylosyl residues released from xylooligosaccharides and reducing end product inhibit xylanases, which makes β-xylosidase a key enzyme in the xylanolytic system with a great potential in many biotechnological applications.
β-Xylosidases (EC 3.2.1.37) have been classified into the glycoside hydrolase (GH) families 3, 39, 43, 52 and 54 based on the amino acid sequence similarities (http://www.cazy.org/Glycoside-Hydrolases.html) [3]. Their catalytic mechanisms are distinct; for instance, those of GH3 employ retaining catalysis and GH43 ones have inverting catalysis. Up to the present days, β-xylosidases have been described from a variety of microorganisms including bacteria and fungi, and fungal β-xylosidases are mostly grouped into GH3 [4–8]. Although β-xylosidase has been a research hotspot in the biofuel field in recent years due to its fermentation ability on pentose sugars [9], few fungal β-xylosidases of GH3 have yet been identified or heterologously expressed. Therefore, a better understanding of the deconstruction of xylan to constituent sugars, mainly xylose, for subsequent fermentation by ethanol producing microbes is critical to the efficient use of plant biomass for biofuel production [10].
Thermophilic and thermostable enzymes from thermophilic fungi are usually used in the conversion of hemicellulose to reduce the risk of contamination and increase the solubility of substrate [11]. Of the fungal sources, thermophilic Humicola is widely considered to be an innocuous and non-toxic fungus that produces a wide variety of GHs. Cellulases and hemicellulases from this genus have favorable characteristics and are utilized in various commercial applications, such as enzyme detergents (Novo Industri A/S., Enzymatic detergent additive, U.S. Patent 4810414) and enzyme preparations (Ultraflo L, Novozymes A/S, mainly composed of β-glucanase and xylanase). Humicola insolens Y1 has been reported to be an excellent producer of xylanolytic enzymes [12, 13], in which three thermophilic GH10 xylanases, one GH11 xylanase, and two bifunctional GH43 xylosidase/arabinosidases have been characterized. In this study, a novel β-xylosidase gene of GH3 was identified in H. insolens Y1 and overexpressed in Pichia pastoris. Biochemical characterization and synergy test with xylanase on xylan degradation were also carried out. This is a prior article for now through exhaustively describing the sequence identity, enzymatic properties, xylose tolerance, transxylosylation activity and synergistic action with xylanase of a fungal GH3 β-xylosidase.
Materials and Methods
Strains, media, vectors and chemicals
H. insolens Y1 GCMCC 4573 was routinely cultured in wheat bran medium as described previously [12]. Escherichia coli Trans1-T1 and vector pEASY-T3 (TransGen, Beijing, China) were used for gene cloning. The heterologous protein expression system of pPIC9 and P. pastoris GS115 (Invitrogen, Carlsbad, CA) was employed as the gene expression vector and expression host, respectively. The DNA purification kit, restriction endonucleases and LA Taq DNA polymerase were purchased from TaKaRa (Otsu, Japan). T4 DNA ligase and the total RNA isolation system kit were purchased from Promega (Madison, WI). The cDNA synthesis kit was purchased from TransGen.
Beechwood xylan, 4-nitrophenyl β-d-xylopyranoside (pNPX), 4-nitrophenyl α-l-arabinofuranoside (pNPAf), 4-nitrophenyl α-d-galactopyranoside (pNPGal), 4-nitrophenyl β-d-glucopyranoside (pNPG), 4-nitrophenyl α-l-arabinopyranoside (pNPAb) and 4-nitrophenyl β-d-cellobioside (pNPC) were purchased from Sigma-Aldrich (St. Louis, MO). Soluble wheat arabinoxylan was obtained from Megazyme (Wicklow, Ireland). All other chemicals used were of analytical grade and commercially available.
