Isolation, Identification Of Feather-Degrading Bacillus Subtilis And Cloning, Expression Of Its Keratinase Gene KerC

Keratinolytic enzymes, called keratinases, are a group of enzymes which are able to hydrolyze insoluble keratins. Keratinases are usually used in degradation of keratin-containing wastes for industrial application, which is important for potential applications in waste treatment, animal feed production and recycling of the insoluble proteins. Therefore, keratinases had shown satisfactory application performance and economic value. A feather-degrading Bacillus subtilis was isolated from the feather waste site of poultry farm of Sichuan agricultural university, and the gene kerC was cloned and expressed in E.coli BL21 (DE3) in this paper.1. Seven feather-degrading bacteria were isolated from the feather waste site of poultry farm of Sichuan agricultural university. B-3 showed the strongest feather-degrading ability among the isolated bacteria, which was choosed for futher study. Based on morphological, physiological and biochemical characteristics, and sequence alignment of 16s rDNA, B-3 was identified as Bacillus subtilis, which was named Bacillus subtilis B-3 in this paper.2. The nucleotide sequence of the keratinase gene was amplified from chromosomal DNA through PCR. The length of kerC gene was 1146 bp encoding 381 amino acids, which had a signal peptide. Sequence alignment of kerC with GenBank database showed it shared a 100% sequence identity with the kerC gene from Bacillus subtilis strain YYW-1 submitted in GenBank (GenBank No.:EU362730). The kerC had two conserved domains belong to subtilisin superfamily, which indicated that kerC might be a member of serine proteases.3. The kerC gene was cloned into pET32a(+) vector and transformed into Escherichia coli BL21 (DE3). The kerC gene was expressed fused with E.coli thioredoxin (trxA) and had an approximate molecular mass of 60.0 kDa. Then, the recombinant kerC was purified through His-tag column. The characters of His-Tag purified recombinant kerC were also determined. The optimal pH and temperature of kerC were 7.0 and 65℃, respectively. The activity of kerC was thermostable below 50℃, while it was stable in the basic buffers. Under the optimal reaction condition, the activity of recombinant kerC was 14.814U/mL, and its specific activity was 0.0852 U/mg. Some basical study of kerC from Bacillus subtilis had done in this paper, which provided foundation for futher basic and applied research. It also provided valuable data for uncovering the evolution history of keratinases.