title: Expression and characterization of Pichia etchellsii beta-glucosidase in Escherichia coli.
abstract: The beta-glucosidase enzyme is important as the terminal enzyme involved in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation. Under controlled reaction conditions the enzyme also displays cello-oligosaccharide synthesizing ability (based on either the thermodynamic or kinetic approach). We present here the purification of the enzyme beta-glucosidase (BGL) of Pichia etchellsii from recombinant pBG55 Escherichia coli clone. The kinetic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported. The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-specificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme. Temperature directed substrate specificity for aryl beta,1-4 linkage, and beta (1-2), beta(1-4), beta(1-6) and beta(2-1) linkages in various natural disaccharides was observed. Glycosylation of the enzyme was found to be unimportant for enzyme activity. With both cellobiose and glucose, oligosaccharide synthesis was detected. The implications of this information with regard to cellulose hydrolysis and oligosaccharide synthesis are discussed.
authors.affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, India.
authors.forename: M
authors.initials: M
authors.lastname: Pandey
authors.forename: S
authors.initials: S
authors.lastname: Mishra
mesh_terms: D015139:Blotting, Southern; D002475:Cellobiose / Q000096:biosynthesis / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D010843:Pichia / Q000201:enzymology*; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002475:Cellobiose; D001617:beta-Glucosidase