title: Purification and characterization of a cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli.
abstract: A cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli was purified 88-fold by chromatography on High Q and hydroxyapatite. N-terminal amino acid sequence analyses confirmed that the cellulase represented the product of the cellulase gene Cel B2. The purified enzyme possessed high activity toward barley beta-glucan, lichenan, carboxymethyl cellulose (CMC), xylan, but not toward laminarin and pachyman. In addition, the cloned enzyme was able to hydrolyze p-nitrophenyl (PNP)-cellobioside, PNP-cellotrioside, PNP-cellotetraoside, PNP-cellopentaoside, but not PNP-glucopyranoside. The specific activity of the cloned enzyme on barley beta-glucan was 297 units/mg protein. The purified enzyme appeared as a single band in SDS-polyacrylamide gel electrophoresis and the molecular mass of this enzyme (58000) was consistent with the value (56463) calculated from the DNA sequence. The optimal pH of the enzyme was 5.5, and the enzyme was stable between pH 5.0 and pH 7.5. The enzyme had a temperature optimum at 40 degrees C. The K(m) values estimated for barley beta-glucan and CMC were 0.32 and 0.50 mg/ml, respectively.
authors.affiliation: Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT Hong Kong, China.
authors.forename: X Y
authors.initials: XY
authors.lastname: Ye
authors.forename: T B
authors.initials: TB
authors.lastname: Ng
authors.forename: K J
authors.initials: KJ
authors.lastname: Cheng
mesh_terms: D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry* / Q000302:isolation & purification*; D002845:Chromatography; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005936:Glucans / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D020085:Neocallimastigales / Q000201:enzymology*; D013696:Temperature; D014990:Xylans / Q000378:metabolism
chemical_list: D005936:Glucans; D014990:Xylans; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium; C015968:lichenin