Cloning of a Novel Gene Encoding β-1,3-Xylosidase from a Marine Bacterium, Vibrio sp. Strain XY-214, and Characterization of the Gene Product

Abstract

The β-1,3-xylosidase gene (xloA) of Vibrio sp. strain XY-214 was cloned and expressed in Escherichia coli. The xloA gene consisted of a 1,608-bp nucleotide sequence encoding a protein of 535 amino acids with a predicted molecular weight of 60,835. The recombinant β-1,3-xylosidase hydrolyzed β-1,3-xylooligosaccharides to d-xylose as a final product.

β-1,3-Xylan is found in the cell walls of red (Porphyra and Bangia spp.) and green (Caulerpa, Bryopsis, and Udotea spp.) algae (12, 16), whereas β-1,4-xylan is contained in the cell walls of land plants (25). The β-1,4-xylanases (1,4-β-d-xylan xylanohydrolase; EC 3.2.1.8) and β-1,4-xylosidases (β-d-xyloside xylohydrolase; EC 3.2.1.37) required for complete breakdown of β-1,4-xylan are useful tools for production of d-xylose, which is expected to be a source of xylitol and bioethanol (5, 26). These two enzymes are produced by many microbes, including aerobic and anaerobic mesophiles and thermophiles, and their biochemical and molecular characteristics have been widely studied (7, 19, 27). However, there have been only a few reports (1, 2, 4, 9, 29) on β-1,3-xylanases (1,3-β-d-xylan xylanohydrolase; EC 3.2.1.32) and no reports on a β-1,3-xylosidase that is able to hydrolyze β-1,3-linked xylooligosaccharides to d-xylose. Vibrio sp. strain XY-214 is a marine bacterium that secretes an extracellular β-1,3-xylanase (TxyA) into the growth medium in the presence of β-1,3-xylan as an inducer (3, 4). Recently, we found that the organism produces an intracellular β-1,3-xylosidase A (XloA).

In this paper, we describe the cloning and sequence analysis of the novel gene encoding XloA and the purification and characterization of recombinant XloA (rXloA) produced by transformed Escherichia coli.

Construction of a probe for cloning of the xloA gene.

The organism was grown in peptone medium (0.5% peptone, 0.1% yeast extract, 3% NaCl, 0.05% MgSO₄, 0.2% K₂HPO₄, 0.04% KH₂PO₄, pH 7.6) containing 0.5% d-xylose for 15 h at 25°C. After centrifugation, the precipitated cells were resuspended in a small volume of 50 mM 2-morpholineethanesulfonic acid (MES) buffer (pH 7.0) and disrupted by sonication. The supernatant resulting from centrifugation of the cell lysate was purified by chromatography with DEAE-Toyopearl 650M (Tosoh, Tokyo, Japan), Ether-Toyopearl 650S (Tosoh, Tokyo, Japan), and MonoQ 5/50 (GE Healthcare) columns. The final enzyme preparation was ascertained to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (13) and then transferred by blotting to a polyvinylidene difluoride membrane (Bio-Rad). The protein band was detected by staining with Coomassie brilliant blue G-250, and a protein of approximately 60 kDa was excised from the membrane. The N-terminal amino acid sequence was determined by automated Edman sequencing with a Procise 49X-clC protein sequencer and a 140D Micro Gradient delivery system (Applied Biosystems). A partial internal amino acid sequence of XloA was also determined by performing the same procedure on peptides obtained by digestion of the purified enzyme with a lysyl endopeptidase (Wako Chemicals). Degenerate primers were designed on the basis of the underlined parts of the N-terminal amino acid sequences of purified XloA (sequence TTTIQNPILKGFNPDPSIVR) and a 22-kDa digested peptide (sequence WLSLSERPGFLRLKGRHYLY). To construct a probe for the cloning of the gene for XloA (xloA), one specifically amplified oligonucleotide was obtained from a template of genomic DNA of Vibrio sp. strain XY-214 by PCR with the degenerate primers. The PCR product ligated into a pT7Blue vector (Novagen) was composed of a 1,128-bp nucleotide sequence encoding 376 amino acid residues. The sequences of the first 20 N-terminal and the last 20 C-terminal amino acids of the fragment were confirmed to be identical to each 20 N-terminal amino acid sequence of native XloA and a 22-kDa peptide. Therefore, the DNA insert in the pT7Blue vector was considered to be derived from the gene encoding XloA. On the basis of the insert nucleotide sequence, a second set of primers was designed. The PCR product (300 bp) that was amplified from the insert nucleotide sequence with the second set of primers was used as the probe for the cloning of xloA after it was labeled with AlkPhos (GE Healthcare).

