Purification and characterization of a 1,2,4-trihydroxybenzene 1,2-dioxygenase from the basidiomycete Phanerochaete chrysosporium.

1,2,4-Trihydroxybenzene (THB) is an intermediate in the Phanerochaete chrysosporium degradation of vanillate and aromatic pollutants. A P. chrysosporium intracellular enzyme able to oxidatively cleave the aromatic ring of THB was purified by ammonium sulfate precipitation, hydrophobic and ion-exchange chromatographies, and native gel electrophoresis. The native protein has a molecular mass of 90 kDa and a subunit mass of 45 kDa. The enzyme catalyzes an intradiol cleavage of the substrate aromatic ring to produce maleylacetate. 18O2 incorporation studies demonstrate that molecular oxygen is a cosubstrate in the reaction. The enzyme exhibits high substrate specificity for THB; however, catechol cleavage occurs at approximately 20% of the optimal rate. THB dioxygenase catalyzes a key step in the degradation pathway of vanillate, an intermediate in lignin degradation. Maleylacetate, the product of THB cleavage, is reduced to beta-ketoadipate by an NADPH-requiring enzyme present in partially purified extracts.     

Soybean peroxidase-catalyzed removal of phenylenediamines and benzenediols from water

Crude soybean peroxidase (SBP), isolated from soybean seed coats (hulls), catalyzes the oxidative polymerization of hazardous aqueous phenylenediamines and benzenediols in the presence of hydrogen peroxide. Experiments were conducted to investigate the optimum operating conditions including pH, hydrogen peroxide-to-substrate concentration ratio and the minimum SBP concentration required to achieve at least 95% conversion of these pollutants in synthetic wastewaters. The substrate conversion and hydrogen peroxide consumption were monitored over the period of the reactions. Polyethylene glycol (PEG) was ineffective as an additive in enhancing the conversion efficiency. The enzymatically generated polymeric products from phenylenediamines could be removed with the aid of a surfactant, sodium dodecyl sulfate (SDS), whereas the polyvalent metal cation salt, aluminum sulfate (alum), was able to remove the products from benzenediols, except hydroquinone. Enzyme-catalyzed polymerization with SBP and subsequent removal of the polymeric products generated can provide an alternative means to the conventional methods for treating many aromatic wastewater pollutants, including the title compounds.     

Crystal structure of a mono- and diacylglycerol lipase from Malassezia globosa reveals a novel lid conformation and insights into the substrate specificity

Most lipases contain a lid domain to shield the hydrophobic binding site from the water environment. The lid, mostly in helical form, can undergo a conformational change to expose the active cleft during the interfacial activation. Here we report the crystal structures of Malassezia globosa LIP1 (SMG1) at 1.45 and 2.60 ? resolution in two crystal forms. The structures present SMG1 in its closed form, with a novel lid in loop conformation. SMG1 is one of the few members in the fungal lipase family that has been found to be strictly specific for mono- and diacylglycerol. To date, the mechanism for this substrate specificity remains largely unknown. To investigate the substrate binding properties, we built a model of SMG1 in open conformation. Based on this model, we found that the two bulky hydrophobic residues adjacent to the catalytic site and the N-terminal hinge region of the lid both may act as steric hindrances for triacylglycerols binding. These unique structural features of SMG1 will provide a better understanding on the substrate specificity of mono- and diacylglycerol lipases and a platform for further functional study of this enzyme.     

