Chemical crosslinking and the stabilization of proteins and enzymes.

The technique of chemical crosslinking has been used to enhance the stability of proteins and enzymes. In this procedure, the molecule is braced with chemical crosslinks either intramolecularly or intermolecularly to another species to reinforce its active structure. Various chemicals have been used for this purpose. The bifunctional reagents are the most prominent. These compounds are derived from group-specific reagents and may be classified into homobifunctional, heterobifunctional, and zero-length crosslinkers. Different physical and chemical characteristics have been incorporated into these chemicals. Their versatility holds great potential in preparing chemically, thermally, and mechanically stable proteins and enzymes for industrial applications.     

Methanol,a potential feedstock for biotechnological processes

A wide variety of bacteria and yeasts is able to grow in inexpensive synthetic media with methanol as the sole or major source of carbon and energy. This is due to the presence of a few unique enzymes which enable these organisms to generate metabolic energy and synthesize cell constituents from this one-carbon substrate. In the chemical industry there is currently much interest in the production of fuels and chemicals from methanol. As a feedstock for industrial fermentations methanol is also attractive because of its low cost, ease of handling and abundant availability. In many countries methanol-utilizing microbes are being studied and their potential utility in biotechnological processes is explored. These studies are aimed at making use of their characteristic properties, exploiting known organisms and new strains for improving existing processes and developing novel products.     

Elimination of Endocrine Disrupting Chemicals using White Rot Fungi and their Lignin Modifying Enzymes: A Review

The ability of white rot fungi (WRF) and their lignin modifying enzymes (LMEs), i.e. laccase and lignin‐ and manganese‐dependent peroxidase, to treat endocrine disrupting chemicals (EDCs) is extensively reviewed in this paper. These chemicals cause adverse health effects by mimicking endogenous hormones in receiving organisms. The alkylphenolic EDCs nonylphenol, bisphenol A and triclosan, the phthalic acid esters dibutylphthalate, diethylphthalate and di‐(2‐ethylhexyl)phthalate, the natural estrogens estrone, 17β‐estradiol, estriol and 17α‐ethynylestradiol and the phytoestrogens genistein and β‐sitosterol have been shown to be eliminated by several fungi and LMEs. WRF have manifested a highly efficient removal of EDCs in aqueous media and soil matrices using both LME and non LME‐systems. The ligninolytic system of WRF could also be used for the elimination of several EDCs and the associated hormone‐mimicking activity. The transformation of EDCs by LMEs and WRF is supported by emerging knowledge on the physiology and biochemistry of these organisms and the biocatalytic properties of their enzymes. Due to field reaction conditions, which drastically differ from laboratory conditions, further efforts will have to be directed towards developing robust and reliable biotechnological processes for the treatment of EDC‐contaminated environmental matrices.     

Chemically Regulated Expression Systems and their Applications in Transgenic Plants

In the past 20 years, several systems have been developed to control transgene expression in plants using chemicals. The components used to construct these systems are derived from regulatory sequences mostly from non-plant organisms such as bacteria, fungi, insects and mammals. These constructs allowed transgene expression to be controlled temporally, spatially and quantitatively with the help of exogenous chemicals, without disturbing endogenous plant gene expression. Various chemically regulated transgene expression systems, their advantages/disadvantages and their potential for large-scale field application are reviewed.     

Biochemical strategies for the detection and detoxification of toxic chemicals in the environment

Addressing the problems related to the widespread presence of an increasing number of chemicals released into the environment by human activities represents one of the most important challenges of this century. In the last few years, to replace the high cost, in terms of time and money, of conventional technologies, the scientific community has directed considerable research towards the development both of new detection systems for the measurement of the contamination levels of chemicals in people's body fluids and tissue, as well as in the environment, and of new remediation strategies for the removal of such chemicals from the environment, as a means of the prevention of human diseases. New emerging biosensors for the analysis of environmental chemicals have been proposed, including VHH antibodies, that combine the antibody performance with the affinity for small molecules, genetically engineered microorganisms, aptamers and new highly stable enzymes. However, the advances in the field of chemicals monitoring are still far from producing a continuous realtime and on-line system for their detection. Better results have been obtained in the development of strategies which use organisms(microorganisms, plants and animals) or metabolic pathway-based approaches(single enzymes or more complex enzymatic solutions) for the fixation, degradation and detoxification of chemicals in the environment. Systems for enzymatic detoxification and degradation of toxic agents in wastewater from chemical and manufacturing industries, such as ligninolytic enzymes for the treatment of wastewater from the textile industry, have been proposed. Considering the high value of these research studies, in terms of the protection of human health and of the ecosystem, science must play a major role in guiding policy changes in this field.     

