INTRODUCTION

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The brown color characteristic of the traditional Japanese fermented food soy sauce, or shoyu, is one of the important parameters of its quality; the lighter brown is preferable. The brown color is formed by the aminocarbonyl reaction between the reducing sugars and amino acids in soy sauce mash. Of the reducing sugars, pentose, especially xylose, has been considered to be mainly responsible for the brown color formation (4). Xylose in soy sauce mash is formed as a result of xylan hydrolysis in soybean and wheat with the xylanolytic enzymes produced by the shoyu koji mold, Aspergillus oryzae. The major xylanolytic enzyme complex includes endo-(1,4)--xylanases (EC 3.2.1.8), the so called xylanases, which hydrolyze the polysaccharide backbone, and -xylosidases (EC 3.2.1.37), which hydrolyze xylo-oligosaccharides to xylose. The use of A. oryzae mutants producing low levels of the xylanolytic enzymes, especially -xylosidase, has been reported to prevent browning of soy sauce (14). Therefore, among the xylanolytic enzymes secreted by A. oryzae, -xylosidase is the key enzyme responsible for browning of soy sauce.

Numerous studies on fungal -xylosidases have been done, but only three fungal -xylosidase genes have been isolated, from Aspergillus nidulans (16), Aspergillus niger (18), and Trichoderma reesei (13). When grown on a shoyu koji composed of soybean and wheat, A. oryzae secretes three -xylosidases (14). However, little is known about the enzymatic properties of -xylosidases and the molecular mechanisms controlling their gene expression in A. oryzae. Therefore, we purified a major -xylosidase (XylA) from an industrial shoyu koji mold strain, A. oryzae KBN616, and characterized its enzymatic properties. The -xylosidase gene (xylA) was cloned and sequenced. In order to assess the role of -xylosidase in the formation of the dark brown color during soy sauce brewing, the sense or antisense xylA gene was expressed in A. oryzae under control of the promoter of the A. oryzae TEF1 gene (10). Expression of the antisense gene reduced the -xylosidase level significantly.

	MATERIALS AND METHODS

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General procedures. Recombinant DNA techniques, double-strand DNA sequencing, A. oryzae cultivation, and genomic DNA isolation were carried out by standard procedures (8, 17). Total RNA was extracted from mycelia grown for 3 days in xylan-peptone (XP) medium (2% birchwood xylan, 1% polypeptone, 0.5% KH₂PO₄, 0.5% KCl, 0.1% NaNO₃, 0.05% MgSO₄ · 7H₂O) by the method of Cathala et al. (3). Reverse transcription-PCR was performed with total RNA by using the Access RT-PCR system (Promega, Madison, Wis.).

Purification of the A. oryzae -xylosidase. A. oryzae KBN616 was grown in XP medium for 10 days. After removal of mycelia through filtration, 250 ml of the culture filtrate was diluted 10 times with 10 mM potassium phosphate buffer, pH 6.0. Protein was adsorbed to a STREAMLINE DEAE (Pharmacia Biotech, Uppsala, Sweden) column (2.6 by 12.0 cm) equilibrated with the same buffer and eluted by pulse elution with the same buffer containing 1.0 M NaCl. The -xylosidase-containing fractions were diluted 10 times with the same buffer and loaded on an HR16/20 Fast Flow Q-Sepharose anion-exchange column (Pharmacia Biotech) followed by elution with a linear gradient of 0 to 0.4 M NaCl. Fractions containing -xylosidase activity were pooled, dialyzed against 10 mM potassium phosphate buffer, pH 6.0, and rechromatographed on an HR16/20 Fast Flow Q-Sepharose anion-exchange column under the same conditions. The active pool was then dialyzed against 10 mM potassium phosphate buffer, pH 6.8, and loaded on a Gigapite (Seikagakukogyo, Tokyo, Japan) hydroxyapatite column (1.0 by 7.0 cm) equilibrated in the same buffer. Elution was performed with a linear gradient of 10 to 300 mM potassium phosphate buffer, pH 6.8. Finally, -xylosidase was purified by using a HiLoad 26/60 Superdex 200-pg gel column (Pharmacia Biotech).

