Cloning, expression and characterization of a Family 48 exocellulase, Cel48A, from Thermobifida fusca

The gene for a 104-kDa exocellulase, Cel48A, formerly E6, was cloned from T hermobifida fusca into Escherichia coli and Streptomyces lividans. The DNA sequence revealed a type II cellulose-binding domain at the N-terminus, fol lowed by a FNIII-like domain and ending with a glycosyl hydrolase Family 48 catalytic domain. The enzyme and catalytic domain alone were each expresse d in and purified from S. lividans and had very low catalytic activity on s wollen cellulose, carboxymethyl cellulose, bacterial microcrystalline cellu lose and filter paper. However, in synergistic assays on Biter paper, the a ddition of Cel48A to a balanced mixture of T. fusca endocellulase and exoce llulase increased the specific activity from 7.9 to 11.7 mu mol cellobiose min(-1) mL(-1), more than 15-fold higher than any single enzyme alone. Cel4 8A retained > 50% of its maximum activity from pH 5 to 9 and from 40 to 60 degrees C. Using SWISSMODEL, the amino-acid sequence of the Cel48Acd was mo deled to the known structure of Clostridium cellulolyticum CelF. Family 48 enzymes are remarkably homologous at 35% identity for all their catalytic d omains and some of the properties of the 10 members are discussed.