Biodegradation of benzo(a)pyrene by a newly isolated Fusarium sp

Benzo(a)pyrene (BaP) is a five-ring polycyclic aromatic hydrocarbon produced by the incomplete combustion of organic materials. It is one of the priority pollutants listed by the US Environmental Protection Agency. This study describes a fungal isolate that is able to biodegrade benzo(a)pyrene. The filamentous fungus, isolated from leaves of Pterocarpus macrocarpus Kurz., was identified as a Fusarium sp. (strain E033). Fusarium sp. E033 was able to survive in the presence of benzo(a)pyrene concentrations up to 1.2 mM (300 mg L(-1)). Biodegradation experiments using 0.4 mM (100 mg L(-1)) benzo(a)pyrene demonstrated that Fusarium sp. E033 was able to degrade 65-70% of the initial benzo(a)pyrene provided, and two transformation products, a dihydroxy dihydro-benzo(a)pyrene and a benzo(a)pyrene-quinone, were detected within 30 days of incubation at 32 degrees C. The factors affecting biodegradation efficiency were also investigated. While increasing aeration promoted better fungal growth and benzo(a)pyrene biodegradation, increasing the glucose concentration from 5 to 50 mM had an adverse effect on biodegradation. Ethanol and methanol, provided at 5 mM to increase benzo(a)pyrene water solubility, increased the fungal biomass yield but did not promote degradation. The Fusarium sp. E033 isolated in this study can tolerate and degrade relatively high concentrations of benzo(a)pyrene, suggesting its potential application in benzo(a)pyrene bioremediation.     

Fungal metabolism of environmentally persistent compounds: Substrate recognition and metabolic response

Mechanism of lignin biodegradation caused by basidiomycetes and the history of lignin biodegradation studies were briefly reviewed. The important roles of fungal extracellular ligninolytic enzymes such as lignin and manganese peroxidases (LiP and MnP) were also summarized. These enzymes were unique in their catalytic mechanisms and substrate specificities. Either LiP or MnP system is capable of oxidizing a variety of aromatic substrates via a one-electron oxidation. Extracellular fungal system for aromatic degradation is non-specific, which recently attracts many people working in a bioremediation field. On the other hand, an intracellular degradation system for aromatic compounds is rather specific in the fungal cell. Structurally similar compounds were prepared and metabolized, indicating that an intracellular degradation strategy consisted of the cellular systems for substrate recognition and metabolic response. It was assumed that lignin-degrading fungi might be needed to develop multiple metabolic pathways for a variety of aromatic compounds caused by the action of non-specific ligninolytic enzymes on lignin. Our recent results on chemical stress responsible factors analyzed using mRNA differential display techniques were also mentioned.     

Halotolerant laccases from Chaetomium sp., Xylogone sphaerospora, and Coprinopsis sp. isolated from a Mediterranean coastal area

Laccases (EC 1.10.3.2) are phenoloxidases involved in the transformation of the recalcitrant fraction of organic matter in soil. These enzymes are also able to transform certain aromatic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and are known to be inhibited by chloride ions. This study aims to test the potential of some fungal strains newly isolated from natural environments subjected to high osmotic pressure such as coastal ecosystems, to produce chloride tolerant laccases. Three strains were identified as Chaetomium sp., Xylogone sphaerospora (two Ascomycota), and Coprinopsis sp. (a Basidiomycota) and the laccases produced by these fungi were weakly inhibited by chloride ions compared with previous data from literature. Moreover, we tested their reactivity towards various PAHs which are widespread anthropic pollutants. They were able to transform anthracene to 9,10-anthraquinone and we determine 7.5 eV as the threshold of ionization potential for PAH oxidation by these laccases.     

Engineering of a psychrophilic bacterium for the bioremediation of aromatic compounds

Microbial degradation of aromatic hydrocarbons has been studied with the aim of developing applications for the removal of toxic compounds. Efforts have been directed toward the genetic manipulation of mesophilic bacteria to improve their ability to degrade pollutants, even though many pollution problems occur in sea waters and in effluents of industrial processes which are characterized by low temperatures. From these considerations the idea of engineering a psychrophilic microorganism for the oxidation of aromatic compounds was developed.In a previous paper it was demonstrated that the recombinant Antarctic Pseudoalteromonas haloplanktis TAC125 (PhTAC/tou) expressing a toluene-o-xylene monooxygenase (ToMO) is able to convert several aromatic compounds into corresponding catechols. In our work we improved the metabolic capability of PhTAC/tou cells by combining action of recombinant ToMO enzyme with that of the endogenous P. haloplanktis TAC125 laccase-like protein. This strategy allowed conferring new and specific degradative capabilities to a bacterium isolated from an unpolluted environment; indeed engineered PhTAC/tou cells are able to grow on aromatic compounds as sole carbon and energy sources. Our approach demonstrates the possibility to use the engineered psychrophilic bacterium for the bioremediation of chemically contaminated marine environments and/or cold effluents.     