Enzyme assay. -Xylosidase activity was assayed as described by Ooi et al. (15). -Galactosidase, -L-arabinofuranosidase, -L-arabinopyranosidase, -L-arabinopyranosidase, -galactosidase, -glucosidase, and -mannosidase activities were determined as described by Margolles-Clark et al. (13). Xylanase activity was assayed as described by Bailey (1).

N-terminal and internal amino acid sequencing. The purified enzyme was blotted onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Richmond, Calif.), and its N-terminal amino acid sequence was determined with an Applied Biosystems 477A-120A sequencer according to the manufacturer's instructions (Applied Biosystems, Foster City, Calif.). The purified enzyme was digested with V8 protease, and the resulting peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Three major peptides, designated P1, P2, and P3, were subjected to amino acid sequencing as described above.

Amplification of -xylosidase genomic DNA sequences by PCR. A genomic DNA fragment encoding a portion of the -xylosidase gene was amplified by PCR with chromosomal DNA of A. oryzae KBN616 as a template. Two oligonucleotide primers were synthesized based on the amino acid sequences: a sense primer [5'-GC(T/C)GA(T/C)CT(T/C)AT(T/C)AT(T/C)TT(T/C)GC(T/C)GG-3'] corresponding to peptide P2 (ADLIIFAG) and an antisense primer [5'-GT (A/G)TA(A/G)AA(A/G)AG(A/G)CC(A/G)TG(A/G)CC(A/G)AA-3'] corre-sponding to peptide P3 (FGHGLFYT). The amplified 419-bp fragment designated BXF1 was cloned on pUC118 and sequenced.

Expression of the A. oryzae xylA gene under control of the A. oryzae TEF1 promoter. To express the xylA gene under control of the A. oryzae TEF1- gene (TEF1) promoter (10), the expression plasmid vector pTFBX200 was constructed as follows. An EcoT22I cleavage site was introduced just before the ATG codon of the xylA gene by PCR in order to ligate the TEF1 promoter fragment precisely next to the xylA coding region. The 2.9-kb EcoT22I xylA DNA fragment was cloned into the EcoT22I site on pTF100 (10) to create plasmid pTFBX100, which contained the xylA gene under control of the TEF1 promoter. The 3.7-kb PstI-XbaI fusion gene fragment excised from pTFBX100 was cloned into the PstI and XbaI sites on pND300 containing the A. oryzae niaD gene on pUC119 (9) to create plasmid pTFBX200. Then, pTFBX200 was introduced to the niaD-deficient A. oryzae strain KBN616-39 by the method described previously (9).

Expression of the partial antisense A. oryzae xylA gene under control of the A. oryzae TEF1 promoter. To express the partial antisense xylA gene by use of the A. oryzae TEF1 promoter, a plasmid vector, pASBX200, was constructed as follows. A 192-bp KpnI fragment from xylA at nucleotides 51 to 141 was cloned in the antisense orientation into the KpnI site on pTF100 to create plasmid pASBX100, which expressed the antisense RNA for the xylA gene under control of the A. oryzae TEF1 promoter. The 1.0-kb PstI-EcoRI fusion fragment excised from pASBX100 was treated with Klenow fragment and cloned into the SmaI site on pND300 to create plasmid pASBX200. Then, pASBX200 was introduced to the niaD-deficient A. oryzae strain KBN616-39 as described above.

Nucleotide sequence accession number. The sequence of the xylA gene has been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB013851.

	RESULTS AND DISCUSSION

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Purification and characterization of the A. oryzae -xylosidase. A. oryzae produced three -xylosidases with different molecular masses, and a major enzyme was purified 65.6-fold with recovery of 11% of the initial activity from culture supernatants of A. oryzae KBN616, as described in Materials and Methods. The purified enzyme showed a single protein band with an apparent molecular mass of 110 kDa on analysis by SDS-PAGE (Fig. 1). The molecular mass of 110 kDa is on the same order of magnitude as reported before for -xylosidases of other Aspergillus spp. (90 to 122 kDa [11, 12, 15, 18]).

