title: Molecular cloning and characterization of a unique beta-glucosidase from Vibrio cholerae.
abstract: We have recently reported the molecular cloning of a gene, gspK, in Vibrio cholerae that encodes a specific glucosamine kinase. We describe here the identification of bglA, a gene contiguous to gspK in a presumptive large chitin catabolic operon. BglA was molecularly cloned into Escherichia coli, and the protein BglA was overexpressed and purified to apparent homogeneity. BglA is 65 kDa (574 amino acids) with an N-terminal amino acid sequence predicted by the gene sequence, suggesting that the enzyme is cytoplasmic. The purified enzyme exhibited optimal activity with p-nitrophenyl beta-glucoside, cellobiose, and higher oligosaccharides of cellulose. No other glucosides or glycosides tested were hydrolyzed, including Glc-Glc disaccharides where the linkage is beta 1-->2, beta 1-->3, and beta 1-->6, respectively. The predicted BglA sequence bears little similarity to other proteins in the data banks. The Henrissat algorithm places BglA sequence in Family 9 of the glycosidases, suggesting it is an endoglucanase. However, the results summarized above suggested that BglA is an exoenzyme yielding Glc at each cleavage step. To resolve this apparent discrepancy, detailed kinetic studies were conducted with cellotetraose. Only exoglucanase activity was detected. The function of this enzyme in V. cholerae remains to be determined, especially because our strain of this organism does not utilize cellobiose.
authors.affiliation: Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: Jae Kweon
authors.initials: JK
authors.lastname: Park
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
authors.forename: Himatkumar V
authors.initials: HV
authors.lastname: Patel
authors.forename: Saul
authors.initials: S
authors.lastname: Roseman
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D007700:Kinetics; D013379:Substrate Specificity; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics; D001617:beta-Glucosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D017931:DNA Primers; D001617:beta-Glucosidase