Cloning and expression of a bacillus cellulase gene in escherichia coli and nucleotide sequence of the gene

Abstract

In order to open a way for economical saccharification of the natural cellulose, amplification of a microbial cellulase gene using recombinant DNA technique was attempted. A cellulolytic spore-former was isolated from heat treated compost, identified as $\underline{Bacillus}$ $\underline{subtilis}$, and used as gene donor. From the $\underline{Bacillus}$ isolate a new cellulase gene was cloned into $\underline{E.}$ $\underline{coli}$ HB101 using pBR322 as a vector. Two different types of recombinant plasmids were isolated from the carboxymethyl cellulase (CMCase) positive transformants and named pBS1 and PBS2. CMCase genes were found to be located in 3.2- and 3.5 kb PstI fragments, of the respective plasmids. The 3.2- and 3.5 kb chromosomal inserts had the same restriction patterns, but their orientations were in opposite. DNA hybridization experiments showed that the two inserts were homologous and the 3.5kb fragment seemed to be derived from the 3.2 kb fragment through recombination with $\underline{E.}$ $\underline{coli}$ chromosomal DNA in vivo. The CMCase gene was expressed independently of its orientation in the cloning vector pBR322 showing enzyme production 40 times more than the original $\underline{Bacillus}$ species. The high production of CMCase in E. coli by the foreign gene did not impede growth of the host cells. The $\underline{E.}$ $\underline{coli}$ transformants secrete only 5% of their total CMCase into the medium and the remaining 95% was shared equally in periplasmic and cytoplasmic compartments. The CMCase produced by $\underline{E.}$ $\underline{coli}$ HB101 (pBS1) hydrolyzed CMC (1,4-β-D-glucan) and lichenan (1,3-1,4-β-D-glucan) powerfully, but did not show any hydrolyzing activity to laminarin (1,3-β-D-glucan), pustulan (1,6-β-D-glucan) and starch (1,4-α-D-glucan). The CMCase showed typical endoglucanase kinetics; caused a rapid decrease in viscosity with linear increase of reducing sugars in CMC solution. The CMCase produced by the E....