A combination of bioinformatics analysis and rational design strategies to enhance keratinase thermostability for efficient biodegradation of feathers.

Keratinase has shown great significance and application potentials in the biodegradation and recycle of keratin waste due to its unique and efficient hydrolysis ability. However, the inherent instability of the enzyme limits its practical utilization. Herein, we obtained a thermostability-enhanced keratinase based on a combination of bioinformatics analysis and rational design strategies for the efficient biodegradation of feathers. A systematical in silico analysis combined with filtering of virtual libraries derived a smart library for experimental validation. Synergistic mutations around the highly flexible loop, the calcium binding site and the non-consensus amino acids generated a dominant mutant which increased the optimal temperature of keratinase from 40 °C to 60 °C, and the half-life at 60 °C was increased from 17.3 min to 66.1 min. The mutant could achieve more than 66% biodegradation of 50 g/L feathers to high-valued keratin product with a major molecular weight of 36 kDa. Collectively, this work provided a promising keratinase variant with enhanced thermostability for efficient conversion of keratin wastes to valuable products. It also generated a general strategy to facilitate enzyme thermostability design which is more targeted and predictable.