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

Abstract

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.