Biosynthesis, purification, and biochemical properties of an alkaline-thermostable keratinase derived from Bacillus cereus J6

In this study, Strain J6, a keratinase producer, was isolated from feather waste and identified as Bacillus cereus J6. The enzyme yield was enhanced from 172 U/mL to 727 U/mL via optimization. The purification of keratinase involved ammonium sulfate fractionation and subsequent anion exchange chromatography with a specific activity of 4215.24 U/mg, achieving 9.34-fold purification. The keratinase was about 35 kDa and had maximal activity at 55°C and pH of 9.0. When the temperature was below 55°C, this enzyme retained over 90% of its activity, indicating its thermal stability. Moreover, Mg²⁺, Ca²⁺, Zn²⁺, and Mn²⁺ promoted the activity differently. Especially, Mg²⁺ increased the keratinase activity by 31%. In contrast, Al³⁺, Fe²⁺, Fe³⁺, Cu²⁺ displayed varying degrees of inhibitory effects on keratinase. The keratinase was stable and even boosted significantly in the presence of multiple surfactants. But EDTA and PMSF inactivated the activity, thereby suggesting that it belongs to the serine metalloprotease. Further, the enzyme had high catalytic efficiency on casein and keratin, but relatively low degradability of wool, and feathers.