EXPRESSION AND CHARACTERIZATION OF PICHIA-ETCHELLSII BETA-GLUCOSIDASEIN ESCHERICHIA-COLI

The beta-glucosidase enzyme is important as the terminal enzyme involv ed in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation. Under controlled reaction con ditions the enzyme also displays cello-oligosaccharide synthesizing ab ility (based on either the thermodynamic or kinetic approach). We pres ent here the purification of the enzyme beta-glucosidase (BGL) of Pich ia etchellsii from recombinant pBG55 Escherichia coli clone. The kinet ic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported. The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-spec ificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme. Temperature directed substrate sp ecificity far aryl beta,1-4 linkage, and beta(1-2), beta(1-4), beta(1- 6) and beta(2-1) linkages in various natural disaccharides was observe d. Glycosylation of the enzyme was found to be unimportant for enzyme activity. With both cellobiose and glucose, oligosaccharide synthesis was detected. The implications of this information with regard to cell ulose hydrolysis and oligosaccharide synthesis are discussed.