Enhancement of keratin-degradation ability of the keratinase KerBL from Bacillus licheniformis WHU by proximity-triggered chemical crosslinking.

Abstract Keratinases are valuable enzymes, given their application in keratin-rich waste recycling. Considering that keratinases usually require reducing agents to efficiently degrade keratin, improving the stability of keratinases under reducing conditions is highly desirable for practical applications. Here, we show that the introduction of several tyrosine derivatives containing para-substituted long-chain haloalkanes into the keratinase KerBL, which enabled proximity-triggered covalent crosslinking by rational design, could improve both the thermostability and autolytic resistance of the enzyme. After screening a series of noncanonical amino acid (ncAA)-based variants generated by rational design, two variants, N159C/Y260BprY and N159C/Y260BbtY, with enhanced keratinolytic activity were obtained. Both variants increased the Tm of the enzyme by approximately 10 °C. The potential mechanism underlying these improvements was investigated by molecular dynamics (MD) analysis. The results indicated that BprY-Cys and BbtY-Cys covalent bonds in the N159C/Y260TAG variant could significantly decrease the flexibility and fluctuations of the long loop (residues 151–162).