微生物降解有机磷农药酶促机制

有机磷农药污染严重,微生物有机磷农药是治理有机磷农药残留的新技术,综述有机磷农药降解酶的研究现状、酶促作用机理、基因工程等方面的研究现状。     

有机磷农药降解菌及其基因工程研究新进展

有机磷农药是目前我国使用量最大的农药,对农业的发展有重要的作用,但同时造成了严重的环境污染.利用微生物及其产生的降解酶来降解农药是行之有效的方法.随着分子生物学技术的深入利用,农药的微生物降解已在基因工程领域取得了很大进展.本文综述了有机磷农药降解微生物的筛选、降解酶和基因的克隆、基因工程菌的构建以及应用等几个方面的研究进展.     

有机磷农药微生物降解研究进展

微生物降解是有机磷农药在环境中去毒降解的主要方式,是治理环境污染的一项有效手段。该文综述了有机磷农药降解菌的分离鉴定、降解机理与代谢途径、降解基因的克隆及表达、降解菌制剂和酶制剂的应用、以及有机磷农药微生物降解研究趋势五个方面的研究现状。     

转基因表达OPH提高番茄果实降解有机磷农药能力的研究

拟通过基因工程提高番茄果实降解有机磷农药残留的能力。构建了E8启动子基因驱动有机磷降解基因(OPD)的植物表达载体pSE8OP,经农杆菌介导遗传转化番茄子叶后,进行GUS染色、PCR和Southern blotting分析。证明OPD基因已整合进转基因植株基因组中,为1个拷贝。HPLC比较分析发现,转基因番茄果实能显著提高降解毒死蜱和对硫磷的能力,大大减少了番茄中的农药残留。     

海洋微生物有机磷降解酶的纯化与性质研究

从长期受有机磷农药污染的海水中分离得到1株能高效降解农药的芽胞杆菌M-1,通过离子交换层析、凝胶过滤层析等方法从发酵液中分离纯化了有机磷农药降解酶,SDS-PAGE测得该酶的分子质量约为45 kD。酶反应最适pH为7.5,最适反应温度为30℃,30℃下保温30 min,酶活力基本不变,高于30℃酶活力则迅速下降;K 、Na 、Ca2 、Mn2 对酶活性有促进作用,Hg2 、Zn2 和Cu2 等对酶有抑制作用。     

有机磷水解酶及其在传感器应用中的研究进展

有机磷农药的大规模使用对环境造成了严重污染, 同时由于其残留严重威胁着人类健康。有机磷水解酶是一种广泛存在于生物体内的可以催化各种有机磷化合物水解的酶。利用有机磷水解酶制成的生物传感器能够有效检测有机磷农药的残留。文章分别从有机磷水解酶的结构、重组表达以及在生物传感器应用等方面进行了综述, 旨在为有机磷农药的检测和降解提供参考。     

微生物降解有机磷农药污染的研究进展

有机磷农药严重污染生态环境,微生物降解是治理有机磷农药污染的新技术,综述了降解有机磷农药污染的微生物种类、降解的机理、应用、存在的问题及今后研究方向。     

有机磷农药降解酶基因工程菌研究新进展

有机磷农药,因其具有杀虫效率高,防治范围广,成本低等特点,而成为我国目前使用量最大的农药,对农业的发展有着重要作用,但同时它也造成了严重的环境污染。利用微生物或酶制剂来降解有机磷农药,是近年来从事酶基因工程研究人员的又一重大课题。在我国,继研发基因工程植酸酶、乳糖酶等酶制剂取得重要突破并实现规模化生产之后,中国农业科学院生物技术研究所范云六院士、伍宁丰研究员课题组,在有机磷农药降解酶制剂基因工程研究方面也取得了重要进展。     

基因工程技术在降解农药中的应用

综述了基因重组、原生质体融合、外源基因、降解酶和固相反应器等基因工程技术在降解农药中的研究及应用进展,提出了几种高效降解农药的基因工程菌的构建策略和构建手段。     

细菌表面展示技术在有机磷农药降解中的应用

通过微生物代谢作用来生物降解有机磷农药被认为是安全有效的途径,而近年来发展的细胞表面展示技术,由于是将农药降解酶定位在细胞表面,具有提高反应效率、可再生和减少对细胞的毒害等多种优点,从而为有机磷农药的生物降解与转化提供了一种新的技术对策,已显示出广阔的应用前景。     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.     

