Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Abstract

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70±13.19 s −1 , which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser) 5 -] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 μL hemolymph from an infected shrimp and differentiated it from a healthy shrimp.

Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

“…Another report stated that over 90% of maximum enzyme activity was retained between pH 7 and 9 and approximately 85% of the maximum activity can be maintained at both pH 6.86 and 9.18 [26], which is obviously different from the results in this manuscript. However Yang et al [35] reported that the optimal pH of MPH is 11.0. This discrepancy may be attributed to the different bioengineering methods used in enzyme preparation and the underlying reasons need further investigation.…”

A S‐Layer Protein of Bacillus anthracis as a Building Block for Functional Protein Arrays by In Vitro Self‐Assembly

“…In particular, it is a monomer in solution and its 3D structure is known. Because of these features, MPH is recommended as a new analytical enzyme …”

Identification of New Genes Related to Virulence of <i>Xanthomonas axonopodis</i> Pv. <i>Citri</i> during Citrus Host Interactions

“…fuscans. The methyl parathion hydrolase (MPH) protein is an enzyme that catalyzes the degradation of a wide range of organophosphate compounds, among them the methyl parathion, which is one of the most commonly used organophosphate fertilizers worldwide, especially in fruit and vegetable farming [54].…”