Characterization of the recombinant cellobiase from celG gene in the beta-glucoside utilization gene operon of Pectobacterium carotovorum subsp. carotovorum LY34

Abstract

A third cel operon containing celE, celF, and celG genes was isolated from Pectobacterium carotovorum subsp. carotovorum LY34 ( Pcc LY34) genomic DNA using a cosmid library. The amino acid sequences of CelE and CelF shared high sequence identity with the cellobiose-specific PTS enzymes IIB and IIC, respectively. CelF contained the disaccharide binding region essential for acquiring cellobiose molecules. The amino acid sequence of CelG shared high sequence identity with various β-glucosidases (cellobiases) belonging to the glycosyl hydrolase family 1. Sequence and structural analysis also demonstrated that this cel operon differs from three operons previously reported from Pcc LY34, including bglTPB (accession number AY542524), ascGFB (accession number AY622309), and bglEFIA (accession number AY769096). In this study, the celF and celG genes were expressed in the presence of cellobiose. Purified CelG was estimated to be approximately 54 kDa by SDS-PAGE and was able to hydrolyze salicin, arbutin, pNPG, cellobiose, and MUG, and exhibited maximal activity at pH 5.0 to 40 °C. Two glutamic acid residues (Glu 172 and Glu 370) were shown to be essential for enzyme activity.