Cutinase Inhibition by Means of Insecticidal Organophosphates and Carbamates. 3. Oxidation of Phosphorothionates by Chloroperoxidase from Caldariomyces fumago

Abstract

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).

Enzymatic detoxification of organophosphorus pesticides and related toxicants

“…126,132) These oxon derivatives are known to be more toxic than the original pesticide. 133) On the other hand, Versatile Peroxidase involved in lignin degradation was able to transform several pesticides mostly halogenated compounds, and no transformation of organophosphorus pesticide could be detected. 134) Thus, the peroxidase capacity to transform pesticides could not be envisaged for OPP detoxification purposes.…”

Tailoring biosensors for Brazil: Detection of insecticides in foods

“…However, this method revealed itself unsuitable for the analysis of food samples, because of the excess of oxidizable compounds like ascorbic acid or other antioxidants present in certain foods (Schulze, Schmid, & Bachmann, 2004). Besides, due to a restricted water solubility of NBS, increasing this oxidant's concentration is rather limited (Walz & Schwack, 2007). This could then justify the absence of stronger inhibition rates in the biosensor analysis than rather expected.…”

Effect of bromine oxidation on high-performance thin-layer chromatography multi-enzyme inhibition assay detection of organophosphates and carbamate insecticides

“…Choline esterases from different animal sources have been used in cuvette, microtiterplate [10][11][12][13], biosensor [14,15], and thin-layer chromatography (TLC) based assays [16]. In addition to choline esterases, microtiterplate multi-enzyme inhibition assays using rabbit liver esterase (RLE), Bacillus subtilis (BS2) esterase, or cutinase (from Fusarium solani pisi) have been previously reported [17][18][19][20], and recently have been successfully incorporated in highperformance thin-layer chromatography-enzyme inhibition assays (HPTLC-EI) [21,22].…”

“…Although N-bromosuccinimide has been used for water sample testing in choline esterase inhibition assays [29], this reagent was not effective in tests of organic matrices, such as plant food [17]. In food sample extracts, enzymatic oxidation by chloroperoxidase has been shown to be a suitable alternative [17], which recently was directly applied for testing fruit juice samples, coupled with biosensor detection [30].…”

“…In food sample extracts, enzymatic oxidation by chloroperoxidase has been shown to be a suitable alternative [17], which recently was directly applied for testing fruit juice samples, coupled with biosensor detection [30]. For TLC/HPTLC based assays, oxidation by both bromine vapour and UV irradiation were the most commonly used procedures, although bromine was described to be more efficient than UV irradiation in converting pesticides to more potent inhibitors [31].…”