PURIFICATION AND CHARACTERIZATION OF A CARBOXYMETHYLCELLULOSE-DEGRADING ENZYME SECRETED BY A YEAST-STRAIN NEWLY ISOLATED FROM SOIL

Eight yeast strains (MUT-series) possessing high abilities to utilize carboxymethylcellulose (CMC) were obtained from 127 strains previously isolated from soil. A strain MUT-6 secreted a carboxymethylcellulose-degrading enzyme (CMCase) and accumulated oligosaccharides in a CMC-medium. The CMCase was purified from the culture fluid of the yeast as a single protein (MW: 34,000 daltons) in SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the enzyme was as follows: H-Lys-Leu-Pro-Tyr-Leu-Gly-Gly-Val-Asn-Leu-Ala-Gly-Thr-Asp-Phe-Gly-Ile-Asp-Ile-Tyr-Gly-. Optimal conditions for enzyme reaction were 65-70-degrees-C for temperature and 4.3 for pH. Heat stability was remarkably high; half-life time at 98-degrees-C (pH 6.0) was 30 min. Products from hydrolysis of CMC by the CMCase were di-, tri-, and tetra-oligosaccharides without mono-mer, and could be metabolized by a transformant of Saccharomyces cerevisiae with a plasmid carrying the BGL1 gene encoding cellobiase of Saccharomycopsis fibuligera. Cellobiose, as well as CMC, was found to be an effective inducer for CMCase production.