Molecular cloning and expression of cellulase gene of bacillus subtilis in escherichia coli

Abstract

Cellulase gene of Bacillus subtilis was cloned in Escherchia coli as a primary step of cloning the same gene in cells of alcohol producing organisms such as Zymomonas strains to let them ferment cellulosic materials into ethanol. The chromosomal DNA isolated from B.subtilis was partially digested with Hind III and ligated to the plasmid pBR 325 which was digested with Hind III and dephosphorylated with BAP. The ligated DNA was transformed into competent E.coli JM 83 cells and selected transformants showing cellulase activity by Congo-Red tests. Around 38,000 colonies were appeared on the selection media having ampicillin and chloramphenicol indicating the colonies are transformants. These transformed colonies were tooth-picked and transferred on LB agar containing 0.5\% of carboxy methyl cellulose (CMC) followed by Congo-Red tests. Among the 38,000 transformants tested only one colony, the 7324thly picked colony, showed clear halo indicating the transformant contains cellulase gene (CMCase gene). The new hybrid plasmid containing CMCase gene was designated as pDH 73. The plasmid pDH73 maintained stably in E.coli and expressed well for the production of CMCase of both intra- and extra-cellular types constitutively. The ratio of the two types was 9 to 1 with advantage to the intra-cellular CMCase. Although the CMCase activity of extra-cellular type was only one tenth of the total activity it may be concluded that the pDH73 contains DNA sequences which code for the secretion of CMCase out of the cell. It is of interest that the pDH 73 can not be digested by restriction endonucleases even with Hind III which was used to transfer CMCase gene on it. Several other resriction endonucleases were tried without success. Further studies to clarify this phenomenon should be followed.