有机磷降解酶在重组大肠杆菌中的表达及一步纯化固定化

【背景】有机磷化合物作为一类广谱杀虫剂,因其用量大、毒性强且不易降解,在自然界中的残留已对环境造成了严重污染。【目的】有机磷降解酶(organophosphohydrolase,OpdA)可降解多种有机磷化合物,探究其被固定到NiCo_2O_4载体上后用于有机磷化合物的降解效果。【方法】构建含组氨酸标签(histidine tag,His-tag)的OpdA,以pET-28a(+)为载体,Escherichia coli Rosetta(DE3)为宿主细胞,在终浓度为1.0 mmol/L的IPTG诱导下表达His-tagged OpdA。采用一步纯化固定化方法,实现固定化酶(OpdA@NiCo_2O_4)的制备。【结果】采用水热处理和煅烧制备了含过渡金属离子的NiCo_2O_4,利用过渡金属离子对酶分子表面组氨酸咪唑基的配位作用,实现了发酵粗酶液中His-taggedOpdA的一步纯化固定化,在优化条件下获得了高稳定性的OpdA@NiCo_2O_4;然后将其用于有机磷化合物的降解,在NaBH_4存在条件下,通过级联反应和降解条件优化,实现了有机磷化合物的高效降解。【结论】该研究不但实现了重组酶的一步分离纯化和固定化,也为有机磷化合物的降解提供了一条安全、高效、环保的新途径。     

有机磷水解酶的表面显示技术研究进展

有机磷化合物在农业中的广泛应用,给农作物带来增产的同时对环境造成污染,严重威胁着人类的健康,其生物解毒已受到高度重视.有机磷水解酶(OPH)是目前处理有机磷化合物最有效的水解酶类,而通过基因工程手段获得高表达、高降解效率的OPH,尤其是OPH的表面显示技术是近年来的研究热点.主要综述了大肠杆菌、假单胞菌、酿酒酵母的OPH表面显示技术研究及应用进展和发展趋势.     

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。     

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。     

多功能降解菌Pseudomonas putida KT2440-DOP的构建与降解特性研究

三唑磷水解酶基因为研究发现的一个新的广谱有机磷水解酶基因,通过PCR从有机磷降解菌株Ochrobactrumsp.mp-4总DNA扩增了tpd,将tpd定向克隆到pBBRMCS-5载体上,构建重组质粒pTPD,在辅助质粒pRK2013的帮助下,通过三亲接合将pTPD转移到模式菌株Pseudomonas putidaKT2440中,获得的工程菌PseudomonasputidaKT2440-DOP可以降解多种有机磷农药及芳香烃化合物;KT2440-DOP的有机磷水解酶活较出发菌株MP-4提高了一倍左右,且遗传性状稳定。     

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。     

一种降解农药和致癌物的土壤细菌

<正> 发现一种叫做缺陷假单胞菌(PD)的常见土壤细菌能够把有机磷农药对硫磷和地亚农降解成毒性较小的产物,并还能把致癌化合物苯并蒽降解成二氧化碳和水溶性产物。美国得克萨斯大学的研究人员在美国微生物学会于3月3日～7日在内华达拉斯维加斯召开的年会上介绍了这些研究结果。     

对氧磷酶及其生理功能的研究进展

对氧磷酶是酯酶的一种,可能和人类的某些心血管疾病,如动脉粥样硬化的发生有关,研究证实,对氧磷酶还具有对抗细菌内毒素的毒性及降解有机磷化合物类神经毒剂的作用。     

有机磷生物修复研究进展

目前,有机磷的生物修复还主要是微生物修复。但是植物修复更具优越性,因其花费更少、对环境更安全。然而植物对生长条件的要求相对较高,修复效率较低,应用还非常有限。本文综述了有机磷微生物修复和植物修复的研究进展,总结了已知的有机磷降解酶及其生物来源。结果表明,植物材料的筛选、土壤与OPs作用机理的研究、植物耐受和消除OPs的基因组学研究、植物-微生物联合降解体系的建立以及降解酶的植物根系分泌系统的利用是提高有机磷植物修复效率的重要途径。     

有机磷农药的微生物降解研究进展

微生物因种类和代谢多样性在有机磷农药降解中表现出独特的优势。对微生物降解有机磷农药的机制、微生物的获得、基因工程菌的构建及研究展望进行了综述。     

Microbial degradation of pollutants at high salt concentrations

Though our knowledge on microbial degradation of organic pollutants at high salt concentrations is still limited, the list of toxic compounds shown to be degraded or transformed in media of high salinity is growing. Compounds transformed aerobically include saturated and aromatic hydrocarbons (by certain archaeobacteria), certain aromatic compounds, organophosphorus compounds, and formaldehyde (by halotolerant eubacteria). Anaerobic microbial transformations of toxic compounds occurring at high salt concentrations include reduction of nitroaromatic compounds, and possibly transformation of chlorinated aromatic compounds.     

