Purification and characterization of a novel fenamiphos hydrolysing enzyme from Microbacterium esteraromaticum MM1.

Fenamiphos is a neurotoxic organophosphorus pesticide used widely to control pests of crops. Fenamiphos and its toxic oxidation products have been detected in surface and groundwaters. A novel enzyme capable of hydrolysing P-O-C bond of fenamiphos is purified from Microbacterium esteraromaticum MM1 total cellular protein using a combination of methods. The purified fenamiphos hydrolysing enzyme (FHE) was identified as enolase (phosphopyruvate hydratase), a housekeeping enzyme with molecular mass and pI value of 45 kDa and 4.5, respectively. The optimum pH and temperature for the activity of the FHE are 7 and 25 °C, respectively. We studied the influence of metal ions and inhibitors on the enzyme activity. The enzyme was strongly activated by Mg2+ whereas Hg2+ and phenylmethyl sulfonyl fluoride (PMSF) inhibited the enzyme. The kinetic parameters, Km and Vmax for fenamiphos hydrolysis were estimated to be 584.15 ± 16.22 μM and 6.46 ± 0.13 μM min-1, respectively. The FHE was functionally active against its original substrate (2-phosphoglycerate) with Km value of 5.82 ± 1.42 μM and Vmax of 4.2 ± 0.1 μM min-1. This enzyme has great potential for its application in the detoxification of fenamiphos and its warfare homologs. To our knowledge, this is the first report on the purification of fenamiphos hydrolysing enzyme.