Remediation and treatment of organopollutants mediated by peroxidases: a review

In this paper an effort has been made to review the literature on the role of peroxidases in the remediation and treatment of a wide spectrum of aromatic pollutants. Peroxidases can catalyse degradation/transformation of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, organochlorines, 2,4,6-trinitrotoluene, phenolic compounds and dyes. These enzymes are also capable of treating various types of recalcitrant aromatic compounds in the presence of redox mediators. Immobilised peroxidases from plant and fungal sources have been used for the remediation of such types of industrial pollutants on a large scale.     

Fungal laccases as tools for the synthesis of new hybrid molecules and biomaterials

Laccase is a ligninolytic enzyme widely distributed in wood-rotting fungi and which is also found in a variety of molds and insects as well as some plants and bacteria. Its biological roles range from depolmerization of lignin, coal and humic acids via the oxidation of various mono- and diaromatic structures, to polymerization reactions and pigment formation in microbial cells or spores. Apart from its action in catabolic, depolymerizing and polymerizing processes, laccases have also been shown to be powerful enzymes for coupling two different molecules to create new low-molecular-weight products in high yield. In addition to their homomolecular coupling capabilities, laccases are also able to couple a hydroxylated aromatic substrate with a nonlaccase substrate of variable structure to create new heteromolecular hybrid molecules. Thus, laccases are increasingly finding applications in biotechnology in the fields of environment-friendly synthesis of fine chemicals and for the gentle derivatization of biologically active compounds e.g., antibiotics, amino acids, antioxidants, and cytostatics. Finally, oligomerization and polymerization reactions can lead to new homo- or heteropolymers and biomaterials. These may be useful in a wide range of applications including the production of polymers with antioxidative properties, the copolymerizing of lignin components with low-molecular mass compounds, the coating of cellulosic cotton fibers or wool, the coloring of hair and leathers, or the cross-linking and oligomerization of peptides.     

Tactic responses to pollutants and their potential to increase biodegradation efficiency

A significant number of bacterial strains are able to use toxic aromatic hydrocarbons as carbon and energy sources. In a number of cases, the evolution of the corresponding degradation pathway was accompanied by the evolution of tactic behaviours either towards or away from these toxic carbon sources. Reports are reviewed which show that a chemoattraction to heterogeneously distributed aromatic pollutants increases the bioavailability of these compounds and their biodegradation efficiency. An extreme form of chemoattraction towards aromatic pollutants, termed ‘hyperchemotaxis’, was described for Pseudomonas putida DOT‐T1E, which is based on the action of the plasmid‐encoded McpT chemoreceptor. Cells with this phenotype were found of being able to approach and of establishing contact with undiluted crude oil samples. Although close McpT homologues are found on other degradation plasmids, the sequence of their ligand‐binding domains does not share significant similarity with that of NahY, the other characterized chemoreceptor for aromatic hydrocarbons. This may suggest the existence of at least two families of chemoreceptors for aromatic pollutants. The use of receptor chimers comprising the ligand‐binding region of McpT for biosensing purposes is discussed.     