Anatomical and chemical defenses of conifer bark against bark beetles and other pests

Conifers are long-lived organisms, and part of their success is due to their potent defense mechanisms. This review focuses on bark defenses, a front line against organisms trying to reach the nutrient-rich phloem. A major breach of the bark can lead to tree death, as evidenced by the millions of trees killed every year by specialized bark-invading insects. Different defense strategies have arisen in conifer lineages, but the general strategy is one of overlapping constitutive mechanical and chemical defenses overlaid with the capacity to up-regulate additional defenses. The defense strategy incorporates a graded response from 'repel', through 'defend' and 'kill', to 'compartmentalize', depending upon the advance of the invading organism. Using a combination of toxic and polymer chemistry, anatomical structures and their placement, and inducible defenses, conifers have evolved bark defense mechanisms that work against a variety of pests. However, these can be overcome by strategies including aggregation pheromones of bark beetles and introduction of virulent phytopathogens. The defense structures and chemicals in conifer bark are reviewed and questions about their coevolution with bark beetles are discussed.     

Environmental contaminants in pathogenesis of breast cancer

This review is an attempt to comprehend the diverse groups of environmental chemical contaminants with a potential for pathogenesis of breast cancer, their probable sources and the possible mechanisms by which these environmental contaminants act and interplay with other risk factors. Estrogens are closely related to the pathogenesis of breast cancer. Oxidative catabolism of estrogen, mediated by various cytochrome P450 enzymes, generates reactive free radicals that can cause oxidative damage. The same enzymes of estrogenic metabolic pathways catalyze biological activation of several environmental (xenobiotic) chemicals. Xenobiotic chemicals may exert their pathological effects through generation of reactive free radicals. Breast tissue can be a target of several xenobiotic agents. DNA-reactive metabolites of different xenobiotic compounds have been detected in breast tissue. Many phase I and II xenobiotic metabolizing enzymes are expressed in both normal and cancerous breast tissues. These enzymes play a significant role in the activation/detoxification of xenobiotic and endogenous compounds including estrogens. More than 30 carcinogenic chemicals are present in tobacco smoke; many of them are fat-soluble, resistant to metabolism and can be stored in breast adipose tissue. Similarly, pesticides are also known to cause oxidative stress; while some act as endocrine disruptor, some are shown to suppress apoptosis in estrogen sensitive cell lines. Reports have shown an association of smoking (both active and passive) and pesticides with breast cancer risk. However, the issues have remained controversial. Different mutagenic substances that are generated in the cooking process e.g., heterocyclic amines and polycyclic aromatic hydrocarbons (PAHs) can be a threat to breast tissue. PAHs and dioxins exert their adverse effects through the aryl hydrocarbon receptor (AhR), which activates several genes involved in the metabolisms of xenobiotic compounds and endogenous estrogens. These chemicals also induce AhR-dependent mitochondrial dysfunction. Many of the environmental pollutants suppress the immune system, which are implicated to risk. A better understanding about the biological effects of different environmental carcinogenic compounds and determination of their impact on rising incidence of breast cancer will be beneficial in improving preventive policy against breast cancer.     

Proteomics in Myzus persicae: effect of aphid host plant switch

Chemical ecology is the study of how particular chemicals are involved in interactions of organisms with each other and with their surroundings. In order to reduce insect attack, plants have evolved a variety of defence mechanisms, both constitutive and inducible, while insects have evolved strategies to overcome these plant defences (such as detoxification enzymes). A major determinant of the influence of evolutionary arms races is the strategy of the insect: generalist insect herbivores, such as Myzus persicae aphid, need more complex adaptive mechanisms since they need to respond to a large array of different plant defensive chemicals. Here we studied the chemical ecology of M. persicae associated with different plant species, from Brassicaceae and Solanaceae families. To identify the involved adaptation systems to cope with the plant secondary substances and to assess the differential expression of these systems, a proteomic approach was developed. A non-restrictive approach was developed to identify all the potential adaptation systems toward the secondary metabolites from host plants. The complex protein mixtures were separated by two-dimensional electrophoresis methods and the related spots of proteins significantly varying were selected and identified by mass spectrometry (ESI MS/MS) coupled with data bank investigations. Fourteen aphid proteins were found to vary according to host plant switch; ten of them were down regulated (proteins involved in glycolysis, TCA cycle, protein and lipid synthesis) while four others were overexpressed (mainly related to the cytoskeleton). These techniques are very reliable to describe the proteome from organisms such as insects in response to particular environmental change such as host plant species of herbivores.     

