Three-step surface design of spore-displayed keratinase improved acid tolerance and feather degradation

The global chicken business has grown rapidly, producing millions of tons of feather waste annually. Keratinase is a special enzyme that catalyzes the degradation of keratin and can be applied to the feed industry. In this study, we initially set the tone for the acid-resistant mutation of spore surface-display keratinase cotG-KERQ7 by replacing base-catalytic residues in the active center. We then performed molecular dynamics simulations of the KERQ7-AAPF, the enzyme-substrate complex, to enhance both acid stability and activity by substituting unstable positively charged amino acids on the surface. Finally, based on the change of protein rigidity-flexibility, a flexible tentacle outside the catalytic pocket was introduced to enhance the feather meal degradation activity of KERQ7 in an acidic environment. The Ding-F was finally obtained through the three-step design of keratinase surface. The mutant Ding-F was able to break down feather meal better in both lactobacillus-fermented feed environments and simulated gastric fluid digestion environments. This study not only establishes a theoretical foundation for the development of keratinases used in lactobacillus-fermented feed, but it also generates a three-step surface design method that can serve as a guide for future general strategies aimed at modifying the acid resistance of serine keratinases.