Molecular cloning of two genes for beta-D-glucosidase in Bacillus sp. GL1 and identification of one as a gellan-degrading enzyme

In the bacterium Bacillus sp. GL1, gellan is depolymerized to give a tetras accharide by extracellular gellan lyase and then the tetrasaccharide is con verted to constituent monosaccharides by intracellular glycosidases. Two ge nes encoding one of the glycosidases, beta-D-glucosidase (Bgl), were cloned in a genomic DNA library of the bacterium constructed in Escherichia coli and nucleotide sequences of the genes were determined. One of the genes, te rmed bglA, contained an open reading frame (ORF) consisting of 1344 base pa irs coding a polypeptide (BglA) with a molecular mass of 51 kDa and the oth er, termed bglB, 2268 base pairs coding a protein (BglB) with a molecular m ass of 82 kDa. By homology analyses of the ORFs against protein sequence da tabases, beta-D-glucosidase A (BglA) and beta-D-glucosidase B (BglB) were f ound to be classified into subfamilies BGA and BGB of cellulase family BG, respectively. BgLA and BglB purified from E. coli were monomeric enzymes wi th molecular masses of 50 and 82 kDa and most active at pH 6.0 and 8.0, res pectively. BglA showed broader substrate specificity than BglB, Only BglA a cted on the tetrasaccharide produced from gellan by gellan lyase and releas ed glucose from the molecule. (C) 1998 Academic Press.