Isolation and identification of feather-degrading bacteria and cloning and expression of the keratinase gene

Abstract

In this study, the strain CY-A, which has high feather degradation efficiency, was screened from the soil of a chicken pen. Bacillus tequilensis was identified by biological methods. The keratinase gene (bta) of Bacillus tequilensis CY-A was cloned by genetic engineering. The gene is 1110 bp in length, contains an open reading frame of 1089 bp and encodes 369 amino acids. The obtained gene sequence and amino acid sequence have been registered to GenBank under the database accession number OR733336.1. The physicochemical properties, secondary structure and tertiary structure of the protein were analysed by bioinformatics tools, and the relative molecular mass was found to be 37.953 kDa. The tertiary structure of keratin was 98.30% similar to that of Bacillus subtilis arpE. Finally, the keratinase gene bta was ligated to the expression vector pET28a (+) to construct the expression plasmid pET28a(+)-bta, which was subsequently transformed into E. coli BL21 (DE3) to generate engineered recombinant bacteria. Recombinant keratinase Bta was purified using Ni2+ column affinity chromatography with a molecular weight of approximately 37.953 kDa. Further studies on its recombinant enzymatic properties revealed that the enzyme activity of Bta was 283.93 U/mL, the optimum pH was 8, and the optimum reaction temperature was 50°C. Ca2+ has a very significant role in promoting Bta. EDTA and SDS can significantly inhibit the enzyme activity of Bta, which indicates that the enzyme activity requires metal ions. At a concentration of 10 mmol. L-1, PMSF almost completely inhibited the enzyme activity of Bta, indicating that Bta is a typical serine protease. The identification of the keratinase gene provides a theoretical basis for further improving keratinase activity via genetic engineering.