Cloning of the cDNA of xyl3A
Total RNA of H. insolens Y1 was extracted from the mycelia after 3 days’ growth in the inducing medium [12], and was reverse transcribed into cDNA by RT-PCR following the protocol of One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen). Based on the partial genome sequence of strain Y1 (whole genome sequencing in progress), a β-xylosidase encoding gene of GH3, xyl3A, was identified, and the gene fragment without the signal peptide-coding sequence was amplified with the specific primer set (Xyl3A-F: 5′-GGGGAATTCGGGGTCCTCCCCGACTGCAGCAAGCCG-3′, and Xyl3A-R: 5′-GGGGCGGCCGCTCAATTCTTGAGGTTGGGGTCAACAG-3′, restriction sites underlined). H. insolens Y1 cDNA was used as the template, and the annealing temperature was 60°C. The PCR products were purified and ligated into the pEASY-T3 vector for sequencing.
Sequence analysis
DNA and protein sequence were aligned using the BLASTn and BLASTp programs (http://www.ncbi.nlm.nih.gov/BLAST/), respectively. Vector NTI Advance 10.0 software (Invitrogen) was used to analyze DNA sequence and to predict the molecular weight of protein. Genes, introns, exons and transcription initiation sites were predicted using the online software FGENESH (http://linux1.softberry.com/berry.phtml). Multiple sequence alignments were performed with the ClustalW software. The signal peptide and the potential N-glycosylation sites were predicted by the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/) and the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/), respectively.
High level expression and purification of recombinant Xyl3A
The cDNA of xyl3A without the signal peptide-coding sequence and the pPIC9 vector were both digested by EcoRI and NotI and ligated into in-frame fusion of the α-factor signal peptide to construct the recombinant plasmid pPIC9-xyl3A. The recombinant plasmid was linearized using BglII and transformed into P. pastoris GS115 competent cells by electroporation using Gene Pulser X cell Electroporation System (Bio-Rad, Hercules, CA). Minimal dextrose medium (MD) plates were prepared for the screening of positive transformants. The positive transformants were transferred to buffered glycerol complex medium (BMGY) and grown at 30°C for 2 days. The cells were collected by centrifugation and resuspended in buffered methanol complex medium (BMMY) containing 0.5% methanol for induction. The β-xylosidase activities of the culture supernatants were assayed as described below, and the transformant exhibiting the highest β-xylosidase activity was subjected to high level expression in 1-L Erlenmeyer flasks according to the Pichia expression manual (Invitrogen).
The culture supernatants of aforementioned recombinant strain were collected by centrifugation (12,000 × g, 4°C, 10 min) to remove cell debris and undissolved materials, followed by concentration through a Vivaflow ultrafiltration membrane (Vivascience, Hannover, Germany) with a molecular weight cut-off of 5 kDa. The crude enzyme was loaded onto a FPLC HiTrap Q Sepharose XL 5 mL column (GE Healthcare, Uppsala, Sweden) that was equilibrated with 20 mM Tris-HCl (pH 8.0). Proteins were eluted using a linear gradient of NaCl (0–1.0 M) in the buffer mentioned above at a flow rate of 3.0 mL/min. Fractions exhibiting β-xylosidase activities were pooled and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined by a Bradford assay with bovine serine albumin as a standard. To remove N-glycosylation during heterologous expression in P. pastoris, purified recombinant Xyl3A was incubated with 500 U of endo-β-N- acetylglucosaminidase H (Endo H) at 37°C for 2 h according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA). The deglycosylated enzyme was also analyzed by SDS-PAGE.
Enzyme activity assay
The β-xylosidase activity was assayed using pNPX as the substrate. The standard reaction system consisted of 250 μL of appropriately diluted enzyme and 250 μL of McIlvaine buffer (pH 6.0) containing 2 mM pNPX. After incubation at 60°C for 10 min, 1.5 mL of 1.0 M Na2CO3 was added into the system to terminate the reaction. The amount of p-nitrophenol released was determined spectrophotometrically by reading the absorbance at 405 nm. One unit of β-xylosidase activity was defined as the amount of enzyme that released 1 μmol of p-nitrophenol per minute under the assay conditions. Each experiment was performed in triplicate.
Biochemical characterization
The optimal pH for Xyl3A activity was determined at 60°C for 10 min over a pH range of 2.0–11.0 using the following buffers: 100 mM Na2HPO4-citric acid (pH 2.0–8.0), 100 mM Tris-HCl (pH 8.0–9.0), and 100 mM glycine-NaOH (pH 9.0–11.0). To estimate pH stability, the enzyme was pre-incubated in the buffers mentioned above without substrate at 37°C (physiological temperature) or 60°C (optimal temperature) for 1 h, and the residual activities were measured under the standard conditions (pH 6.0, 60°C, 10 min).