细菌Rieske非血红素铁环羟化酶在多环芳烃降解中的研究进展

多环芳烃是一种常见的持久性有机污染物,因具有致癌、致突变等毒性而被广泛关注。其微生物降解过程通常由羟化起始,随后脱氢、开环、一步步去除支链,最终进入三羧酸循环。Rieske 非血红素铁环羟化酶(Rieske-type non-heme iron aromatic ring-hydroxylating oxygenases , RHOs , 又称 aromatic ring-hydroxylating dioxygenases) 或细胞色素 P450 氧化酶负责将羟基加成到多环芳烃环上,将疏水性的多环芳烃转化为亲水性的衍生物,这一过程是多环芳烃降解转化的起始步骤,也是关键步骤和限速步骤之一。文中主要介绍 RHOs 的分布、底物特异性、底物识别机制以及研究 RHOs 与多环芳烃的一些技术和方法等,并对 RHOs 在环境修复技术中的潜在应用进行了展望。     

靶向5-LOX中药黄酮类化合物的活性筛选及其作用方式和共性规律研究

筛选靶向结合炎症相关蛋白5-LOX(5-lipoxygenase,5-脂氧合酶)的中药黄酮类天然产物,分析与5-LOX结合的黄酮类成分及其来源中药的共性规律。本研究借助Discovery Studio 2017 R2分子对接和药效团构建模块,结合SPR分子筛选实验,以及关联网络构建的方法进行研究。研究结果显示,来源于17种中药的18个黄酮类小分子中有11个能够与5-LOX结合,并从分子对接以及药效团构建研究中发现其作用的3种方式和共性特征:(1)部分中药黄酮成分(如木犀草素等)通过结构中的B环与5-LOX在活性位点ASP243形成静电中心相结合;(2)部分中药黄酮成分(如芹菜素等)是通过结构中的A环与活性位点VAL520形成疏水键、与活性位点ASP243形成氢键与5-LOX结合;(3)杨梅苷等黄酮类成分由于极性较强,在没有形成疏水键的情况下,也是通过形成静电中心与5-LOX在活性位点ASP243产生相互作用。此外还发现靶向5-LOX的活性中药黄酮类化合物,大多来源于具有利湿、退黄等功效,性味甘苦寒的景天科中药中。本研究发现了部分靶向5-LOX的中药黄酮类成分及其作用方式和共性规律,为开发靶向5-LOX抗肿瘤新药提供思路和方法。     

Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia

Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems. Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora. Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria. These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further. The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack. Sequencing anaerobic and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants. The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g. biofilm, soil aggregate, etc.). Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers. The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm.Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments. Consequently, an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances. The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation. Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups. The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step.     

Endolithic growth of two Lecidea lichens in granite from continental Antarctica detected by molecular and microscopy techniques

Through the combined use of molecular and microscopy techniques, the endolithic lichens Lecidea cancriformis and Lecidea sp. were identified, even in the absence of fruiting bodies, and positioned under epilithic lichens. Cells of both algal and fungal symbionts were observed in fissures and cracks of the lithic substrate with no clear heteromerous structure. At the ultrastructural level, the two lichens differed in terms of their algal-fungal relationships. Only one genotype of Trebouxia ITS sequence was identified from specimens of Lecidea sp., Umbilicaria aprina and Buellia frigida from the same zone, which could be mainly determined by low availability of alga in these extreme environments. These lichens showed features typical of both chasmoendolithic and euendolithic microorganisms. Signs of biogeophysical and biogeochemical action on the substrate were detected close to fungal cells. This action seemed to be mainly conditioned by the local physico-chemical features of the substrate. Evidence for the biomobilization of elements by these endolithic lichens was found. L. cancriformis was observed to accumulate substantial amounts of calcium-rich biominerals. The combined approach proposed is useful for mapping the distribution of endolithic lichens and analysing the processes that occur in their microscopic environment.     