Cloning of the xloA gene.

To obtain the complete xloA gene, Southern hybridization with the probe was carried out. Digestion of Vibrio sp. strain XY-214 genomic DNA with both XbaI and SpeI gave a 4.2-kbp fragment that hybridized with the AlkPhos-labeled probe. When XbaI-SpeI fragments were ligated into pBluescriptII KS(−) (Stratagene) and transfected into E. coli DH5α (Stratagene), 1 of the 40 colonies was selected as positive by direct PCR with the second set of primers. The cloned plasmid was named xloA/pBluescript(XbaI-SpeI). Nucleotide sequence analysis of the positive DNA fragment inserted into the vector was carried out on a Beckman CEQ2000XL sequencer (Beckman Coulter) with a GenomeLab DTCS-Quick Start kit. The nucleotide sequence data were analyzed with GENETYX-WIN computer software (Software Development, Tokyo, Japan). Homology searches were carried out with the BLAST program at the National Center for Biotechnology Information website.

The plasmid xloA/pBluescript(XbaI-SpeI) contained a 4,206-bp DNA fragment including the xloA gene and three possible open reading frames. The xloA gene consisted of a 1,608-bp nucleotide sequence encoding a protein of 535 amino acids with a predicted molecular mass of 60,835 Da. The sequences TTCATA and TACCCT with a 17-bp spacing, having a certain homology to the −35 and −10 promoter consensus sequences of E. coli, were identified upstream of the coding region (21). A possible transcription terminator that consisted of a 36-bp palindrome sequence corresponding to an mRNA hairpin loop, followed by a T-rich portion, was found downstream of the TAA termination codon (23).

Similarity of the deduced amino acid sequence encoded by the xloA gene.

The sequence of the XloA protein was scanned with BLAST, and the protein showed similarity to the β-xylosidases that belong to family 43 of glycoside hydrolases (GH43). XloA exhibited the highest protein similarities to the GH43 β-xylosidases (identity percentages are in parentheses) from Bacillus sp. strain KK-1 (47%) (GenBank accession no. AF045479), Bacillus halodurans C-125 (47%) (AP001519), B. subtilis (46%) (U66480), Selenomonas ruminantium GA192 (45%) (AF040720), Geobacillus stearothermophilus T-6 (45%) (AY690618), Clostridium acetobutylicum (44%) (AE007842), Bacillus pumilus IPO (44%) (X05793), and the GH43 α-l-arabinofuranosidase from Chromohalobacter salexigens DSM 3043 (46%) (CP000285). This suggested that XloA should be classified into the GH43 family, which is a component of the superfamily clan GH-F together with the GH62 family, by forming a fivefold β-propeller architecture (20). Recently, the catalytic residues of GH43 β-xylosidase (XynB3) from G. stearothermophilus T-6 were revealed to be Asp15, Glu187, and Asp128 (8). From the sequence similarities between XloA and XynB3, we surmised that the active-site amino acids in XloA are Asp16, Glu189, and Asp130. Although GH43 glycosidases from different organisms were found to be either intra- or extracellular (15, 17, 24), the xloA gene was found not to include any recognizable signal peptides of gram-negative bacteria, as determined by the SignalP server (18), which therefore suggested that XloA is an intracellular enzyme.

Expression and purification of rXloA.

