Cloning and characterization of two genes from Bacillus polymyxa expressing β-glucosidase activity in Escherichia coli

Abstract

DNA fragments from Bacillus polymyxa which encode β-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-β-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably as phosphorylated sugar.