CLONING OF GENES ENCODING ALPHA-L-ARABINOFURANOSIDASE AND BETA-XYLOSIDASE FROM TRICHODERMA-REESEI BY EXPRESSION IN SACCHAROMYCES-CEREVISIAE

A cDNA expression library of Trichoderma reesei RutC-30 was constructe d in the yeast Saccharomyces cerevisiae, Two genes, abf1 and bxl1, wer e isolated by screening the yeast library for extracellular alpha-L-ar abinofuranosidase activity with the substrate p-nitrophenyl-alpha-L-ar abinofuranoside. The genes abf1 and bxl1 encode 500 and 758 amino acid s, respectively, including the signal sequences, The deduced amino aci d sequence of ABFI displays high-level similarity to the alpha-L-arabi nofuranosidase B of Aspergillus niger, and the two can form a new fami ly of glycosyl hydrolases, The deduced amino acid sequence of BXLI sho ws similarities to the beta-glucosidases grouped in family 3. The yeas t-produced enzymes were tested for enzymatic activities against differ ent substrates, ABFI released L-arabinose from p-nitrophenyl-alpha-L-a rabinofuranoside and arabinoxylans and showed some beta-xylosidase act ivity toward p-nitrophenyl-beta-D-xylopyranoside. BXLI did not release L-arabinose from arabinoxylan, It showed alpha-L-arabinofuranosidase, alpha-L-arabinopyranosidase, and beta-xylosidase activities against p -nitrophenyl-alpha-L-arabinofuranosidase, p-nitrophenyl-alpha-L-arabin opyranoside, and p-nitrophenyl-beta-D-xylopyranoside, respectively, wi th the last activity being the highest, It was also able to hydrolyze xylobiose and slowly release xylose from polymeric xylan, ABFI and BXL I correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T. reesei, respectively, as confirmed by partia l amino acid sequencing of the Trichoderma-produced enzymes. Both enzy mes produced in yeasts displayed hydrolytic properties similar to thos e of the corresponding enzymes purified from T. reesei.