A cDNA expression library of Trichoderma reesei RutC-30 was constructed in the yeast Saccharomyces cerevisiae. Two genes, abf1 and bxl1, were isolated by screening the yeast library for extracellular alpha-L-arabinofuranosidase activity with the substrate p-nitrophenyl-alpha-L-arabinofuranoside. The genes abf1 and bxl1 encode 500 and 758 amino acids, respectively, including the signal sequences. The deduced amino acid sequence of ABFI displays high-level similarity to the alpha-L-arabinofuranosidase B of Aspergillus niger, and the two can form a new family of glycosyl hydrolases. The deduced amino acid sequence of BXLI shows similarities to the beta-glucosidases grouped in family 3. The yeast-produced enzymes were tested for enzymatic activities against different substrates. ABFI released L-arabinose from p-nitrophenyl-alpha-L-arabinofuranoside and arabinoxylans and showed some beta-xylosidase activity 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-arabinopyranoside, and p-nitrophenyl-beta-D- xylopyranoside, respectively, with the last activity being the highest. It was also able to hydrolyze xylobiose and slowly release xylose from polymeric xylan. ABFI and BXLI correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T. reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes. Both enzymes produced in yeasts displayed hydrolytic properties similar to those of the corresponding enzymes purified from T. reesei.
|Journal||Applied and Environmental Microbiology|
|Publication status||Published - 1996|
|MoE publication type||A1 Journal article-refereed|
Margolles-Clark, E., Tenkanen, M., Nakari-Setälä, T., & Penttilä, M. (1996). Cloning of genes encoding alfa-L-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 62(10), 3840-3846. http://aem.asm.org/content/62/10/3840.abstract