Paraoxon organophosphorus hydrolase

Figure 1 Proposed mechanism for paraoxon hydrolysis by organophosphorus hydrolase (X and X are unspecified ligands).

The LbL technique is undoubtedly one of the best methods to incorporate biological components into man-made devices. Therefore, sensor applications must be one of the most promising subjects for LbL assemblies of biomaterials. For example, Leblanc and coworkers used several bilayers of chitosan and poly(thiophene-3-acetic acid) as cushion layers for stable enzyme films [187]. The first five bilayers of the cushion layer allowed for better adsorption of organophosphorus hydrolase than the corresponding adsorption on a quartz slide. The immobilized enzyme becomes more stable and can be used under harsher conditions. The assembled LbL films can be used for spectroscopic detection of paraoxon, an organophosphorus compound. This cushion layer strategy provides a well-defined substrate-independent interface for enzyme immobilization, in which the bioactivity of the enzyme is not compromised. This leads to fast detection of paraoxon and quick recovery times. 

Organophosphorus hydrolase (OPH, EC 3.1.8.1) is a homodimer with a binuclear metal center. OPH has broad substrate specificity and can hydrolyze organophosphate pesticides such as methyl paradiion, ediyl parathion, paraoxon, chlorpyrifos, coumaphos, cyanophos and diazinoa Table I 9,12-14). The enzymatic hydrolysis rates are 40 - 2450 times faster than chemical hydrolysis at pH 7.0 and the enzyme is reported to be stable at ten ratures of up to 4S-S0°C (3). However, hydrolysis rates varied from very fast for phosphotriesters and phosphothiolester pesticides (P-0 bond) such as paraoxon (ken > 3800s ) and coumaphos (kcat = 800s ) to limited hydrolysis for Diazinon (kcat == 176 s ) and fensulfothion (l a, = 67 s ) (14). 

Improved activity upon immobilization in mesoporous silicates was also observed by electrochemical studies. It was demonstrated that the specific activity of organophosphorus hydrolase entrapped in mesoporous amine functionalized ordered-silicate powder could reach more than twice as high as that of the free enzyme, and in a subsequent article it was reported that enzymatic sensor for paraoxon based on the immobilization of enzyme-grafted mesoporous silicate particulates in Nation film was reported [334]. 

Lei, C. H., M. M. Valenta, K. P. Saripalli, E. J. Ackerman, 2007. Biosensing paraoxon in simulated environmental samples by immobilized organophosphorus hydrolase in functionalized mesoporous silica. J Environ Quality 36 233-38. 

Other enzymes have also been immobilized on CNTs for the construction of electrochemical biosensors. Deo et al. [115] have described an amperometric biosensor for organophosphorus (OP) pesticides based on a CNT-modified transducer and OP hydrolase, which is used to measure as low as 0.15 pM paraoxon and 0.8 pM parathion with... 

The inactivation and detoxification of paraoxon and congeners are catalyzed by the so-called A-esterases, which, as discussed, comprise aryleste-rase (sometimes still called paraoxonase, EC 3.1.1.2) and phosphoric triester hydrolases (phosphotriesterases, EC 3.1.8) subdivided into aryldialkylphos-phatase (organophosphate hydrolase, paraoxonase, EC 3.1.8.1) and organophosphorus acid anhydrolases (EC 3.1.8.2 see Sect. 9.3.7) [65][69][106-108], These activities, which occur mostly in the mammalian liver and... 

Carboxylamidase activity toward p-nitroacetanilide has been detected in different insect species from the orders Lepidoptera, Orthoptera, and Dictyoptera. The carboxylamidase from fall army worm larvae has been purified. The purified enzyme is a monomer with a molecular weight of 59,000-60,000 Da. The enzyme is inhibited by the hydrolase inhibitors paraoxon, triphenyl phosphate, eserine, and phenylmethylsulfonyl fluoride, showing I50 values of 4.7 iM, 0.2 mM, 16 iM, and 90 iM, respectively. Activity is also completely inhibited by the organophosphorus insecticides profenfos and dichlorvos at 0.1 mM. The enzyme is active toward other amides, such as acetanilide and phenacetin, and various a- and p-naphtholic esters. Based on the purification factor, substrate specificity, and sensitivity to hydrolase inhibitors, the carboxylamidase appears to be different from carboxy-lesterases in the fall army worm (Yu and Nguyen, 1998). 

Arvlester Hydrolase and Parathion Hydrolase. Arylester hydrolase in mammalian serum (42.431 catalyzes very efficient hydrolysis of the organophosphorus oxons such as paraoxon (Table III). This enzyme does not hydrolyze the parent phosphorothioate insecticides such as methyl parathion, but only the oxon metabolites such as methyl paraoxon (441. This enzyme is has been referred to as phosphotriesterase however, a triester is not required as seen in the rapid hydrolysis of 10 organophosphinates by rabbit serum arylester hydrolase (44). This mechanism appears only rarely in birds... 

Organophosphorus reagents that inhibit hydrolases (serine hydrolases) diethyl p-nitrophenyl phosphate (paraoxon) diisopropyl phosphofluoridate (DFP) dimethyl(dichlorovinyl) phosphate (dichlovos) O -methylphosphoramidothioate 3-hydroxy-N-methyl-cis-crotonamide dimethylphosphate tetraethyl pyrophosphate (TEPP)... 

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