THE CLONING, EXPRESSION AND CHARACTERIZATION OF A CELLOBIASE GENE ENCODING A SECRETORY ENZYME FROM CELLULOMONAS-BIAZOTEA

A 4.7-kb DNA insert encoding a secretory cellobiase (Cba) was cloned f rom Cellulomonas biazotea in Escherichia coli using an excretion vecto r, pM. Host cells transformed with the recombinant construct, designat ed pBZ4.7, were able to utilize cellobiose as the sole carbon source. Part of the Cba activity encoded by pBZ4.7 could be detected in the pe riplasm and even in the culture supernatant. The Cba protein was purif ied from the culture supernatant and analysed by SDS-PAGE to have an a pparent M-r of 86 000. The insert consisted of two PstI fragments with lengths of 0.75 and 3.95 kb, both of which were found to be crucial f or expressing the Cba activity. Sequencing of the first 3.95 kb of the insert revealed that the coding sequence for Cba, designated the cba gene, was 2484 bp long. Comparison of the deduced Cba sequence with th ose of published beta-glucosidases revealed a potential active site lo cated at the N-terminal portion of the former. The cba gene has a high G+C content of 76.4% and is flanked by a putative ribosome-binding si te and potential transcriptional termination signals upstream and down stream from its coding sequence, respectively. (C) 1998 Elsevier Scien ce B.V.