Purification and properties of an organophosphorus acid anhydrase from a halophilic bacterial isolate.

A moderately halophilic bacterial isolate has been found to possess high levels of enzymatic activity against several highly toxic organophosphorus compounds. The predominant enzyme, designated organophosphorus acid anhydrase 2, has been purified 1,000-fold to homogeneity and characterized. The enzyme is a single polypeptide with a molecular weight of 60,000. With diisopropylfluorophosphate as a substrate, the enzyme has optimum activity at pH 8.5 and 50 degrees C, and it is stimulated by manganese and cobalt.     

Nothing lasts forever: understanding microbial biodegradation of polyfluorinated compounds and perfluorinated alkyl substances

Poly- and perfluorinated chemicals, including perfluorinated alkyl substances (PFAS), are pervasive in today’s society, with a negative impact on human and ecosystem health continually emerging. These chemicals are now subject to strict government regulations, leading to costly environmental remediation efforts. Commercial polyfluorinated compounds have been called ‘forever chemicals’ due to their strong resistance to biological and chemical degradation. Environmental cleanup by bioremediation is not considered practical currently. Implementation of bioremediation will require uncovering and understanding the rare microbial successes in degrading these compounds. This review discusses the underlying reasons why microbial degradation of heavily fluorinated compounds is rare. Fluorinated and chlorinated compounds are very different with respect to chemistry and microbial physiology. Moreover, the end product of biodegradation, fluoride, is much more toxic than chloride. It is imperative to understand these limitations, and elucidate physiological mechanisms of defluorination, in order to better discover, study, and engineer bacteria that can efficiently degrade polyfluorinated compounds.     

Molecular bases of aerobic bacterial degradation of dioxins: involvement of angular dioxygenation

In the last decade, extensive investigation has been done on the bacterial degradation of dioxins and its related compounds, because this class of chemicals is highly toxic and has been widely distributed in the environment. These studies have revealed the primary importance of a novel dioxygenation reaction, called angular dioxygenation, in the aerobic bacterial degradation pathway of dioxin. Accompanied by the electron transport proteins, Rieske nonheme iron oxygenase catalyzes the incorporation of oxygen atoms to the ether bond-carrying carbon (the angular position) and an adjacent carbon, resulting in the irreversible cleavage of the recalcitrant aryl ether bond. The 2,2',3-trihydroxybiphenyl or 2,2',3-trihydroxydiphenyl ether derivatives formed are degraded through meta cleavage. In addition to the degradation system of dibenzofuran and dibenzo-p-dioxin (the nonchlorinated model compounds of dioxin), those of fluorene and carbazole were shown to function in dioxin degradation. Some dioxin degradation pathways have been studied biochemically and genetically. In addition, feasibility studies have shown that some dioxin-degrading strains can function in actual dioxin-contaminated soil. These studies provide useful information for the establishment of a bioremediation method for dioxin contamination. This review summarizes recent progress on molecular and biochemical bases of the bacterial aerobic degradation of dioxin and related compounds.     

Microbiological and Biotechnological Aspects of Metabolism of Carbamates and Organophosphates

Abstract

Several carbamate and organophosphate compounds are used to control a wide variety of insect pests, weeds, and disease-transmitting vectors. These chemicals were introduced to replace the recalcitrant and hazardous chlorinated pesticides. Although newly introduced pesticides were considered to be biodegradable, some of them are highly toxic and their residues are found in certain environments. In addition, degradation of some of the carbamates generates metabolites that are also toxic. In general, hydrolysis of the carbamate and organophosphates yields less toxic metabolites compared with the metabolites produced from oxidation. Although microorganisms capable of degrading many of these pesticides have been isolated, knowledge about the biochemical pathways and respective genes involved in the degradation is sparse. Recently, a great deal of interest in the mechanisms of biodegradation of carbamate and organophosphate compounds has been shown because (1) an efficient mineralization of the pesticides used for insect control could eliminate the problems of environmental pollution, (2) a balance between degradation and efficacy of pesticides could result in safer application and effective insect control, and (3) knowledge about the mechanisms of biodegradation could help to deal with situations leading to the generation of toxic metabolites and bioremediation of polluted environments. In addition, advances in genetic engineering and biotechnology offer great potential to exploit the degradative properties of microorganisms in order to develop bioremediation strategies and novel applications such as development of economic plants tolerant to herbicides. In this review, recent advances in the biochemical and genetic aspects of microbial degradation of carbamate and organophosphates are discussed and areas in need of further investigation identified.     

Microbiological and biotechnological aspects of metabolism of carbamates and organophosphates.

