A BETA-GLUCOSIDASE GENE (BGL3) FROM STREPTOMYCES SP STRAIN-QM-B814 - MOLECULAR-CLONING, NUCLEOTIDE-SEQUENCE, PURIFICATION AND CHARACTERIZATION OF THE ENCODED ENZYME, A NEW MEMBER OF FAMILY 1 GLYCOSYL HYDROLASES

A beta-glucosidase gene (bgl3) from Streptomyces sp. QM-B814 (American Type Culture Collection 11238) has been cloned by functional compleme ntation of a beta-glucosidase-negative mutant of Streptomyces lividans . An open-reading frame of 1440 nucleotides encoding a polypeptide of 479 amino acids was found by sequencing. The encoded protein (Bgl3) sh ows extensive similarity (over 45% identity) with beta-glycosidases fr om family-1 glycosyl hydrolases. The cloned enzyme, purified following ammonium sulphate precipitation and two chromatographic steps, is mon omeric with molecular mass 52.6 kDa, as determined by mass spectrometr y, and an isoelectric point of pI 4.4. The enzyme appears to be a beta -glucosidase with broad substrate specificity, is active on cellooligo mers, and performs transglycosylation reactions. The estimated apparen t K-m values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose a re 0.27 mM and 7.9 mM, respectively. The K-i values for glucose and de lta-gluconolactone, using p-nitrophenyl-beta-D-glucopyranoside as a su bstrate, are 65 mM and 0.08 mM, respectively. The purified enzyme has a pH optimum of pH 6.5 and the temperature optimum for activity is 50 degrees C.