Optimization Of Fermentation Conditions Of Bacillus Aquimaris JM7 And Cloning And Expression Of The Functional Gene

With the development of the poultry industry, accumulation of feather which is produced from poultry industry not only cause environmental problems, but also cause serious waste of protein resources. In this study, microbial process was used to degrade feather keratin. We used liquid fermentation to explore the optimal conditions of feather degradation. Then the keratinase gene Ker02562 was further cloned, expressed and purified.Bacillus aquimaris JM7 was isolated from the deep sea of the South China Sea with feather as the sole carbon and nitrogen source. The effects of fermentation conditions and medium composition on feather degradation by Bacillus aquimaris JM7 were investigated, and the results showed that the addition of appropriate carbon(sucrose) and nitrogen sources would inhibit the production of keratinase. The optimal fermentation conditions for chicken feather powder degradation are as follows: initiative medium pH value 9.0; cultivate temperature 40℃; shaking speed 150rpm; quantity of added chicken feather powder 0.2%(m/v). Under the conditions above, Bacillus aquimaris JM7 was able to degrade chicken feather powder with the degradation rate of 79.4 % within 30 h, which was higher than most of other microorganisms reported capable of degrading feather keratin. 17 amino acids such as glutamic acid, serine, proline were detected.The keratinase gene Ker02562 of keratin-degrading bacteria was cloned, expressed and purified. The keratinase gene was amplified through PCR, the length of it was 963 bp, encoding 320 amino acids. Then the gene was cloned into pCold I vector and transformed into Escherichia coli BL21(DE3). SDS-PAGE analysis showed that the recombinant keratinase had an approximate molecular mass of 38 kDa, which was the same as predicted value. Finally, the recombinant keratinase was purified through His-tag column.The purified enzyme exhibited optimum activity at 40℃ and was highly active at various pH ranging from 5.0-12.0, with maximum activity observed at p H 8.0-9.0. And the keratinase was stable at 10-50℃. The enzyme could be activated in the presence of Ca2+, Fe2+ and Sr2+. It was inhibited by PMSF and EDTA, which allowed its identification as metallo-serine keratinase. Furthermore, it was also inhibited by organic solvents like DMSO, acetonitrile and isopropanol.