View larger version (38K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE of purified -xylosidase XylA. XylA was purified as described in Materials and Methods, subjected to SDS-PAGE on a 10% gel, and stained with Coomassie brilliant blue. Lane 1, molecular size markers (rabbit muscle phoshorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; hen egg white ovalbumin, 42.7 kDa; bovine carbonic anhydrase, 31.0 kDa); lane 2, purified XylA.

Amino acid sequencing of -xylosidase and amplification of a partial -xylosidase gene. N-terminal amino acid sequencing of the purified enzyme failed, indicating that the N terminus might be blocked. V8 protease digestion of the purified enzyme, followed by SDS-PAGE, yielded three major peptides: P1, P2, and P3. Their N-terminal amino acid sequences were SFHDQFVSRQDL, ADLIIFAGGIDNTLETEAQD, and FGHGLFYT, respectively. Upon comparison of these sequences to those of other -xylosidases, the amino acid sequences of the P1, P2, and P3 peptides showed high homologies to those of A. niger XlnD and T. reesei BxlI. Therefore, two oligonucleotide mixtures were designed based on the N-terminal amino acid sequence from residues 1 to 8 of the P2 peptide and the P3 peptide amino acid sequence. The amplified 419-bp fragment, designated BXF1, was sequenced and found to contain an open reading frame encoding 139 amino acids bearing high homology to the sequences of A. niger XlnD and T. reesei BxlI.

Characterization of the A. oryzae xylA gene. For amplification of the 5' and 3' regions of BXF1, four specific primers, termed I-1, I-2, II-1, and II-2, were synthesized on the basis of the nucleotide sequence of BXF1. By CLM-PCR with a Sau3AI cassette-ligated genomic DNA as the template, a 2.5-kb fragment for the 5' region, designated BXF2, and a 1.5-kb fragment for the 3' region, designated BXF3, were amplified as described in Materials and Methods. BXF2 has the nucleotide sequence of the 5' region of BXF1 and the ATG start codon of the xylA gene. BXF3 has the nucleotide sequence of the 3' region of BXF1 and the TAG stop codon of the xylA gene. Based on the sequences of BXF1, BXF2, and BXF3, the entire nucleotide sequence of the xylA gene was determined (Fig. 2).

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Restriction map of the xylA gene. The sequencing strategy for the xylA gene is represented below the restriction map by arrows. Solid and hatched boxes indicate the coding regions of the xylA gene and the BXF1 fragment amplified by PCR, respectively.

View larger version (82K): [in this window] [in a new window]   FIG. 3.   Nucleotide and deduced amino acid sequences of the xylA gene. Numbers on the right refer to the nucleotide sequence (negative numbers refer to nucleotides upstream from the xylA ATG) and the amino acid sequence. The TATA and CCAAT sequences are double underlined, and CreA consensus binding sites are underlined. The GGCTAAA sequence is boxed. An asterisk (*) marks the translation stop codon. A putative termination sequence [TAG...TA(T)GT...TTT] is underlined. The amino acids determined by sequencing of the P1, P2, and P3 peptides are also underlined, in the derived amino acid sequence. #, potential N-glycosylation site.

Comparison of the amino acid sequence of A. oryzae XylA with those of other -xylosidases. Many of the -xylosidases known so far have been classified by hydrophobic cluster analysis into three distinct groups: families 39, 43, and 52 (5). Although XylA shows no amino acid similarity to these families of -xylosidases, it exhibits significant similarity to A. nidulans XlnD (70%), A. niger XlnD (64%), and T. reesei BxlI (63%), which have high sequence similarity to family 3 -glucosidases. Several highly conserved sequences found among XylA, A. nidulans XlnD, A. niger XlnD, and T. reesei BxlI might be involved in the catalytic reaction, binding of the substrate, or both. One putative active-site Asp residue which might take part in the catalytic activity, as determined for the Aspergillus wentii -glucosidase A3 (2), was also conserved in XylA (Asp-310).

