Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method.

ABSTRACT: In the present study, a feruloyl esterase DLFae4 identified in our previous research was modified by error-prone PCR and site-directed saturation mutation to enhance the catalytic efficiency and acyltransferase activity further. Five mutants with 6.9-118.9% enhanced catalytic activity toward methyl ferulate (MFA) were characterized under the optimum conditions. Double variant DLFae4-m5 exhibited the highest hydrolytic activity (270.97 U/mg), the Km value decreased by 83.91%, and the Kcat/Km value increased by 6.08-fold toward MFA. Molecular docking indicated that a complex hydrogen bond network in DLFae4-m5 was formed, with four of five bond lengths being shortened compared with DLFae4, which might account for the increase in catalytic activity. Acyl transfer activity assay r

SUBMITTER: Li L 

PROVIDER: S-EPMC10048530 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature