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<p>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 <i>Bacillus velezensis</i> 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 <i>Escherichia coli</i> BL21(DE3) cells, demonstrating high hydrolytic activity against short-chain <i>p</i>-nitrophenyl esters and carbaryl. The enzyme exhibits an optimum pH of 6.5 and an optimal temperature of 25&#xa0;°C, with a <i>K</i><sub><i>m</i></sub> of 139.70 µM and a <i>V</i><sub><i>max</i></sub> 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 <i>p</i>-nitrophenyl acetate, an extended half-life at 25&#xa0;°C from 13.6&#xa0;h to 40.6&#xa0;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 <i>Pseudomonas</i> 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.</p>

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

  • 期刊论文

  • 发表日期：

    2025年10月23日

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  • Jiaoqing Li,
  • Guohao Huang,
  • Jie Zeng,
  • Xiaodong Wang,
  • Tan Wang,
  • Lin Li

摘要

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 Km of 139.70 µM and a Vmax 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.