Cloning, Sequencing, and Expression of the Gene Encoding a Family 9 Cellulase from Bacillus licheniformis F11 in Escherichia coli and Bacillus megaterium, and Characterization of the Recombinant Enzymes

Abstract

A gene encoding cellulase belonging to the glycosyl hydrolase family 9 along with its native promoter was isolated from Bacillus licheniformis F11, cloned in Escherichia coli DH5 α and subcloned by transconjugation to Bacillus megaterium MS941. Functionality of the encoded protein was proven both in heterologous hosts, E. coli and B. megaterium . In the latter, the gene product was found in the extracellular fraction expressing a high specific activity; whereas in E. coli the protein was not secreted into the medium, and rather, showed a lower specific activity. The optimum temperature of the recombinant enzyme expressed in the hosts range from 65-75 oC; whereas the optimum pH is 6. The recombinant enzyme was stable between 50-60 oC and in a broad pH range (pH 5 - 9). Addition of Ca 2+ and Fe 3+ enhanced the enzyme activity, whereas EDTA and Cu 2+ had the opposite effect. Lichenin, rather than carboxyl methyl cellulose, is the preferred substrate.