Biocatalytic chlorination of aromatic hydrocarbons by chloroperoxidase of Caldariomyces fumago

Chloroperoxidase from Caldariomyces fumago was able to chlorinate 17 of 20 aromatic hydrocarbons assayed in the presence of hydrogen peroxide and chloride ions. Reaction rates varied from 0.6 min(-1) for naphthalene to 758 min(-1) for 9-methylanthracene. Mono-, di- and tri-chlorinated compounds were obtained from the chloroperoxidase-mediated reaction on aromatic compounds. Dichloroacenaphthene, trichloroacenaphthene, 9,10-dichloroanthracene, chloropyrene, dichloropyrene, dichlorobiphenylene and trichlorobiphenylene were identified by mass spectral analyses as products from acenaphthene, anthracene, pyrene and biophenylene respectively. Polycyclic aromatic hydrocarbons with 5 and 6 aromatic rings were also substrates for the chloroperoxidase reaction. The importance of the microbial chlorination of aromatic pollutants and its potential environmental impact are discussed.     

Rational redesign of the 4-chlorobenzoate binding site of 4-chlorobenzoate: coenzyme a ligase for expanded substrate range

Environmental aromatic acids are transformed to chemical energy in bacteria that possess the requisite secondary pathways. Some of these pathways rely on the activation of the aromatic acid by coenzyme A (CoA) thioesterification catalyzed by an aromatic acid: CoA ligase. Adaptation of such pathways to the bioremediation of man-made pollutants such as polychlorinated biphenyl (PCB) and dichlorodiphenyltrichloroethane (DDT) requires that the chlorinated benzoic acid byproduct that is formed be able to be eliminated by further degradation. To take advantage of natural benzoic acid degrading pathways requiring initial ring activation by thioesterification, the pathway aromatic acid:CoA ligase must be an effective catalyst with the chlorinated benzoic acid. This study, which focuses on the 4-chlorobenzoate:CoA ligase (CBL) of the 4-monochlorobiphenyl degrading bacterium Alcaligenes sp. strain ALP83, was carried out to determine if the 4-chlorobenzoate binding site of this enzyme can be transformed by rational design to recognize the chlorobenzoic acids formed in the course of breakdown of other environmental PCB congeners. The fundamental question addressed in this study is whether it is possible to add or subtract space from the substrate-binding pocket of this ligase (to complement the topology of the unnatural aromatic substrate) without causing disruption of the ligase catalytic machinery. Herein, we report the results of a substrate specificity analysis that, when interpreted within the context of the X-ray crystal structures, set the stage for the rational design of the ligase for thioesterification of two PCB-derived chlorobenzoic acids. The ligase was first optimized to catalyze CoA thioesterification of 3,4-dichlorobenzoic acid, a poor substrate, by truncating Ile303, a large hydrophobic residue that packs against the ring meta-C(H) group. The structural basis for the approximately 100-fold enhancement in the rate of 3,4-dichlorobenzoate thioesterification catalyzed by the I303A and I303G CBL mutants was validated by determination of the crystal structure of the 3,4-dichlorobenzoate-bound enzymes. Determinations of the structures of I303 mutant complexes of 3-chlorobenzoate, a very poor substrate, revealed nonproductive binding as a result of the inability of the substrate ring C(4)H group to fill the pocket that binds the C(4)Cl group of the native substrate. The C(4)Cl pocket of the CBL I303A mutant was then reduced in size by strategic amino acid replacement. A 54-fold improvement in catalytic efficiency was observed for the CBL F184W/I303A/V209T triple mutant. The results of this investigation are interpreted as evidence that the plasticity of the ligase catalytic scaffold is sufficient to allow expansion of substrate range by rational design. The combination of structural and kinetic analyses of the constructed mutants proved to be an effective approach to engineering the ligase for novel substrates.     

Hydroxylations of substituted naphthalenes by Escherichia coli expressing aromatic dihydroxylating dioxygenase genes from polycyclic aromatic hydrocarbon-utilizing marine bacteria

Bioconversion experiments of various mono- or di-substituted naphthalenes such as dimethylnaphthalenes were carried out using the cells of Escherichia coli that expressed aromatic dihydroxylating dioxygenase genes (phnA1A2A3A4 and phdABCD) from polycyclic aromatic hydrocarbon-utilizing marine bacteria, Nocardioides sp. KP7 and Cycloclasticus sp. A5, respectively. We found that the former dioxygenase PhnA1A2A3A4 had broad substrate preference for these compounds and often was able to hydroxylate their methyl groups. Specifically, 1,4-dimethylnaphthalene was predominantly bioconverted into 1,4-dihydroxymethylnaphthalene.     

