Expression Of Keratinase Gene KerC From Bacillus Subtilis In Pichia Pastoris

Keratinases are a group of proteolytic enzymes that can hydrolyze insoluble keratins.In recent years,keratinases have been used in the fields of leather processing,detergent manufacture and feed production.Therefore,keratinases have displayed satisfactory application performance and economic value. Particularly in animal feed additives,the bioconversion of chicken feather keratin into animal feedstuffs will ameliorate the shortage of protein feed.The keratinase gene kerC without leader sequence from Bacillus subtilis B-3 was cloned into vector pPICZaA for extracellular expression in Pichia pastoris X-33.KerC was expressed successfully in the Eukaryotic expression system, and the expression condition was highly improved.1.According to the sequence of keratinase gene kerC from Bacillus subtilis B-3,a pair of primers containing the restriction sites of EcoRI and Xball was designed and synthesized.The sequence of kerC without peptide sequence was amplified by PCR and cloned into vector pPICZaA,recombinant plasmid pPICZaA-kerC was constructed successfully.The amplified sequence of kerC generated three bases missense mutation that resulted in the change of three amino acids.According to the analysis of spatial structure for kerC,the site where the three amino acids changed was not in the active region of kerC.2.The recombinant plasmid pPICZaA-kerC was transformed into Pichia pastoris X-33,and the recombinant strains was screened by the zeocin,by PCR using genomic DNA as template and by keratinase activity assay.A maximum keratinase yield of 32.31 U/mL with specific activity of 95.43 U/mg was obtained in the culture supernatant when P.X.KC-9 transformant had grown for 108h with 1% methanol induction.3.The recombinant keratinase kerC showed activity at 30℃-65℃temperature range and the pH range is 7-8.5. However,the optimal temperature and pH value was respectively 60℃and 7.5. For all the tested metal ions,the presence of 0.33mM of Fe2+,Co2+and Ca2+ could improve significantly the keratinase activity of kerC.About 1364.44% enhancement of keratinase activity was observed in the presence of Fe2+.Fe3+could inhibit completely the activity of kerC,and Na+,Mn2+,Zn2+,Mg2+and Cu2+had negative influence on the keratinase activity.The recombinant keratinase showed a molecular mass of 31 KDa as determined by SDS-PAGE.