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, X ylA, and its encoding gene were characterized. beta-Xylosidase XylA was pur ified to homogeneity from culture filtrates of A. oryzae KBN616, The optimu m pH and temperature of the enzyme were found to be 4.0 and 60 degrees C, r espectively, and the molecular mass was estimated to be 110 kDa based on so dium dodecyl sulfate-polyacrylamide gel electrophoresis. The xylA gene comp rises 2,397 bp with no introns and encodes a protein consisting of 798 amin o acids (86,475 Da) with 14 potential N-glycosylation sites. The deduced am ino 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. oryz ae TEF1 promoter. One of the ii. oryzae transformants produced approximatel y 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 th e host strain.