The optimal temperature was examined at the optimal pH by measuring the enzyme activity over the temperature range of 30 and 90°C. The thermostability was investigated by determining residual enzyme activities after preincubation at 60°C or 70°C and optimal pH without substrate for various periods. The samples were collected at 0, 2, 5, 10, 20, 30 and 60 min, respectively. Enzyme activity assays were performed under the standard conditions.
The enzyme activity of Xyl3A was measured in the presence of 5 mM of various metal ions and chemical reagents (Ag+, Ca2+, Li+, Co2+, Cr3+, Ni2+, Cu2+, Mg2+, Fe3+, Mn2+, Hg2+, Pb2+, EDTA, SDS or β-mercaptoethanol) to estimate their effect on Xyl3A activity. The reaction without any additive was used as a blank control.
Substrate specificity and kinetic parameters
To investigate the substrate specificity of Xyl3A, activity assays were performed under optimum conditions with 2 mM of p-nitrophenyl derivatives (pNPX, pNPAf, pNPG, pNPGal, pNPAb, pNPC) or 1% (w/v) polysaccharides (beechwood xylan, water-soluble wheat arabinoxylan, sugar beet arabinan and debranched AZCL-arabinan) as the substrate.
The Km, Vmax and Kcat values of Xyl3A were determined at pH 6.0 and 60°C for 5 min in 100 mM Na2HPO4-citric acid containing 1–10 mM pNPX as the substrate. The data were plotted according to the Lineweaver-Burk method.
Xyl3A tolerance to xylose
The tolerance of Xyl3A to xylose was investigated as described by Yan et al. [14]. The β-xylosidase activity of Xyl3A in each reaction system containing xylose of different concentrations (5–50 mM) was determined in the presence of two final concentrations of pNPX (0.75 mM and 1 mM). The residual β-xylosidase activities were measured according to the standard assay method. The data were analyzed by a Dixon plot. The Ki value of xylose was defined as the amount of xylose required to inhibit 50% of the Xyl3A activity.
Transxylosylation of Xyl3A
For transxylosylation assay, pNPX was added to serve as both the donor and acceptor. The reaction system containing 75 mM of pNPX and 3 U/mL of Xyl3A was incubated at pH 6.0 and 37°C for 0–24 h. After the removal of Xyl3A by ultrafiltration, the transxylosylation products were analyzed by thin layer chromatography (TLC) using the mixture of xylose, xylobiose and xylotriose as standards. TLC was performed on aluminum-coated silica gel 60 sheets (Merck, Darmstadt, Germany), developed in a system of n-butyl alcohol/acetic acid/water 2:1:1 (v/v/v), and colored by saturating the plate in 5% sulphuric acid (v/v) and then heating at 110°C for 5 min.
Enzyme synergy
Xyl3A and Xyn11A of the same microbial source were selected to study their synergic actions on the hydrolysis of beechwood xylan and water soluble wheat arabinoxylan [13]. Basic manipulation consulted to the method reported by Raweesri et al. [15]. Each reaction containing 900 μL of 0.5% (w/v) substrate and 100 μL of enzyme(s) in 100 mM Na2HPO4-citric acid (pH 6.0) was incubated at 37°C for 12 h. Reactions containing substrate only were treated as blank controls. Enzymes, 0.25 U of Xyl3A and/or 0.25 U of Xyn11A, were added alone, simultaneously or sequentially. For sequential reactions, the first reactions were terminated by 10-min boiling water bath, and the second reactions were performed as described above. The xylose equivalents (mM) released were determined using the DNS method at room temperature, with xylose as the standard.
The synergy degree was defined as the ratio of xylose equivalents released by enzyme combinations to the sum of the xylose equivalents released by each enzyme alone. One-way ANOVA with a Tukey’s test was used for statistical analysis in OriginPro 8.
