The biochemical properties of a novel paraoxonase-like enzyme in Trichoderma atroviride strain T23 involved in the degradation of 2,2-dichlorovinyl dimethyl phosphate

Abstract

Dichlorvos, is a broad-spectrum organophosphorus pesticide that is widely applied in the agricultural industry and considered a pollutant to fish and bees. T. atroviride strain T23, an efficient DDVP-degrading strain, could convert DDVP to dichloroacetic acid, 2,2-dichloroethanol and phosphoric acid through mineralization. RT-qPCR analysis showed TaPon1-like encoding an organophosphorus hydrolase, is continuously highly expressed in the process of degrading DDVP. TaPon1-like contained an open reading frame of 1317 bp, and the deduced amino acid sequence shared 21% homology with HuPON1, which also exhibits excellent hydrolysis of organophosphate-oxons compounds. By analysis of gene knockout, we found the {Delta}TaPon1-like knockout strain KO1 lost 35.6% of its DDVP-degradation capacity at 24 h, but this loss of degradation activity was recovered when the gene was complemented. Furthermore, the purified recombinant protein reTAPON1-LIKE, could transform DDVP only to dimethyl phosphate and showed significant paraoxonase activity (1028 U L-1). The reTAPON1-LIKE enzyme showed a broad degradation spectrum, degrading not only DDVP but also organophosphate-oxons and lactone. The kinetic parameters (Km and kcat) of the purified reTAPON1-LIKE were determined to be 0.23 mM and 204.3 s-1 for DDVP, respectively. The highest activity was obtained at 35 {degrees}C, and the optimal pH was 8.5. The activity of reTAPON1-LIKE was enhanced most significantly when 1.0 mM Ca2+ was added but declined when 1.0 mM Cu2+ was added. These results showed TAPON1-LIKE play an important role for DDVP degradation in the first step by T23 and provided clue to comprehensively understanding the degradation mechanism of organophosphate-oxons pesticides by filamentous fungi.\n\nImportanceThe large amounts of residues of organophosphate pesticides in agroecological system has become a great threat to the safety of environment and humans. Bioremediation in association with microbial is innovative technology having a potential to alleviate such pollution problems. The genus Trichoderma is genetically diverse with capabilities to degrade chemical pesticides among different strains with agricultural significance. As a typical organophosphorus pesticide, it is one of the most employed compounds of the family. Though it was classified as a highly toxic pesticide by WHO due to its hazardous properties, it plays an important role in the control of plant pests, food storage and homes, as well as to treat infections in livestock. Therefore, we use DDVP as a model of organophosphate pesticide to study the mechanism of Trichoderma degrading organophosphate pesticides, for the aim of globally understanding molecular mechanism of enzymatic degradation of organophosphate pesticides by beneficial fungi.