For the production of rXloA in E. coli, the xloA region was subcloned into the pET22b(+) vector (Novagen) as follows. The full-length xloA gene was amplified from xloA/pBluescript(XbaI-SpeI) as the template by PCR with primers 5′-GGAATTCCATATGACAACTACGATTCAG-3′ and 5′-AAGCGGCCGCATGCTCTAGGTACTCAAAGT-3′, which contained artificial NdeI and NotI sites (italicized), respectively. The PCR product was digested with NdeI and NotI and then ligated into a pET22b(+) vector linearized with the same enzymes to construct xloA/pET22b. This plasmid provides rXloA with a six-His tag fused at its C terminus. xloA/pET22b was transformed into E. coli BL21(DE3) competent cells (Novagen) and used for the production of rXloA. The absence of undesired mutations in the amplified DNA fragment was verified by DNA sequencing.

E. coli BL21(DE3) transformants carrying xloA/pET22b were cultivated at 37°C in 800 ml of Luria-Bertani medium in the presence of ampicillin (100 μg ml⁻¹). When the optical density at 600 nm of the culture reached about 0.6, the temperature was changed to 25°C and isopropyl-β-d-galactopyranoside (IPTG) was added to the culture to give a final concentration of 1 mM for induction of gene expression. After an additional incubation of 20 h at 25°C, the cells harvested by centrifugation were suspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4) and disrupted on ice by sonication. The supernatant of the cell lysate was collected by centrifugation and fractionated by a HiTrap chelating HP column (GE Healthcare) precharged with Ni²⁺. The final preparation of purified rXloA was used in all of the experiments in this study. β-Xylosidase activity was measured by using p-nitrophenyl-β-d-xylopyranoside (PNPX) (Sigma) as a substrate. One unit of β-xylosidase activity was defined as the amount of enzyme releasing 1 μmol p-nitrophenol per min from the substrate. The protein concentration was measured by the method of Lowry et al. (14) with bovine serum albumin as the standard.

Characterization of rXloA.

The final recombinant enzyme was purified 37-fold by using a HiTrap chelating HP column and ascertained to be a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its relative molecular mass was estimated to be approximately 60 kDa, which is almost the same size as a monomer of native XloA from Vibrio sp. strain XY-214. However, the relative molecular mass of the native enzyme was estimated to be 251 kDa by gel filtration chromatography on a Superdex 200 HR 10/30 column (GE Healthcare) (data not shown). According to the nucleotide sequence, the relative molecular mass of the XloA monomer should be 60,835 Da. These results suggest that XloA is a tetramer. Similarly, the GH43 β-xylosidase from G. stearothermophilus T-6 (PDB code 2EXH) (8) has been reported to be a tetramer in the crystal state, whereas the GH43 β-xylosidases from B. halodurans C-125 (PDB code 1YRZ) (22) and B. pumilus IPO (28) were revealed to be a dimer and a trimer, respectively, by gel filtration chromatography.

Properties of rXloA.

The optimal pH was observed to be around 7.0 when the enzyme activity was assayed with 2 mM PNPX at 37°C for 20 min at various pHs (3.0 to 12.0) in Britton and Robinson's universal buffer (40 mM phosphoric acid, 40 mM boric acid, 40 mM acetic acid). The enzyme was stable at pHs 6.0 to 9.0, retaining more than 80% of the original activity after preincubation in the buffer at various pHs at 4°C for 12 h. The enzyme was optimally active at around 35°C and stable at up to 30°C for 20 min. The enzymatic activity was inhibited more than 90% by Ag⁺, Cu²⁺, Hg²⁺, Mn²⁺, Pb²⁺, Zn²⁺, and p-chloromercuric benzoic acid. The K_m and V_max values of rXloA for PNPX were determined to be 0.244 mM and 1.82 μmol min⁻¹ mg⁻¹, respectively. To examine the substrate specificity of rXloA, enzymatic reactions were performed after incubation with various substrates at 30°C for 12 h, and hydrolysis products were analyzed by thin-layer chromatography (TLC) on a silica gel 60-plastic sheet (Merck) with a solvent of n-butanol-acetic acid-water (10:5:1 for β-1,3-xylooligosacharides, 2:1:1 for β-1,4-xylooligosacharides). β-1,3-Xylooligosacharides (xylobiose to xylotetraose) were prepared by fractionation of the products released from β-1,3-xylan by the β-1,3-xylanase of Vibrio sp. strain XY-214 by using a charcoal column and TLC. β-1,3-Xylan was prepared from a green alga, Caulerpa racemosa var. laetevirens according to the method of Iriki et al. (12). β-1,4-Xylooligosacharides were purchased from Biocon (Japan). Oligosaccharides were visualized by spraying the plate with diphenylamine-aniline-phosphate reagent (6). rXloA completely hydrolyzed β-1,3-xylobiose (TX2), β-1,3-xylotriose (TX3), and β-1,3-xylotetraose (TX4) to xylose as a final hydrolysis product (Fig. 1A). The enzyme also cleaved β-1,4-xylobiose (FX2), β-1,4-xylotriose (FX3), and β-1,4-xylotetraose (FX4) weakly to form a small amount of xylose (Fig. 1B). Thus, rXloA preferred to hydrolyze β-1,3-xylooligosaccharides rather than β-1,4-xylooligosaccharides. Although there have been many reports of β-xylosidases, all of them act on β-1,4-xylooligosaccharides. Therefore, XloA of Vibrio sp. strain XY-214 is the first β-1,3-xylosidase described.

