Purification and characterization of a cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli

A cellulase from the luminal fungus Orpinomyces joyonii cloned in Escherich ia coli was purified 88-fold by chromatography on High Q and hydroxyapatite . N-terminal amino acid sequence analyses confirmed that the cellulase repr esented the product of the cellulase gene Cel B2. The purified enzyme posse ssed high activity toward barley beta -glucan, lichenan, carboxymethyl cell ulose (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-glucop yranoside. The specific activity of the cloned enzyme on barley beta -gluca n was 297 units/mg protein. The purified enzyme appeared as a single band i n SDS-polyacrylamide gel electrophoresis and the molecular mass of this enz yme (58 000) was consistent with the value (56 463) 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 degre esC. The K-m values estimated for barley B-glucan and CMC were 0.32 and 0.5 0 mg/ml, respectively. (C) 2001 Elsevier Science Ltd. All rights reserved.