Expression of cellulomonas fimi β-glucosidase gene in bacillus subtilis using chloramphenicol inducibility

Abstract

To make Cellulomonas fiji $\beta$ -glucosidase gene express in Bacillus subtilis, the gene which was previously cloned in our laboratory, was subcloned in a Bacillus expression vector, pPL 708. Plasmid pPL708 contains bacteriophage SpoII promoter followed by multiple cloning sites and CAT gene from B. pumilus. The CAT gene contains chloramphenicol control site in the upstream 200 base pair region. A shuttle vector between Escherichia coli and B. subtilis was constructed using pUC19 and pPL708. A $\beta$ -glucosidase gene-containing pCF16 was treated with appropriate restriction enzymes and Kllenow fragment, and ligated to the shuttle vector digested with HpaI. This ligation mixture was used to transform E. coli HB101 cells. From the transformants, recombinant plamids were isolated and designated pPCF84. The $\beta$ -glucosidase gene was relocated under the control region of cAT-86 gene from pPL708. B. subtilis RM125 was transformed with pPCF84 again. The $\beta$ -glucosidase activity was tested both in E. coli and B. subtilis. Recombinant E. coli HB101 (pPCF84) was induced with Chloramphenicol when 0.D600 value reached 0.4. The $\beta$ -glucosidase production was increased up to about 5 times of that of uninduced cells. Expression of $\beta$ -glucosidase gene by chloramphenicol induction did not turn on well in E. coli. When the culture of B. subtilis RM125 (pPCF84) was induced with Chloramphenicol, the enzyme activity was increased up to about 17 times higher than that of pCF18, and about 10,000 times than that of gene donor, Cellulomonas fimi. The $\beta$ -glucosidase activity in uninduced B. subtilis (pPCF84) was near to zero, while in uninduced E. coli (pPCF84) enzyme activity was 50\% of that of pCF18.