FIG. 1.

Thin-layer chromatograms of hydrolysis products of β-1,3-xylooligosaccharides and β-1,4-xylooligosaccharides with rXloA. The reaction mixture contained 20 μl of rXloA (0.928 U/ml) and 20 μl each of β-1,3-xylooligosaccharide or β-1,4-xylooligosaccharide in 50 mM MES buffer (pH 7.0). The reaction was carried out at 30°C for 12 h. (A) Lanes M, standard oligosaccharides; TX2, TX3, and TX4, β-1,3-xylobiose, -triose, and -tetraose, respectively. (B) FX2, FX3, and FX4, β-1,4-xylobiose, -triose, and -tetraose, respectively. X, d-xylose.

β-Xylosidases (EC 3.2.1.37) are classified into several families (GH3, -39, -43, -52, and -54) by a glycoside hydrolase classification system based on the amino acid sequences of their catalytic domains (http://afmb.cnrs-mrs.fr/CAZY) (10, 11). The GH3, -39, -52, and -54 β-xylosidases are reported to be retaining enzymes and able to perform both hydrolysis and transglycosylation reactions, whereas GH43 β-xylosidases are inverting enzymes and able to perform only hydrolysis (22). In concordance with this, rXloA was confirmed not to perform transglycosylation based on the activity pattern of β-1,3-xylooligosaccharides by TLC (Fig. 1A). This result also supported the hypothesis that XloA belongs to the GH43 family.

Furthermore, the enzyme did not act on laminarioligosaccharides comprising β-1,3-linked d-glucose units (Biocon), polysaccharides such as β-1,3-xylan and β-1,4-xylan (Sigma), carboxymethyl cellulose (Wako Chemicals), or laminaran (Nacalai Tesque). Examination of the activities of rXloA on various synthetic substrates composed of p-nitrophenyl (PNP) attached to different sugar units (Sigma) showed only β-d-xylopyranosidase activity, and no activities were detected against other PNP substrates like PNP-α-d-xylopyranoside, PNP-α-d-mannopyranoside, PNP-β-d-mannopyranoside, PNP-α-d-galactopyranoside, PNP-α-d-glucopyranoside, PNP-β-d-glucopyranoside, PNP-α-l-fucopyranoside, PNP-α-l-arabinofuranoside, PNP-α-l-arabinopyranoside PNP-β-d-glucuronide, and PNP-β-d-cellobioside.

Recently, a mutant species of Caulerpa taxifolia has continued to multiply abnormally in the Mediterranean; the mutant algae have destroyed the ecosystem of the sea environment, and fisheries have suffered great damage. Because β-1,3-xylosidase can produce d-xylose efficiently from β-1,3-xylan contained in the cell walls of the algae by combining with β-1,3-xylanase, it will play an important role in mitigating the damage caused by the harmful mutant alga.

Nucleotide sequence accession number.

The nucleotide sequence of the xloA gene described here has been submitted to the DDBJ, EMBL, and GenBank databases and assigned accession no. AB300564.