Display of a novel carboxylesterase CarCby on Escherichia coli cell surface for carbaryl pesticide bioremediation

Carbamate pesticides have been widely used in agricultural and forestry pest control. The large-scale use of carbamates has caused severe toxicity in various systems because of their toxic environmental residues. Carbaryl is a representative carbamate pesticide and hydrolase/carboxylesterase is the initial and critical enzyme for its degradation. Whole-cell biocatalysts have become a powerful tool for environmental bioremediation. Here, a whole cell biocatalyst was constructed by displaying a novel carboxylesterase/hydrolase on the surface of Escherichia coli cells for carbaryl bioremediation. The carCby gene, encoding a protein with carbaryl hydrolysis activity was cloned and characterized. Subsequently, CarCby was displayed on the outer membrane of E. coli BL21(DE3) cells using the N-terminus of ice nucleation protein as an anchor. The surface localization of CarCby was confirmed by SDS–PAGE and fluorescence microscopy. The optimal temperature and pH of the engineered E. coli cells were 30 °C and 7.5, respectively, using pNPC4 as a substrate. The whole cell biocatalyst exhibited better stability and maintained approximately 8-fold higher specific enzymatic activity than purified CarCby when incubated at 30 °C for 120 h. In addition, ~ 100% and 50% of the original activity was retained when incubated with the whole cell biocatalyst at 4 ℃ and 30 °C for 35 days, respectively. However, the purified CarCby lost almost 100% of its activity when incubated at 30 °C for 134 h or 37 °C for 96 h, respectively. Finally, approximately 30 mg/L of carbaryl was hydrolyzed by 200 U of the engineered E. coli cells in 12 h. Here, a carbaryl hydrolase-containing surface-displayed system was first constructed, and the whole cell biocatalyst displayed better stability and maintained its catalytic activity. This surface-displayed strategy provides a new solution for the cost-efficient bioremediation of carbaryl and could also have the potential to be used to treat other carbamates in environmental bioremediation.