Sequence analysis, overexpression, and antisense inhibition of a beta-xylosidase gene, xylA, from Aspergillus oryzae KBN616.

beta-Xylosidase secreted by the shoyu koji mold, Aspergillus oryzae, is the key enzyme responsible for browning of soy sauce. To investigate the role of beta-xylosidase in the brown color formation, a major beta-xylosidase, XylA, and its encoding gene were characterized. beta-Xylosidase XylA was purified to homogeneity from culture filtrates of A. oryzae KBN616. The optimum pH and temperature of the enzyme were found to be 4.0 and 60 degrees C, respectively, and the molecular mass was estimated to be 110 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The xylA gene comprises 2,397 bp with no introns and encodes a protein consisting of 798 amino acids (86,475 Da) with 14 potential N-glycosylation sites. The deduced amino acid sequence shows high similarity to Aspergillus nidulans XlnD (70%), Aspergillus niger XlnD (64%), and Trichoderma reesei BxII (63%). The xylA gene was overexpressed under control of the strong and constitutive A. oryzae TEF1 promoter. One of the A. oryzae transformants produced approximately 13 times more of the enzyme than did the host strain. The partial-length antisense xylA gene expressed under control of the A. oryzae TEF1 promoter decreased the beta-xylosidase level in A. oryzae to about 20% of that of the host strain.