Results and Discussion
Gene cloning and sequence analysis
The full-length cDNA of xyl3A from H. insolens Y1 contains 2334 bp and encodes a polypeptide of 777 amino acid residues and a termination codon. Deduced Xyl3A consists of a putative signal peptide at the N-terminus (residues 1–19) and a catalytic domain of GH3 (residues 20–777). The calculated molecular mass and pI value were estimated to be 83.2 kDa and 6.21, respectively. The pI value of Xyl3A is higher than that of most fungal β-xylosidases (4.0–5.0) [16]. Five potential N-glycosylation sites (Asn110, Asn252, Asn314, Asn555, and Asn570) were identified in deduced Xyl3A.
Deduced Xyl3A exhibits highest sequence identities of 64% with a putative (trans)glycosidase from Glarea lozoyensis ATCC 20868 and relative low sequence identities with functionally characterized GH3 β-xylosidases (Fig. 1), i.e. 41% with Xyl from Aspergillus awamori K4 [4] and XylA from Aspergillus japonicas [5], 40% with XlnD from Aspergillus nidulans [6], Bxl1 from Talaromyces emersonii [7] and XylA from Aspergillus oryzae [8]. Blast analysis against the PDB database indicates that Xyl3A shares a fairly low sequence similarity to crystal-resolved counterparts (29% identity the highest), thus no available template was found to predict the tertiary structure of Xyl3A.
The putative catalytic residues are marked with asterisks.
The catalysis mechanism of GH3 β-xylosidases has been proved to proceed via a retaining reaction mechanism under the operation of a nucleophile Glu and an acid/base residue Asp. Based on the amino acid sequence alignments, the two putative catalytic residues of Xyl3A were predicted to be Asp282 and Glu488, corresponding to Asp307 and Glu516 of XylA from A. japonicas [5] and Asp242 and Glu441 of Bxl1 from T. emersonii [7].
Overexpression and purification of recombinant Xyl3A
Bacterial enzymes are generally heterologously expressed in E. coli [17], while that of fungi are usually purified from original cultures [18] or produced in high-level expression systems like P. pastoris [19] or Aspergillus [8]. In this study, we successfully overexpressed mature Xyl3A without the signal peptide in P. pastoris GS115 with methanol induction, which was secreted into culture supernatant by the α-factor signal peptide. Without fermentation optimization, the yield of Xyl3A reached approximately 100 mg/L. The result suggests its great potential for cost-saving mass production.
Crude Xyl3A was concentrated by nearly ten fold in volume and purified by a one-step anion exchange chromatography. The purified enzyme solution exhibited 8.1 U/mL of β-xylosidase activity under its optimum condition with a concentration of 0.698 mg/mL. Purified Xyl3A migrated one single band of about 85.0 kDa on SDS-PAGE (Fig. 2). After deglycosylation with Endo H, Xyl3A showed a slight decrease in molecular mass, which was in agreement with the calculated molecular weight. Similar molecular masses have been reported for some GH3 β-xylosidases [4, 18]. Mozolowski and Connerton [20] purified a GH3 β-xylosidase from H. insolens culture, which was composed of two protein subunits of 64 kDa and 17 kDa with a total molecular mass of approximately 81 kDa. Deduced Xyl3A showed high sequence identities (91–100%) to the digested peptide fragments of both subunits, but no supposed fracture occurred at the C-terminus of recombinant Xyl3A when heterologously expressed in P. pastoris GS115.
Enzymatic properties of purified recombinant Xyl3A
Similar to the two GH43 β-xylosidases of H. insolens Y1 [13] but different from other fungal β-xylosidases that function at pH 3.5–5.0 (Table 1), purified recombinant Xyl3A was active in weak acid pH, showing optimal activity at pH 6.0, 65.5% activity at pH 6.5 and 27.4% at pH 7.0 (Fig. 3A). The pH stability of Xyl3A was assayed at both 37°C and 60°C (Fig. 3B). The enzyme had good pH stability at 37°C, retaining more than 80% activity after incubation at pH 4.0–9.0 for 1 h. After pre-incubation at 60°C for 1 h, the enzyme showed a narrower pH stability range (pH 5.07.0). When assayed Xyl3A activity at pH 6.0 (the optimal pH), it acted the best at a relatively high temperature of 60°C and remained 60% activity even at 70°C (Fig. 3C). This temperature optimum falls within the range of other fungal GH3 β-xylosidases (50–70°C; Table 1), but higher than that of β-xylosidase from Neocallimastix frontalis [21]. Xyl3A was thermostable at 60°C, retaining about 80% activity after 1 h incubation at 60°C without substrate, and lost activity rapidly when treated at 70°C (Fig. 3D). The enzymatic properties of Humicola GHs are much similar [12, 14, 20, 22], which might be due to their grown surroundings of neutral pH and high temperature.