Several carbamate and organophosphate compounds are used to control a wide variety of insect pests, weeds, and disease-transmitting vectors. These chemicals were introduced to replace the recalcitrant and hazardous chlorinated pesticides. Although newly introduced pesticides were considered to be biodegradable, some of them are highly toxic and their residues are found in certain environments. In addition, degradation of some of the carbamates generates metabolites that are also toxic. In general, hydrolysis of the carbamate and organophosphates yields less toxic metabolites compared with the metabolites produced from oxidation. Although microorganisms capable of degrading many of these pesticides have been isolated, knowledge about the biochemical pathways and respective genes involved in the degradation is sparse. Recently, a great deal of interest in the mechanisms of biodegradation of carbamate and organophosphate compounds has been shown because (1) an efficient mineralization of the pesticides used for insect control could eliminate the problems of environmental pollution, (2) a balance between degradation and efficacy of pesticides could result in safer application and effective insect control, and (3) knowledge about the mechanisms of biodegradation could help to deal with situations leading to the generation of toxic metabolites and bioremediation of polluted environments. In addition, advances in genetic engineering and biotechnology offer great potential to exploit the degradative properties of microorganisms in order to develop bioremediation strategies and novel applications such as development of economic plants tolerant to herbicides. In this review, recent advances in the biochemical and genetic aspects of microbial degradation of carbamate and organophosphates are discussed and areas in need of further investigation identified.     

High-rate continuous biodegradation of concentrated chlorinated aliphatics by a durable enrichment of methanogenic origin under carrier-dependent conditions

The simultaneous biodegradation of toxic compounds in mixtures is a major current concern. To bioremediate a toxic mixture, we designed a strategy combining an ad-sorbent carrier with an ecological and nutritional system which allowed work close to heavily polluted conditions in nature. Starting from a methanogenic community, we developed a microbial consortium acclimated to a mixture of about 30 chlorinated aliphatics in a fixed-film stationary-bed bioreactor. Prior to the establishment of a durable period of dechlorination, an interval of progressive dechlorination of the toxic mixture was observed during which the excess of the toxic compounds was stored on the carrier. The latter, consisting of activated carbon in a polyurethane foam, allowed us to work at concentrations far above the solubility of the toxic compounds (apparent concentrations of about 10 g/L). The complete disappearance of hexachloroethane as well as its lower homologues, penta-, tetra-, and trichloroethane, present in the toxic mixture, was observed. Additionally, octachlorocyclopentene, carbon tetrachloride, trichloro-ethylene, tetrachloroethylene, and hexachloro-1,3-butadiene also completely disappeared. For the four latter compounds, from mass balances in the bioreactor, degradation rates around 10 mumol/L per day were determined with total dechlorination. The enrichment culture thus developed exhibited high degradation performances similar to those reported in the literature for pure or enriched anaerobic microbial cultures in contact with a single toxic compound. The results demonstrate the possibility of concurrent high-rate degradation of several highly chlorinated toxic compounds, under conditions approximating field situations.(c) 1995 John Wiley & Sons, Inc.     

Use of NMR techniques for toxic organophosphorus compound profiling

This review presents with selected examples the versatility of nuclear magnetic resonance (NMR) spectroscopy in the analysis of toxic organophosphorus (OP) compounds, i.e. OP pesticides and chemical warfare agents (CWAs). Several NMR applications of biological importance, like studies on inhibition mechanism, metabolism, and exposure determination, are presented. The review also concerns with the environmental analysis of OP compounds by NMR spectroscopy. Residue analysis of environment and food samples as well as characterization of degradation in environment is discussed. Some of the NMR studies that have been done to support the Chemical Weapons Convention, i.e. the development of suitable CWA detoxification means and the method development of verification analysis for CWAs and their degradation products, are outlined.     

人血清中的对氧磷酶

对氧磷酶能够水解强毒性农药对氧磷而受到许多学者的重视,但国内尚未见报道.文章综述了人血清中对氧磷酶的分布、特异性、分离纯化、多态性、遗传学特性、与疾病的关系以及对有机磷化合物毒性的防护作用．这有利于进一步研究对氧磷酶的生物学性质、生理功能及其应用．     

Studies on the Mode of Action of Organophosphorus Compounds

In order to further elucidate the mechanism of metabolic difference between sumithion and methylparathion, distribution of sumithion and methylparathion into several tissues, activation, that is, conversion into more toxic oxygen analogs, and degradation into non-toxic compounds were examined in vivo following the intravenous administration of the phosphorothioates to Guinea pigs and white rats. Sumioxon and methylparaoxon were detected in all tissues tested, among which lung and liver were richest in them. More sumioxon than methylparaoxon was found. Chese organophosphorus compounds were found to be decomposed to non-toxic desmethyl compounds and dimethyl phosphorothioic acid mainly in liver and kidney. From these results it seems improbable that the lower toxicity of sumithion than that of methylparathion results from the different in their rate of metabolism.