The Influence of Enzymatic Removal of Chlorpyrifos from Feed Grain Mixes on Biochemical Parameters of Rat Blood

Organophosphorus pesticides (OP) are used to protect crops from pests. Treatment of plants and animals with pesticides can be done during their growth or creation of conditions required for the long-shelf life of the agricultural products. Currently, many remedies exist for prevention and removal of intoxication consequences developed in living organisms exposed to OPs. The development of biologics for degradation of OPs and biotechnologies for their application in agriculture still represents an important task. New biologics based on stabilized forms of such enzyme as hexahistidine-tagged organophosphorus hydrolase (His₆-OPH) have been in the form of nano-sized particles for OPs detoxification. They represent enzyme-polyelectrolyte complexes (EPC) obtained by mixing solutions of His₆-OPH and polyanion under certain conditions. The main purpose of this work was to evaluate the usage efficiency of EPC based on His₆-OPH and polyglutamic acid for OPs detoxification by analyzing biochemical blood parameters of rats fed with a grain mix containing chlorpyrifos. The experiment was conducted using female Sprague-Dowly albino rats. Treatment of the feeding grain mix initially containing chlopyrifos (48 mg/kg of the mix) with EPC based on His₆-OPH (1000 U/kg of the mix) for 24 h was the most effective. The results showed that acetyl cholinesterase activity in blood of rats from the group consuming food after the enzymatic removal of chlorpyrifos, was comparable to acetyl cholinesterase activity in blood of rats consuming pure food.     

Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters

Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.     

Biochemistry of Esterases Associated with Organophosphate Resistance in Lucilia cuprina with Comparisons to Putative Orthologues in Other Diptera

Esterase activities associated with organophosphate insecticide resistance in the Australian sheep blowfly, Lucilia cuprina, are compared with similar activities in other Diptera. The enzymes making the major contribution to methyl butyrate hydrolysis (ali-esterase) in L. cuprina, M. domestica, and D. melanogaster comigrate during electrophoresis. The enzymes in L. cuprina and D. melanogaster correspond to the naphthyl acetate hydrolyzing E3 and EST23 isozymes of those species. These and previously published data suggest that the ali-esterases of all three species are orthologous. Strains of L. cuprina fall into four groups on the basis of quantitative determinations of their ali-estesterase, OP hydrolase, and malathion carboxylesterase activities and these groups correspond to their status with respect to two types of OP resistance. Strains susceptible to OPs have high ali-esterase, low OP hydrolase, and intermediate MCE activities; those resistant to malathion but not diazinon have low ali-esterase, intermediate OP hydrolase, and high MCE activities; those resistant to diazinon but not malathion have low ali-esterase, high OP hydrolase, and low MCE activities; those resistant to both OPs have low ali-esterase, high OP hydrolase, and high MCE activities. The correlated changes among the three biochemical and two resistance phenotypes suggest that they are all properties of one gene/enzyme system; three major allelic variants of that system explain OP susceptibility and the two types of OP resistance. Models are proposed to explain the joint contribution of OP hydrolase and MCE activities to malathion resistance and the invariant association of low ali-esterase and elevated OP hydrolase activities in either type of resistance.     

