Engineered pro-peptide enhances the catalytic activity of keratinase to improve the conversion ability of feather waste.

Keratinase is an attractive industrial enzyme that can specifically catalyze keratin waste to obtain value-added products. A challenge to the application of keratinase is improving catalytic capacity to achieve efficient hydrolysis. In this study, we effectively expressed the keratinase gene from Bacillus licheniformis BBE11-1 in Bacillus subtilis WB600 based on pro-peptide engineering. Partial deletion of the pro-peptide sequence and the substitution of amino acid at the pro-peptide cleavage site (P1) suggested that the "chaperone effect" and "cleavage efficiency" of the pro-peptide determine the activity of the mature enzyme. Subsequently, seven target sites that can increase the activity of the mature enzyme by 16%-66% were obtained through the multiple sequence alignment of pro-peptides and site-directed mutation. We further performed combinatorial mutations at six sites based on the design principle of three-codon saturation mutations and obtained mutant 2-D12 (236.8 KU/mg) with a mature enzyme activity of 186% of the original (127.6 KU/mg). Finally, continuous fermentation was carried out in a 5-L bioreactor for 22 h, and the activity of the 2-D12 mature enzyme was increased to 391.6 KU/mg. Most importantly, 2-D12 could degrade more than 90% of feather waste into amino acids and peptides within 12 h with the aid of sulfite.