Cloning, Expression and Purification of Carboxypeptidase Enzyme from Bacillus halodurans

Microbial proteases have a large portion of the market of industrial enzymes, because of their wide application in detergents, drug enzymes and animal feed processing. Bacillus strains potentially produce, due to their physiological properties, a significant quantity of proteases. The kind of the protease which was cloned and expressed at the present study is Carboxypeptidase from Bacillus halodurans. The coding sequence of the gene from above mentioned strain was amplified with PCR using primer pair containing restriction sites NdeI and BamHI. After digestion of the PCR product, it was cloned into the appropriate site at PET۲۸a+ vector, then recombinant plasmids transformed into E. coli BL۲۱ strain. After confirmation of the recombinant plasmids using colony PCR and Sanger sequencing, recombinant enzyme expression was optimized at different conditions, then purified with Nickel embedded Agarose affinity chromatography. IPTG concentration of ۰.۲ mM, expression temperature of ۲۸ Co and incubation time of ۲۰ hours provided the maximum amount of enzyme expression. Molecular weight of the carboxypeptidase was estimated approx. ۵۵ KDa using SDS-PAGE. With easily optimized expression and production of substantial amount of recombinant enzyme in E. coli system, it can be hoped that proteases originating from native bacterial strains of Iran, including carboxypeptidases derived from these strains, have a high potential to become suitable enzymes for industrial use. Following the steps of the present research in the second phase, which includes measuring the activity of the enzyme in different temperature conditions, salt concentration and pH, in case of competitive results with existing commercial enzymes, it is possible to introduce the obtained enzyme as a suitable candidate for industrial use.

کلیدواژه ها:

Recombinant ، Protease ، pET۲۸a+