Fungal metabolism and detoxification of the nitropolycyclic aromatic hydrocarbon 1-nitropyrene

Nitropolycyclic aromatic hydrocarbons are ubiquitous environmental pollutants, many of which are potent mutagens in bacterial and mammalian cells and carcinogenic to rodents. In this study, we investigated the fungal metabolism of 1-nitropyrene and determined the mutagenic activity of the metabolites toward Salmonella typhimurium TA98, TA98NR, and TA100. Cunninghamella elegans metabolized 1-nitropyrene to form glucoside conjugates of 6-hydroxy-1-nitropyrene and 8-hydroxy-1-nitropyrene. The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by application of UV absorption, 1H-nuclear magnetic resonance, and mass spectroscopy. Mutagenicity assays performed on samples extracted from incubations of C. elegans with 1-nitropyrene indicated that mutagenic activity decreased with time. Consistent with the loss in mutagenic activity, the glucoside conjugates of 6- and 8-hydroxy-1-nitropyrene were nonmutagenic in the Salmonella reversion assay. The results indicate that the fungus C. elegans metabolizes 1-nitropyrene to detoxified products.     

Fungal metabolism and detoxification of the nitropolycyclic aromatic hydrocarbon 1-nitropyrene.

Nitropolycyclic aromatic hydrocarbons are ubiquitous environmental pollutants, many of which are potent mutagens in bacterial and mammalian cells and carcinogenic to rodents. In this study, we investigated the fungal metabolism of 1-nitropyrene and determined the mutagenic activity of the metabolites toward Salmonella typhimurium TA98, TA98NR, and TA100. Cunninghamella elegans metabolized 1-nitropyrene to form glucoside conjugates of 6-hydroxy-1-nitropyrene and 8-hydroxy-1-nitropyrene. The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by application of UV absorption, 1H-nuclear magnetic resonance, and mass spectroscopy. Mutagenicity assays performed on samples extracted from incubations of C. elegans with 1-nitropyrene indicated that mutagenic activity decreased with time. Consistent with the loss in mutagenic activity, the glucoside conjugates of 6- and 8-hydroxy-1-nitropyrene were nonmutagenic in the Salmonella reversion assay. The results indicate that the fungus C. elegans metabolizes 1-nitropyrene to detoxified products.     

Oxidoreductases for the remediation of organic pollutants in water – a critical review

Water contamination by various recalcitrant organic aromatic compounds is an emerging environmental issue that is increasingly attracting the attention of environmental scientists. A great majority of these recalcitrant pollutants are industrial wastes, textile dyes, pharmaceuticals, hormones, and personal care products that are discharged into wastewater. Not surprisingly, various chemical, physical, and biological strategies have been proposed and developed to remove and/or degrade these pollutants from contaminated water bodies. Biological approaches, specifically using oxidoreductase enzymes (such as peroxidases and laccases) for pollutant degradation are a relatively new and a promising research area that has potential advantages over other methods due to their higher efficiency and the ease of handling. This review focuses on the application of different classes of oxidoreductase enzymes to degrade various classes of organic pollutants. In addition to classifying these enzymes based on structural differences, the major factors that can affect their remediation ability, such as the class of peroxidases employed, pH, molecular structure of the pollutant, temperature, and the presence of redox mediators are also examined and discussed. Interestingly, a literature survey combined with our unpublished data suggests that “peroxidases” are a very heterogeneous and diverse family of enzymes and have different pH profiles, temperature optima, thermal stabilities, requirements for redox mediators, and substrate specificities as well as varying detoxification abilities. Additionally, remediation of real-life polluted samples by oxidoreductases is also highlighted as well as a critical look at current challenges and future perspectives.