Each value in the panel represents the means ± SD (n = 3).
The effects of metal ions and chemical reagents on the activity of Xyl3A were determined at the concentrations of 5 mM (Table 2). Xyl3A was highly resistant to EDTA, β-mercaptoethanol and almost all of tested cations except for Ag+ and SDS. Mn2+ enhanced the activity slightly.
Substrate specificity and kinetic parameters
Xyl3A exhibited the highest activity towards pNPX (100%), moderate on pNPAf (26.9%) and weak on pNPGal (2.57%). It has been reported that GH3 β-xylosidases have broad substrate specificity. For example, when the activity against pNPX was defined as 100%, β-xylosidase from Asperigus pullulans ATCC20524 showed 35.8% relative activity towards pNPAf [5], and β-xylosidase from A. japonicus strain MU-2 showed 31.6% β-glucosidase activity and 17.7% α-arabinfuranosidase activity [18]. However, the 64 kDa subunit of H. insolens β-xylosidase had no activity towards pNPAf [20], which was probably due to the deletion of the C-terminus. No activity was detected when using other polysaccharides as the substrate. In contrast, several bifunctional GH43 β-xylosidase-arabinosidases have higher α-arabinfuranosidase and xylanase activities [13]. The underlying mechanism might be ascribed to the spatial similarity of d-xylopyranose and l-arabinofuranose [23].
Using pPNX as the substrate, the specific activity of purified recombinant Xyl3A was determined to be 11.6 U/mg, which was higher than that of GH3 β-xylosidases from Neocallimastix frontalis (4.3 U/mg) [21] and Trichoderma koningii G-39 (3.02 U/mg) [23], but lower than that from Aspergillus phoenicis (820.6 U/mg) [24] and Penicillium herquei IFO 4674 (353.6 U/mg) [25]. The same conclusion applied to the comparison among four β-xylosidases produced by H. insolens [13, 20].
The Km and Vmax values of purified recombinant Xyl3A were 2.51 mM and 37.33 μmol/min/mg, respectively, when pNPX was used as the substrate. The Km value of Xyl3A is similar to that of β-xylosidases from Talaromyces thermophiles (2.37 mM) [26] and A. phoenicis (2.36 mM) [24], suggesting its moderate affinity to substrate. In comparison with the kinetic values of other H. insolens isoenzymes (Xyl43A, 12.2 mM and 203.8 μmol/min/mg; Xyl43B, 1.29 mM and 2.18 μmol/min/mg; a 64 kDa β-xylosidase subunit, 1.74 mM and 22.17 μmol/min/mg), Xyl3A had a middle affinity and Vmax towards pNPX.
Xyl3A tolerance to xylose
d-Xylose is the principal end-product of xylooligosaccharide hydrolysis. It performs competitive inhibition on the activity of β-xylosidase. Thus a xylose-tolerant β-xylosidase is essential to maintain the efficiency of hemicellulose conversion by removing xylobioses, the natural substrate of β-xylosidase [27]. Based on the Dixon plot, Xyl3A had a Ki value of 29 mM, which is significantly higher than almost all reported fungal GH3 β-xylosidases (2 to 10 mM) [23, 28, 29]. Higher tolerance to xylose has been reported in fungal GH43 xylosidases, such as 79 mM and 292 mM for Xyl43A and Xyl43B of H. insolens Y1, respectively [13] and 139 mM for PtXyl43 [17]. However, the GH3 TthXynB3 from archaebacterium Thermotoga thermarum has an extremely high Ki value of 1 M [30]. Moreover, the enzymatic activity was even activated by xylose at concentrations less than 500 mM.
