Engineering a novel carbaryl-degrading esterase from Bacillus velezensis for enhanced degradability via semi-rational design and whole-cell biocatalysis

The carbamate pesticide carbaryl poses significant ecological and health risks due to its widespread misuse and improper long-term disposal. In this study, we engineered a novel bacterial carbaryl-degrading esterase to enhance enzymatic activity, thermostability, and carbaryl degradation through semi-rational design and whole-cell biocatalysis strategies. An esterase, designated Est03320, was identified from Bacillus velezensis MB01B. Phylogenetic analysis revealed it resides in a distinct clade. However, it contains a catalytic domain similar to those of the well-characterized carbaryl-hydrolyzing esterase CarH and the chlorpyrifos-degrading esterase MPH. Est03320 was expressed and purified from Escherichia coli BL21(DE3) cells, demonstrating high hydrolytic activity against short-chain p-nitrophenyl esters and carbaryl. The enzyme exhibits an optimum pH of 6.5 and an optimal temperature of 25 °C, with a K_m of 139.70 µM and a V_max of 29.47 µM/min. The mutant Est03320-L230G, engineered through a combined semi-rational design and site-directed mutagenesis approach, exhibited a 68.7% increase in enzymatic activity toward the substrate p-nitrophenyl acetate, an extended half-life at 25 °C from 13.6 h to 40.6 h, and a 96.5% enhancement in carbaryl degradation efficiency. To construct a whole-cell biocatalyst, this mutant esterase was further fused with the autotransporter EhaA and displayed on the surface of engineered Pseudomonas sp. MB04R-14-03320-L230G-EhaA cells. The resulting biocatalyst showed high efficiency in degrading and mineralizing carbaryl, retaining over 83% of its activity after five consecutive degradation cycles. This integrated approach significantly enhances both enzymatic performance and reusability, offering a sustainable and effective approach for mitigating carbaryl pollution and advancing environmental protection.