Overexpression of the A. oryzae -xylosidase gene. In order to obtain high-level expression of the xylA gene in A. oryzae, A. oryzae KBN616-39 (niaD) was transformed with pTFBX200, which contained the xylA gene under control of the A. oryzae TEF1 promoter. Of 100 transformants screened on plates containing 2% glucose and 0.5 mM 4-MUX (4-methylumbelliferyl--D-xyloside), 95 transformants showed -xylosidase activity after 24 h of growth, whereas the reference strain showed no -xylosidase activity in the presence of glucose because of carbon catabolite repression. Two highly XylA-producing strains selected by the plate assay produced extracellularly about 18 U of -xylosidase per ml (about 250 mg/liter) when grown in glucose-peptone medium for 10 days. A 110-kDa XylA was detected as a prominent band on an SDS-polyacrylamide gel stained with Coomassie brilliant blue (Fig. 4, lanes 4 and 5). In XP medium, these transformants produced 23 U of the enzyme per ml, about 13 times more than the reference strain. Southern blot analysis revealed that multiple copies of plasmid pTFBX200 were integrated into chromosomal DNAs of these transformants (Fig. 5).

View larger version (80K): [in this window] [in a new window]   FIG. 4.   SDS-PAGE of XylA overexpressed by A. oryzae transformants. One milliliter of culture filtrate was precipitated with 10% trichloroacetic acid, solubilized in a small volume, and subjected to SDS-PAGE on a 10% gel. Molecular size markers (lane 1) are as described in the legend to Fig. 1. Lane 2, purified XylA; lane 3, culture supernatant of A. oryzae KBN616-39; lanes 4 and 5, culture supernatant of A. oryzae carrying pTFBX200.

View larger version (46K): [in this window] [in a new window]   FIG. 5.   Southern blot analysis of chromosomal DNAs isolated from A. oryzae KBN616 and two representative transformants. DNA (about 5 µg) was digested with PstI and processed for Southern blot hybridization. Hybridization was done with the AlkPhos Direct system (Amersham, Little Chalfont, United Kingdom) with a 300-bp fragment (+1 to +300, with the translation start site at +1) as a probe. Lane 1, A. oryzae KBN616; lanes 2 and 3, transformants.

Antisense inhibition of A. oryzae -xylosidase gene expression. In order to inhibit the expression of the xylA gene in A. oryzae, the partial-length antisense xylA gene was expressed under control of the A. oryzae TEF1 promoter by introducing pASBX200 into A. oryzae KBN616-39. Of 84 transformants screened on plates containing 2% xylan and 0.5 mM 4-MUX, two transformants, designated ASBX13 and ASBX33, showed barely detectable levels of -xylosidase activity after 24 h of growth. Quantitative assays revealed low -xylosidase activities in ASBX13 and ASBX33 (99 and 115 mU/ml), indicating that 75 to 80% inhibition was achieved (Table 2). Southern blot analysis of ASBX13 and ASBX33 revealed that the antisense construct was integrated into their genomes (Fig. 6). Since the A. oryzae TEF1 gene promoter is a strong constitutive promoter, as described previously (10), a high level of the partial-length antisense RNA could be accumulated in the transformed cells, leading to a reduction in sense xylA mRNA levels, probably as a consequence of degradation of sense-antisense complexes (7, 22). However, xylanase activity in these transformants remained unaltered (Table 2). This indicates that the antisense gene specifically inhibited xylA gene expression.

View this table: [in this window] [in a new window]   TABLE 2.   Antisense inhibition of -xylosidase expression in A. oryzae carrying the antisense construct pASBX200a

View larger version (46K): [in this window] [in a new window]   FIG. 6.   Southern blot analysis of chromosomal DNAs isolated from A. oryzae KBN616 and two transformants, ASBX13 and ASBX33. DNA (about 5 µg) was digested with EcoRI and PstI and processed for Southern blot hybridization as described in the legend to Fig. 5. Lane 1, A. oryzae KBN616; lane 2, transformant ASBX13; lane 3, transformant ASBX33.