Transxylosylation of Xyl3A
Preparation of useful oligosaccharides and glycosyl compounds has attracted broad attention in recent years. Compared with chemical approach, transglycosylation activities of glycosidases have advantages on the high specificity for substrate, the selectivity of the bonding, and the mildness of reaction conditions. Therefore transglycosylation ability has been regarded as an important characteristic of a glycosidase for potential applications [31]. Several studies have addressed the transxylosylation capacities of several GH3 β-xylosidases with oligosaccharides, alcohols and even pNPX as acceptors [32–34]. In the present study, the transxylosylation ability of Xyl3A was determined by TLC (Fig. 4). After co-incubation of Xyl3A and pNPX for 1 h, two products appeared on the TLC plate, which are conjectured to be pNPX2 and xylobiose. Along with time, the amount of xylobiose increased while that of pNPX2 decreased. The possible reason might be that pNPX acts as the primary xylosyl acceptor when the concentration of xylose is relatively low in the early stage of the reaction, but is slowly hydrolyzed when pNPX runs out. As the time went on, pNPX2 was hydrolyzed, and the released xylosyl residues formed xylobiose in turn. No larger oligosaccharide or pNP-oligosaccharide was observed, indicating that transxylosylation by Xyl3A tended to produce short chain oligosaccharides with low polymerization degree. It was quite different from Aspergillus β-xylosidase that was able to catalyze the synthesis of p-nitrophenyl (pNP)-(1,4)-d-xylooligosaccharides with a degree of polymerization of 2–7 [33].
Xylan degradation by enzyme synergy
β-Xylosidase is usually used in combination with xylanase to fully deconstruct hemicelluloses and β-1,4-xylooligosaccharides, which products can be fermented to biofuels like ethanol and butanol [35]. In the current study, a synergy value was used to quantify the cooperation between Xyl3A and Xyn11A (Table 3). Significant differences (p < 0.05) were observed among different enzyme combinations on xylan degradation. Birchwood xylan with less side chains acted as a better substrate for synergism. No matter which substrate was used, the sequential enzyme combination of Xyn11A followed by Xyl3A exhibited the highest synergy degrees (1.25 and 1.08, respectively), and Xyl3A alone released the least xylose equivalents. This result suggests that Xyl3A works better on xylooligosacchardies rather than on xylan polysaccharides.
In comparison with Xyl43A and Xyl43B of H. insolens Y1 [13], Xyl3A displayed almost equal synergistic effects with xylanase at a quarter of enzyme dosage. Considering the absence of standard signal peptides in Xyl43A and Xyl43B, Xyl3A may act as the primary extracellular β-xylosidase for xylan degradation in H. insolens Y1 under natural conditions.
Conclusions
A novel GH3 β-xylosidase gene was identified in H. insolens Y1, and its gene fragment without the signal peptide-coding sequence was overexpressed in P. pastoris GS115 with a high level of 100 mg/L. Recombinant Xyl3A exhibited β-xylosidase, α-arabinosidase and transxylosylation activities. Using pPNX as the substrate, the enzyme had maximum activity at pH 6.0 and 60°C and retained stable over pH 4.0−9.0 (after incubation at 37°C for 1 h) and at temperatures of 60°C and below. Moreover, it was highly tolerant to xylose (up to 29 mM) and had significant synergistic action with xylanase on the degradation of various xylans. These properties make Xyl3A prospective for application in the bioconversion of hemicellulose and biofuel industry.
Acknowledgments
The authors are grateful to lab assistants Zhaohui Zhang and Rui Ma at the Key Laboratory for Feed Biotechnology of the Ministry of Agriculture for their help in protein purification and English polishing, respectively.
Author Contributions
Conceived and designed the experiments: WX PS. Performed the experiments: WX YC MX. Analyzed the data: WX PS XX. Contributed reagents/materials/analysis tools: WX PS LQ. Wrote the paper: WX PS. Guided the experiment design: BY. Provided the research platform: BY.
References
- 1.
