The cloning, expression and characterization of a cellobiase gene encoding a secretory enzyme from Cellulomonas biazotea

Abstract

A 4.7-kb DNA insert encoding a secretory cellobiase (Cba) was cloned from Cellulomonas biazotea in Escherichia coli using an excretion vector, pM. Host cells transformed with the recombinant construct, designated 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 periplasm and even in the culture supernatant. The Cba protein was purified from the culture supernatant and analysed by SDS–PAGE to have an apparent 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 for 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 those of published β-glucosidases revealed a potential active site located 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 site and potential transcriptional termination signals upstream and downstream from its coding sequence, respectively.