title: Purification and characterization of a major secretory cellobiase, Cba2, from Cellulomonas biazotea.
abstract: A novel cellobiase (Cba2) was purified from the culture supernatant of Cellulomonas biazotea and characterized. Cba2 appeared to be a major secretory cellobiase in C. biazotea as its enzymatic activity was estimated to represent over 40% of the total extracellular beta-glucosidase activity. The enzyme was purified over 260-fold subsequent to ammonium sulfate precipitation, gel-filtration chromatography, anion-exchange chromatography, and reversed-phase high-performance liquid chromatography. Cba2 was shown by SDS-PAGE to have a large molecular mass of 109 kDa, which makes it one of the largest secretory cellobiases characterized. Its homogeneity was confirmed by N-terminal amino acid sequencing. The K(m) and V(max) values were 0.025 mM and 0.0048 mM min(-1), respectively, for the Cba2 hydrolysis of p-nitrophenyl-beta-d-glucopyranoside, and 0.73 mM and 0.00033 mM min(-1), respectively, for the hydrolysis of cellobiose (at 37 degrees C and pH 7.0). The purified enzyme has a pH optimum of 4.8 and the optimum temperature for activity is 70 degrees C. In view of the secretory nature of Cba2 and the fact that it is a major component of secretory cellobiases of C. biazotea, it is potentially important in the enzymatic degradation of cellulose, and its availability as a recombinant protein may facilitate the studies of its biotechnological applications.
authors.affiliation: Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: A T
authors.initials: AT
authors.lastname: Lau
authors.forename: W K
authors.initials: WK
authors.lastname: Wong
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D013696:Temperature; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001617:beta-Glucosidase