Puls J, Schuseil J (1993) Chemistry of hemicelluloses: relationship between hemicellulose structure and enzymes required for hydrolysis. Portland Press London. pp. 1–27. pmid:10133702
- 2. Rahman AK, Sugitani N, Hatsu M, Takamizawa K (2003) A role of xylanase, α-l-arabinofuranosidase, and xylosidase in xylan degradation. Can J Microbiol 49: 58–64. pmid:12674349
- 3. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7: 637–644. pmid:9345621
- 4. Kurakake M, Osada S, Komaki T (1997) Transglycosylation of β-xylosidase from Aspergillus awamori K4. Biosci Biotechnol Biochem 61: 2010–2014.
- 5. Wakiyama M, Yoshihara K, Hayashi S, Ohta K (2008) Purification and properties of an extracellular β-xylosidase from Aspergillus japonicus and sequence analysis of the encoding gene. J Biosci Bioeng 106: 398–404. pmid:19000618
- 6. Kumar S, Ramón D (1996) Purification and regulation of the synthesis of a β-xylosidase from Aspergillus nidulans. FEMS Microbiol Lett 135: 287–293.
- 7. Reen FJ, Murray PG, Tuohy MG (2003) Molecular characterization and expression analysis of the first hemicellulase gene (bxl1) encoding β-xylosidase from the thermophilic fungus Talaromyces emersonii. Biochem Bioph Res Co 305: 579–585.
- 8. Kitamoto N, Yoshino S, Ohmiya K, Tsukagoshi N (1999) Sequence analysis, overexpression, and antisense inhibition of a β-xylosidase gene, xylA, from Aspergillus oryzae KBN616. Appl Environ Microbiol 65: 20–24. pmid:9872754
- 9. Li Z, Qu H, Li C, Zhou X (2013) Direct and efficient xylitol production from xylan by Saccharomyces cerevisiae through transcriptional level and fermentation processing optimizations. Bioresour Technol 149: 413–419. pmid:24128404
- 10. Dodd D, Cann IK (2009) Enzymatic deconstruction of xylan for biofuel production. GCB Bioenergy 18: 2–17.
- 11. Turner P, Mamo G, Karlsson EN (2007) Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb Cell Fact 6: 9. pmid:17359551
- 12. Du Y, Shi P, Huang H, Zhang X, Luo H, et al. (2013) Characterisation of three novel thermophilic xylanases from Humicola insolens Y1 with application potentials in the brewing industry. Bioresour Technol 130: 161–167. pmid:23306124
- 13. Yang X, Shi P, Huang H, Luo H, Wang Y, et al. (2014) Two xylose-tolerant GH43 bifunctional β-xylosidase/α-arabinosidases and one GH11 xylanase from Humicola insolens and their synergy in the degradation of xylan. Food Chem 148: 381–387. pmid:24262572
- 14. Yan QJ, Wang L, Jiang ZQ, Yang SQ, Zhu HF, et al. (2008) A xylose-tolerant β-xylosidase from Paecilomyces thermophila: characterization and its co-action with the endogenous xylanase. Bioresour Technol 99: 5402–5410. pmid:18180153
- 15. Raweesri P, Riangrungrojana P, Pinphanichakarn P (2008) α-L-Arabinofuranosidase from Streptomyces sp. PC22: purification, characterization and its synergistic action with xylanolytic enzymes in the degradation of xylan and agricultural residues. Bioresour Technol 99: 8981–8986. pmid:18606539
- 16. Knob A, Terrasan CRF, Carmona EC (2010) β-Xylosidases from filamentous fungi: an overview. World J Microbiol Biotechnol 26: 389–407.
- 17. Teng C, Jia H, Yan Q, Zhou P, Jiang Z (2011) High-level expression of extracellular secretion of a β-xylosidase gene from Paecilomyces thermophila in Escherichia coli. Bioresour Technol 102: 1822–1830. pmid:20970996
- 18. Ohta K, Fujimoto H, Fujii S, Wakiyama M (2010) Cell-associated β-xylosidase from Aureobasidium pullulans ATCC 20524: Purification, properties, and characterization of the encoding gene. J Biosci Bioeng 110: 152–157. pmid:20547381
- 19. Chen X, Meng K, Shi P, Bai Y, Luo H, et al. (2012) High-level expression of a novel Penicillium endo-1,3(4)-β-d-glucanase with high specific activity in Pichia pastoris. J Ind Microbiol Biotechnol 39: 869–876. pmid:22354732
- 20. Mozolowski GA, Connerton IF (2009) Characterization of a highly efficient heterodimeric xylosidase from Humicola insolens. Enzyme Microb Tech 5: 436–442.