The tritrophic trinity: a source of pollutant-degrading enzymes and its implications for phytoremediation

Barring bioavailability and nutritional limitations, virtually all organic anthropogenic chemicals can be naturally biodegraded. It is to this phenomenon we owe thanks to the long established 'tritrophic trinity' of microbe-plant-insect interactions. Over hundreds of millennia these organisms have coevolved, producing hundreds of thousands of different chemicals that are used to attract, defend, antagonize, monitor and misdirect one another. In comparison, the numbers of truly novel chemicals of anthropogenic origin are negligible. It is only now that we are beginning to appreciate the fortuitous evolution of xenobiotic-degrading enzymes from these interactions. We argue that success in phytoremediation can be hastened through understanding the structure, sources, uses and targets of these secondary metabolites. Owing to recent developments in molecular biology, particularly stable isotope probing, we eagerly anticipate highly significant insights into trophic interactions, particularly in the rhizosphere, providing phytoremediation with a solid mechanistic understanding.     

藻类对多环芳香烃(PAHs)的富集和代谢

概述了藻类对PAHs的富集和代谢的研究进展。环境中多环芳香烃(PAHs)的污染能导致严重的健康问题,利用生物特别是微生物去除污染环境中的PAHs是一项新的技术。藻类对PAHs的富集与有机污染物的类型、藻类的种类及藻类的生物量有关,活细胞和死细胞对PAHs均有富集能力。还阐述了PAHs在真菌、细菌和藻类体内代谢的途径以及代谢过程中起关键作用的酶,PAHs在藻类中的代谢途径和细菌及真菌都不同,谷胱甘肽转移酶(GST)在藻类代谢PAH过程中起重要作用,但细胞色素P450酶所起的作用则不详。     

Efficient,environmentally-friendly and specific valorization of lignin: promising role of non-radical lignolytic enzymes

Lignin is the second most abundant bio-resource in nature. It is increasingly important to convert lignin into high value-added chemicals to accelerate the development of the lignocellulose biorefinery. Over the past several decades, physical and chemical methods have been widely explored to degrade lignin and convert it into valuable chemicals. Unfortunately, these developments have lagged because of several difficulties, of which high energy consumption and non-specific cleavage of chemical bonds in lignin remain the greatest challenges. A large number of enzymes have been discovered for lignin degradation and these are classified as radical lignolytic enzymes and non-radical lignolytic enzymes. Radical lignolytic enzymes, including laccases, lignin peroxidases, manganese peroxidases and versatile peroxidases, are radical-based bio-catalysts, which degrade lignins through non-specific cleavage of chemical bonds but can also catalyze the radical-based re-polymerization of lignin fragments. In contrast, non-radical lignolytic enzymes selectively cleave chemical bonds in lignin and lignin model compounds and, thus, show promise for use in the preparation of high value-added chemicals. In this mini-review, recent developments on non-radical lignolytic enzymes are discussed. These include recently discovered non-radical lignolytic enzymes, their metabolic pathways for lignin conversion, their recent application in the lignin biorefinery, and the combination of bio-catalysts with physical/chemical methods for industrial development of the lignin refinery.     

The nitrile-degrading enzymes: current status and future prospects

Nitrile-converting enzymes are becoming commonplace in the synthesis of pharmaceuticals and commodity chemicals. These versatile biocatalysts have potential applications in different fields including synthetic biocatalysis and bioremediation. This review attempts to describe in detail the three major classes of nitrile-converting enzymes, namely nitrilases, nitrile hydratases and amidases. Various aspects of these enzymes including their occurrence, mechanism of action, characteristics and applicability in different sectors have been elaborately elucidated. Cloning of genes related to nitrile-converting enzymes is also discussed.     

Enzyme promiscuity – A light on the “darker” side of enzyme specificity

Abstract

Enzyme promiscuity can be defined as the capability of enzymes to catalyse side reaction in addition to its main reaction. The side reaction of an enzyme is termed as promiscuous or sometimes as the “darker” side of enzyme cross-reactivity/specificity. This unique property of enzyme allows organisms to adapt under varying environmental conditions. Promiscuous enzymes can modify their catalytic activities with altered substrates and can adjust their catalytic and kinetic mechanisms according to substrate properties. This group of enzymes evolved from ancestral proteins found in primitive organisms like archaea that survive under extreme environmental conditions. Such ancestral proteins possessed the potential to catalyse a wide range of reactions at low levels, hence create families or superfamilies of highly specialized enzymes. Further, some enzymes were identified which have non-catalytic functions in addition to their major catalytic activities. These enzymes are referred to as moonlighting enzymes. The study of these enzymes will provide important information regarding enzyme evolution and will help in optimizing protein engineering applications.     