An isofenphos-methyl hydrolase (Imh) capable of hydrolyzing the P–O–Z moiety of organophosphorus pesticides containing an aryl or heterocyclic group

Organophosphorus pesticide (OP) hydrolases play key roles in the degradation and decontamination of agricultural and household OPs and in the detoxification of chemical warfare agents. In this study, an isofenphos-methyl hydrolase gene (imh) was cloned from the isocarbophos-degrading strain of Arthrobacter sp. scl-2 using the polymerase chain reaction method. Isofenphos-methyl hydrolase (Imh) showed 98% sequence identity with the isofenphos hydrolase from Arthrobacter sp. strain B-5. Imh was highly expressed in Escherichia coli BL21 (DE3), and the His(6)-tagged Imh was purified (1.7 mg/ml) with a specific activity of 14.35 U/mg for the substrate isofenphos-methyl. The molecular mass of the denatured Imh is about 44 kDa, and the isoelectric point (pI) value was estimated to be 3.4. The optimal pH and temperature for hydrolysis of isofenphos-methyl were pH 8.0 and 35 °C, respectively. The secondary structure of Imh shows that Imh is a metallo-dependent hydrolase, and it was found that Imh was completely inhibited by the metalloprotease inhibitor 1,10-phenanthroline (0.5 mM), and the catalytic activity was restored by the subsequent addition of Zn(2+). Interestingly, Imh had a relatively broader substrate specificity and was capable of hydrolyzing 12 of the tested oxon and thion OPs with the P-O-Z moiety instead of the P-S(C)-Z moiety. Furthermore, it was found that the existence of an aryl or heterocyclic group in the leaving group (Z) is also important in determining the substrate specificity. Among all the substrates hydrolyzed by Imh, it was assumed that Imh preferred P-O-Z substrates still with a phosphamide bond (P-N), such as isofenphos-methyl, isofenphos, isocarbophos, and butamifos. The newly characterized Imh has a great potential for use in the decontamination and detoxification of agricultural and household OPs and is a good candidate for the study of the catalytic mechanism and substrate specificity of OP hydrolases.     

Enhanced biodegradation of toxic organophosphate compounds using recombinant Escherichia coli with sec pathway-driven periplasmic secretion of organophosphorus hydrolase

Although Escherichia coli can be genetically engineered to degrade environmental toxic organophosphate compounds (OPs) to nontoxic materials, a critical problem in such whole cell systems is limited substrate diffusion. The present work examined whether periplasmic expression of organophosphorus hydrolase (OPH) resulted in better whole cell enzymatic activity compared to standard cytosolic expression. Recombinant OPH periplasmic expression was achieved using the general secretory (sec) pathway with the pelB signal sequence. We found that while total OPH activity in periplasmic-expressing cell lysates was lower compared to that in cytosolic-expressing cell lysates whole cell OPH activity was 1.8-fold greater at 12 h post-induction in the periplasmic-expressing cells as a result of OPH translocation into the periplasmic space ( approximately 67% of whole cell OPH activity was found in the periplasmic fraction). These data suggest that E. coli engineered to periplasmically secrete OPH via the sec pathway may provide an improved whole cell biodegradation system for destruction of environmental toxic OPs.     

Organophosphorus hydrolase multilayer modified microcantilevers for organophosphorus detection

We report a biosensor based on organophosphorus hydrolase (OPH) multilayer modified microcantilever (MCL) for detection of organophosphorus compounds (OPs). The assay is based on substrate-dependent bending of the OPH functionalized MCLs. The cantilever bending amplitude at equilibrium was a function of the concentration of paraoxon with the dynamic range extending from 10(-7) to 10(-3)M. The lower detection limit of approximately 10(-7)M for paraoxon was an order of magnitude better than the OPH-based potentiometric and optical biosensors based on pH modulation. There was a good intra-sensor and an acceptable inter-sensor reproducibility as evidenced by the standard errors of 5% and 15%, respectively. OPs measured using this technique included parathion and diisopropyl fluorophosphate (DFP) in the order of paraoxon>DFP>parathion. The conformational change of the OPH was most likely the main origin of MCL bending.     