- 21. Hebraud M, Fevre M (1990) Purification and characterization of an extracellular β-xylosidase from the rumen anaerobic fungus Neocallimastix frontalis. FEMS Microbiol Lett 60: 11–16. pmid:2126511
- 22. Almeida EM, Polizeli MDL, Terenzi HF, Atilio JJ (1995) Purification and biochemical characterisation of β-xylosidase from Humicola grisea var. thermoidea. FEMS Microbiol Lett 130: 171–175.
- 23. Li Y, Yao H, Cho Y (2000) Effective induction, purification and characterization of Thrichoderma koningii G-39 β-xylosidase with high transferase activity. Biotechnol Appl Biochem 31: 119–125. pmid:10744957
- 24. Rizzatti A, Jorge J, Terenzi H, Rechia C, Polizeli M (2001) Purification and properties of a thermostable extracellular β-xylosidase produced by a thermotolerant Aspergillus phoenicis. J Ind Microbiol Biotechnol 26: 1–5. pmid:11548743
- 25. Ito T, Yokoyama E, Sato H, Ujita M, Funaguma T, et al. (2003) Xylosidases associated with the cell surface of Penicillium herquei IFO 4674. J Biosci Bioeng 96: 354–359. pmid:16233536
- 26. Guerfali M, Gargouri A, Belghith H (2008) Talaromyces thermophilus β-D-xylosidase: purification, characterization and xylobiose synthesis. Appl Biochem Biotechnol 150: 267–279. pmid:18592411
- 27. Sunna A, Antranikian G (1997) Xylanolytic enzymes from fungi and bacteria. Crit Rev Biotechnol 17: 39–67. pmid:9118232
- 28. Saha BC (2001) Purification and characterization of an extracellular β-xylosidase from a newly isolated Fusarium verticillioides. J Ind Microbiol Biotechnol 27: 241–245. pmid:11687937
- 29. Saha BC (2003) Purification and properties of an extracellular β-xylosidase from a newly isolated Fusarium proliferatum. Bioresour Technol 90: 33–38. pmid:12835054
- 30. Shi H, Li X, Gu H, Zhang Y, Huang Y, et al. (2013) Biochemical properties of a novel thermostable and highly xylose-tolerant β-xylosidase/α-arabinosidase from Thermotoga thermarum. Biotechnol Biofuels 6: 27. pmid:23422003
- 31. Wang L, Huang W (2009) Enzymatic transglycosylation for glycoconjugate synthesis. Curr Opin Chem Biol 13: 592–600. pmid:19766528
- 32. Dilokpimol A, Nakai H, Gotfredsen CH, Appeldoorn M, Baumann MJ, et al. (2011) Enzymatic synthesis of β-xylosyl-oligosaccharides by transxylosylation using two β-xylosidases of glycoside hydrolase family 3 from Aspergillus nidulans FGSC A4. Carbohyd Res 346: 421–429. pmid:21215963
- 33. Eneyskaya VE, Brumer H, Backinowsky LV, Ivanen DR., Kulminskaya AA, et al. (2003) Enzymatic synthesis of β-xylanase substrates: transglycosylation reactions of the β-xylosidase from Aspergillus sp. Carbohyd Res 338: 313–325. pmid:12559729
- 34. Puchart V, Biely P (2007) A simple enzymatic synthesis of 4-nitrophenyl β-1,4-d-xylobioside, a chromogenic substrate for assay and differentiation of endoxylanases. J Biotechnol 128: 576–586. pmid:17223215
- 35. Jordan DB, Wagschal K (2010) Properties and applications of microbial β-d-xylosidases featuring the catalytically efficient enzyme from Selenomonas ruminantium. Appl Microbiol Biotechnol 86: 1647–1658. pmid:20352422