Study of P450 function using gene knockout and transgenic mice

The xenobiotic-metabolizing P450s have been extensively studied for their ability to metabolize endogenous and exogenous chemicals. The latter include drugs and dietary and environmentally derived toxicants and carcinogens. These enzymes also metabolize endogenous steroids and fatty acids. P450s are thought to be required for efficient removal of most xenobiotics from the body and to be responsible for the hazardous effects of toxicants and carcinogens based on their ability to convert chemicals to electrophilic metabolites that can cause cellular damage and gene mutations. P450 catalytic activities have been extensively studied in vitro and in cell culture, yielding considerable information on their mechanisms of catalysis, substrate specificities, and metabolic products. Targeted gene disruption has been used to determine the roles of P450s in intact animals and their contributions to the mechanisms of toxicity and carcinogenesis. The P450s chosen for study, CYP1A1, CYP1B1, CYP1A2, and CYP2E1, are conserved in mammals and are known to metabolize most toxicants and chemical carcinogens. Mice lacking expression of these enzymes do not differ from wild-type mice, indicating that these P450s are not required for development and physiological homeostasis. However, the P450 null mice have altered responses to the toxic and carcinogenic effects of chemicals as compared with wild-type mice. These studies establish that P450s mediate the adverse effects of drugs and dietary, environmental, and industrial chemicals and serve to validate molecular epidemiology studies that seek to determine links between P450 polymorphisms and susceptibility to chemically associated diseases. More recently, P450 humanized mice have been produced.     

Actinomycete cytochromes P‐450 involved in oxidative metabolism: Biochemistry and molecular biology∗

The diverse metabolic capability of actinomycetes, together with their widespread presence in terrestrial and aquatic environments, has rendered them instrumental in the breakdown of both man‐made and natural chemicals and in the recycling of carbon in nature. Actinomycetes are also well known for their ability to synthesize a wide variety of novel chemicals of pharmaceutical and industrial value. Cytochromes P‐450, a class of oxidative enzymes found in all organisms, play a central role in both biosynthetic and biodegradative reactions performed by actinomycetes. Herein, we describe recent studies of actinomycetes cytochromes P‐450 made possible by advances realized in biochemistry and molecular biology.     

Some like it cold: biocatalysis at low temperatures

In the last few years, increased attention has been focused on a class of organisms called psychrophiles. These organisms, hosts of permanently cold habitats, often display metabolic fluxes more or less comparable to those exhibited by mesophilic organisms at moderate temperatures. Psychrophiles have evolved by producing, among other peculiarities, "cold-adapted" enzymes which have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. Thermal compensation in these enzymes is reached, in most cases, through a high catalytic efficiency associated, however, with a low thermal stability. Thanks to recent advances provided by X-ray crystallography, structure modelling, protein engineering and biophysical studies, the adaptation strategies are beginning to be understood. The emerging picture suggests that psychrophilic enzymes are characterized by an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. Due to their attractive properties, i.e., a high specific activity and a low thermal stability, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications.     

Zinc proteomes, phylogenetics and evolution

Evolution has not been studied in detail with reference to the changing environment. This requires a study of the inorganic chemistry of organisms, especially metalloproteins. The evolution of organisms has been analysed many times previously using comparative studies, fossils, and molecular sequences of proteins, DNA and 16s rRNA (Zhang and Gladyshev, Chem. Rev., 2009, 109, 4828). These methods have led to the confirmation of Darwin's original proposal that evolution followed from natural selection in a changing environment often pictured as a tree. In all cases, the main tree in its upper later reaches has been well studied but its lower earlier parts are not so well defined. To approach this topic we have treated evolution as due to the intimate combination of the effect of chemical changes in the environment and in the organisms (Williams and da Silva, The Chemistry of Evolution, 2006, Elsevier). The best chemicals to examine are inorganic ions as they are common to both. As a more detailed example of the chemical study of organisms we report in this paper a bioinformatic approach to the characterization of the zinc proteomes. We deduce them from the 821 totally sequenced DNA of organisms available on NCBI, exploiting a published method developed by one of us (Andreini, Bertini and Rosato, Acc. Chem. Res., 2009, 42, 1471). Comparing the derived zinc-finger-containing proteins and zinc hydrolytic enzymes in organisms of different complexity there is a correlation in their changes during evolution related to environmental change.     

Fungi in acidic fire: A potential source of industrially important enzymes

The microbial life that exists in harsh habitats of low pH possess several unique characteristics, which assign interesting qualities to these microorganisms and enable them to thrive in such a harsh environment. Among microorganisms inhabiting low pH environments, fungi are the second largest reported organisms. These acidophilic fungi are the main source of acid–stable enzymes that could be utilized in many industries including paper, leather making, food and feed industries, where the efficacy of commonly available enzymes is limited by challenges like stability and functional kinetics. The current review discusses the acidophilic fungi with emphasis on their diversity and pH homeostasis mechanisms adopted against low pH environments. In addition, an overview about the acid–stable enzymes obtained from these acidophilic fungi, their main sources and potential applications have also been discussed.