Cutinase inhibition by means of insecticidal organophosphates and carbamates.: 3.: Oxidation of phosphorothionates by chloroperoxidase from Caldariomyces fumago

Chloroperoxidase (CPO) from Caldariomyces fumago combined with hydrogen peroxide and chloride proved to be most efficient for the transformation of organophosphorothionate pesticides, i.e., chlorpyrifos, chlorpyrifos-methyl, parathion, and parathion-methyl, into their more potent serine esterase inhibiting oxon analogues. Following CPO pre-oxidation steps, the detection limit of a recently described spectrophotometric cutinase assay could be increased by about 2 orders of magnitude as a consequence of increased inhibition rates of the organophosphates. This type of enzymatic oxidation is easier to perform and more efficient, as compared to bromine or N-bromosuccinimide, used for acetylcholine esterase (AChE) assay in water analyses, but is insufficient for complex matrices such as plant sample extracts. The performance of a complete assay, including sample preparation, oxidation, and inhibition, takes about 3 h. Performing oxidations of organophosphorus compounds, two significant anomalies were observed. Upon CPO oxidation, chlorpyrifos-methyl showed a very strong cutinase inhibition as compared to the corresponding oxon standard, and oxidized malathion, contrarily to malaoxon, revealed cutinase inhibition, which however obeyed a reversible reaction mechanism in contrast to the usually irreversible reactions of organophosphates. Except for methomyl, no significant effects of CPO oxidation on the inhibition strength of insecticidal carbamates could be detected. The applicability of the assay was tested with fruit samples spiked with chlorpyrifos at 0.2-0.5 mg/kg, thereby regarding the role of the latter as the pesticide detected most often in fruits. Mean recoveries ranged between 30-50%. An enhanced recovery of 84% was obtained for an apple juice sample spiked with parathion-methyl (0.5 mg/L).