Structural and Enzymatic Characterization of the Phosphotriesterase OPHC2 from Pseudomonas pseudoalcaligenes

Background

Organophosphates (OPs) are neurotoxic compounds for which current methods of elimination are unsatisfactory; thus bio-remediation is considered as a promising alternative. Here we provide the structural and enzymatic characterization of the recently identified enzyme isolated from Pseudomonas pseudoalcaligenes dubbed OPHC2. OPHC2 belongs to the metallo-β-lactamase superfamily and exhibits an unusual thermal resistance and some OP degrading abilities.

Principal findings

The X-ray structure of OPHC2 has been solved at 2.1 Å resolution. The enzyme is roughly globular exhibiting a αβ/βα topology typical of the metallo-β-lactamase superfamily. Several structural determinants, such as an extended dimerization surface and an intramolecular disulfide bridge, common features in thermostable enzymes, are consistent with its high T_m (97.8°C). Additionally, we provide the enzymatic characterization of OPHC2 against a wide range of OPs, esters and lactones.

Significance

OPHC2 possesses a broad substrate activity spectrum, since it hydrolyzes various phosphotriesters, esters, and a lactone. Because of its organophosphorus hydrolase activity, and given its intrinsic thermostability, OPHC2 is an interesting candidate for the development of an OPs bio-decontaminant. Its X-ray structure shed light on its active site, and provides key information for the understanding of the substrate binding mode and catalysis.     

Occurrence of osmiophilic particles is correlated to elongation growth of higher plants

Summary The occurrence of elongation growth-related osmiophilic particles (OPs) was investigated in hypocotyls of sunflower, bean, and spruce as well as in pea epicotyls and in cress roots of intact seedlings. In all analyzed species, OPs were found to occur specifically within the periplasmic space between plasma membrane and the outer epidermal cell walls of elongating parts of hypocotyls, epicotyls, and roots, whereas cells of nonelongating parts were devoid of OPs. Auxin (IAA) markedly increased the number of OPs in epicotyl and hypocotyl segments. Treatment of pea epicotyl segments with the lectin concanavalin A inhibited their elongation growth in the presence of IAA. At a subcellular level this effect was characterized by the occurrence of a pronounced osmiophilic layer in the periplasmic space of the outer periclinal and the outer part of the anticlinal epidermal cell walls. Treatment of IAA-incubated segments with the secretion inhibitor brefeldin A inhibited both elongation growth and periplasmic occurrence of OPs. This effect was accompanied by complementary accumulation of OPs in the peripheral cytoplasm of epidermal cells. Together the results indicate that IAA-induced epidermis-specific secretion of OPs is closely related to cell elongation growth not only in organs of monocotyledonous species, but also in dicotyledonous angiosperms as well as in gymnosperms.Abbreviations OPs osmiophilic particles - ConA concanavalin A - BFA brefeldin A - IAA -indolyl acetic acid     

Mass spectral characterization of organophosphate-labeled lysine in peptides

Organophosphate (OP) esters bind covalently to the active site serine of enzymes in the serine hydrolase family. Recently, mass spectrometry identified covalent binding of OPs to tyrosine in a wide variety of proteins when purified proteins were incubated with OPs. In the current work, manual inspection of tandem mass spectrometry (MS/MS) data led to the realization that lysines also make a covalent bond with OPs. OP-labeled lysine residues were found in seven proteins that had been treated with either chlorpyrifos oxon (CPO) or diisopropylfluorophosphate (DFP): human serum albumin (K212, K414, K199, and K351), human keratin 1 (K211 and K355), human keratin 10 (K163), bovine tubulin alpha (K60, K336, K163, K394, and K401), bovine tubulin beta (K58), bovine actin (K113, K291, K326, K315, and K328), and mouse transferrin (K296 and K626). These results suggest that OP binding to lysine is a general phenomenon. Characteristic fragments specific for CPO-labeled lysine appeared at 237.1, 220.0, 192.0, 163.9, 128.9, and 83.9 amu. Characteristic fragments specific for DFP-labeled lysine appeared at 164.0, 181.2, and 83.8 amu. This new OP-binding motif to lysine suggests new directions to search for mechanisms of long-term effects of OP exposure and in the search for biomarkers of OP exposure.