Gene Cloning, Expression, Purification, and Enzymatic Properties Study of Keratinase from Bacillus velezensis

Abstract:

In order to obtain a better feather meal degrading enzyme, a keratinase gene (baker1) was screened from the genome of Bacillus velezensis SYBC H47. Subsequently, baker1 was cloned and its heterologous expression was achieved in Escherichia coli BL21 (DE3). Finally, the recombinant keratinase (BaKer1) was isolated and purified, characterized, and was studied for its practical application. The results showed that the optimal pH and temperature for BaKer1 were 9.5 and 55 ℃, respectively, and it exhibited good stability under moderately-temperature and alkaline conditions. BaKer1 was significantly activated by 1 mmol/L Ca²⁺ ion, slightly inhibited by 5 mmol/L Mn²⁺ ion, and insignificantly affected by other metal ions. BaKer1 had significant inhibitory effect on the activity of PMSF, indicating that BaKer1 is a typical serine protease. The kinetic parameters of BaKer1 were determined using feather meal, azocasein and casein as substrates. The K_m values were 5.06, 1.09, 1.39 mmol/L, respectively, and their k_cat/K_m values were 1 058.24, 2 536.54, 3 733.79 s·mmol/L, respectively. An acid hydrolysis efficiency of 33.51% could be achieved during the degradation of feather meal by BaKer1, indicating its potential application value in the degradation of feather meal.