Improving the hydrolysis and acyltransferase activity of bifunctional feruloyl esterases DLFae4 by multiple rational predictions and directed evolution

In the present study, the hydrolysis and acyltransferase activities of bifunctional feruloyl esterases DLFae4 were enhanced by a combination of multiple mutation strategies based on rationally predicting the potential beneficial mutation sites. Results showed that V149D&I172G was the strongest double mutant (589.67 U/mg), and the triple mutant N173G&N169G&I148G exhibited 3.60 and 5.57 times higher hydrolytic activity and catalytic efficiency towards MFA, respectively. Furthermore, the triple mutant releases 16.16 mg/g ferulic acid (FA) from de-starched wheat bran (DSWB) in synergy with xylanase, which was increased by 9.34 times than that released by DLFae4 alone, and was the highest reported release amount. The double mutant V251Y&A341G with weakened hydrolytic activity but exhibited 4.30 times higher acyltransferase activity than that of DLFae4, and the corresponding acyl transfer efficiency improved by 11.37 times, representing the highest acyltransferase activity to date. The results from the current investigation demonstrate that combining multiple rational design methods is a robust pipeline for improving enzyme activities and provides new insights for enzyme modification.