Subcloning and Expression of a Protease Gene from Bacillus halodurans CM1 in Bacillus subtilis DB104

Abstract

ABSTRACT: Proteases are potential enzymes that utilized in various industrial fields, and the demand of these enzymes is increasing. Bacillus halodurans CM1 is Indonesia indigenous bacterium which is detected to be able to produce alkalotermophilic protease enzyme. In this study, we subcloned the protease gene consist of Open Reading Frame of protease gene and its promoter from Bacillus halodurans CM1 in Bacillus subtilis DB104 via conjugation, and analyzed the expression of the recombinant protease. The protease gene is 1 417 bp length including the open reading frame and the promoter, and obtained by PCR and cloned into pGEM T easy. After confirmed by sequencing, the gene was subcloned into vector pBBRE194, then the recombinant plasmid was transformed into E. coli S17-1. This E.coli was then conjugated to Bacillus subtilis DB104. The target recombinant B. subtilis DB104 has been obtained confirmed by plasmid verification and erythromycin resistance. The recombinant protease produced showed the highest enzyme activity at 50oC and pH 9 (with pH range 5-9) which with protease activity 13.66 U/mL.