Molecular Cloning of Two Genes for β-d-Glucosidase inBacillussp. GL1 and Identification of One as a Gellan-Degrading Enzyme

In the bacteriumBacillussp. GL1, gellan is depolymerized to give a tetrasaccharide by extracellular gellan lyase and then the tetrasaccharide is converted to constituent monosaccharides by intracellular glycosidases. Two genes encoding one of the glycosidases, β-d-glucosidase (Bgl), were cloned in a genomic DNA library of the bacterium constructed inEscherichia coliand nucleotide sequences of the genes were determined. One of the genes, termedbglA,contained an open reading frame (ORF) consisting of 1344 base pairs coding a polypeptide (BglA) with a molecular mass of 51 kDa and the other, termedbglB,2268 base pairs coding a protein (BglB) with a molecular mass of 82 kDa. By homology analyses of the ORFs against protein sequence databases, β-d-glucosidase A (BglA) and β-d-glucosidase B (BglB) were found to be classified into subfamilies BGA and BGB of cellulase family BG, respectively. BglA and BglB purified fromE. coliwere monomeric enzymes with molecular masses of 50 and 82 kDa and most active at pH 6.0 and 8.0, respectively. BglA showed broader substrate specificity than BglB. Only BglA acted on the tetrasaccharide produced from gellan by gellan lyase and released glucose from the molecule.