Overexpression Of Keratinase From Bacillus Amyloliquefaciens And Construction Of Engineered Bacterium With Significant Feather-Degrading Capacity

Keratinase has capacity of degrading keratin and shows broad application prospects in feed industry, textiles disposal, detergent industry, medical field etc., bringing enormous social and economic benefit. This study aims to obtain a Bacillus amyloliquefaciens strain K11 strain with rapid feather-degrading-ability and construct a recombinant strain pBSlac-kerK-SCK6 with high keratinase expression level and an engineered strain K1127 with improved feather-degrading-ability through genetic manipulation. It will lay a theoretical foundation and provide technical support for the industrial application of keratinase.Bacillus amyloliquefaciens strain K11 grew rapidly in the medium with chicken feathers as the only source of carbon and nitrogen and completely degraded whole feathers within 24 h. The peptide sequence was determined by SDS-PAGE and LC-ESI-MS/MS, and the full-length gene(kerK) was obtained. The recombinant vector pUB110-kerK was constructed by using POE-PCR and was successfully expressed in the Bacillus subtilis SCK6. Assays on skimmed milk plates and in feather medium indicated that kerK is the key functional gene for feather degradation. Biochemical characterization indicated that purified recombinant KerK had maximum activity at pH 11.0 and 50°C, and retained stable over a wide pH range from 6.0 to 12.0, the optimum temperature of recombinant Ker K at pH 11.0 was, the KerK has well thermal stability and 70% of the maximal activity after pre-treatment at 50°C for 30 min.In vitro chicken feather degrading experiment showed that sterilized chicken feather(~5 cm) was completely degraded within 0.5 h at 50°C with incubation of 150 U purified KerK and 2 mM DTT. Hydrolysis product analysis by HPLC-Chip/ESI-QTOF-MS indicated that KerK mainly acted on the linkages between serine and hydrophobic amino acids of feathers and released polypeptides VVIQPSPVVVTLPGPILSS, IQPSPVVVTLPGPILSS, and ILSEEGVPISSGGF as the main products, which were rich in essential amino acids and had molecular masses of 1 to 3 kDa. The results suggested the possibility for recycling of feathers and utilization, especially as feed components in feed industry.To improve the KerK expression level, an inducible recombinant vector pBSlac-kerK was constructed and transformed into Bacillus subtilis SCK6 for heterolgous expression. The keratinase activity of recombinant B. subtilis SCK6 reached 3500 U/ml in 15 L fermentor, which is higher than that of most reported counterparts from bacteria. Constitutive vector pUB110-kerK was also extracted from B. subtilis SCK6 and transformed into B. amyloliquefaciens K11 to increase the copy number of kerK. As results, the recombinant strain K1127 exhibited enhanced feather-degrading capacity: shortened reaction time within 12 h and increased keratinolytic activity(1500 U/ml) by 6-fold. This efficient and rapid feather-degrading character makes the recombinant strain B. amyloliquefaciens K1127 potential for applications in feather meal preparation and waste feather disposal.In summary, B. amyloliquefaciensK11 strain with significant feather-degrading capacity was reported in this study. The functional gene for feather degradation, kerK, was then cloned and the constitutive expression vector pUB110-kerK and inducible expression vector pBSlac-kerK were successfully constructed and expressed in B. subtilis SCK6. The recombinant enzyme was purified and analyzed the biochemical characterization, and the enzymatic hydrolysis products were analyzed. Meanwhile the constitutive expression vector pUB110-kerK was transformed into B. amyloliquefaciens K11 to obtain the recombinant strain K1127 with improved feather-degrading capacity. This study provides theoretical and application foundations for large-scale waste feather disposal and recycling.