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Alison Butler - One of the best experts on this subject based on the ideXlab platform.
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Reactivity of Haloperoxidases and Functional Mimics of Haloperoxidase
2004Co-Authors: Alison ButlerAbstract:Abstract : The objective of the proposal was 1) to investigate oxidative coupling of nitro-toluene compounds, catalyzed by vanadium bromoperoxidase or its functional mimics and 2) to investigate catalytic properties of silicate-encapsulated enzymes, in general, and Haloperoxidase enzymes, in particular, or their functional mimics to assess their utility as catalysts in relevant synthetic schemes. It was found that V-BrPO catalyzed bromination reactions with TNT do not effect the oxidative coupling reaction required to form FINS. FeHeme chloroperoxidase, which functions optimally at low pH is also not suitable for catalyzing the oxidative coupling of TNT to FINS because if only chlorinates at low pH. It was also found that Haloperoxidases immobilized in mesoporous silicate matrices retain their catalytic and functional properties. These enzymes may well have applications as anti-biofouling catalysts.
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the role of vanadium bromoperoxidase in the biosynthesis of halogenated marine natural products
Natural Product Reports, 2004Co-Authors: Alison Butler, Jayme N CarterfranklinAbstract:Covering: 1998–2003 Halogenated natural products are frequently reported metabolites in marine seaweeds. These compounds span a range from halogenated indoles, terpenes, acetogenins, phenols, etc., to volatile halogenated hydrocarbons that are produced on a very large scale. In many cases these halogenated marine metabolites possess biological activities of pharmacological interest. Given the abundance of halogenated marine natural products found in marine organisms and their potentially important biological activities, the biogenesis of these compounds has intrigued marine natural product chemists for decades. Over a quarter of a century ago, a possible role for Haloperoxidase enzymes was first suggested in the biogenesis of certain halogenated marine natural products, although this was long before Haloperoxidases were discovered in marine organisms. Since that time, FeHeme- and Vanadium-Haloperoxidases (V-HPO) have been discovered in many marine organisms. The structure and catalytic activity of vanadium Haloperoxidases is reviewed herein, including the importance of V-HPO-catalyzed bromination and cyclization of terpene substrates.
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on the regiospecificity of vanadium bromoperoxidase
Journal of the American Chemical Society, 2001Co-Authors: Jennifer S Martinez, G L Carroll, Richard A Tschirretguth, G Altenhoff, R.d. Little, Alison ButlerAbstract:Vanadium Haloperoxidase enzymes catalyze the oxidation of halide ions by hydrogen peroxide, producing an oxidized intermediate, which can halogenate an organic substrate or react with a second equivalent of hydrogen peroxide to produce dioxygen. Haloperoxidases are thought to be involved in the biogenesis of halogenated natural products isolated from marine organisms, including indoles and terpenes, of which many are selectively oxidized or halogenated. Little has been shown concerning the ability of the marine Haloperoxidases to catalyze regioselective reactions. Here we report the regiospecific bromoperoxidative oxidation of 1,3-di-tert-butylindole by V-BrPO from the marine algae Ascophyllum nodosum and Corallina officinalis. Both enzymes catalyze the regiospecific oxidation of 1,3-di-tert-butylindole in a reaction requiring both H2O2 and Br- as substrates, but which produce the unbrominated 1,3-di-tert-butyl-2-indolinone product exclusively, in near quantitative yield (i.e. one H2O2 consumed per produc...
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Mechanistic considerations of the vanadium Haloperoxidases
Coordination Chemistry Reviews, 1999Co-Authors: Alison ButlerAbstract:Abstract Haloperoxidases are enzymes which catalyze the oxidation of halide ions (i.e. chloride, bromide and iodide) by hydrogen peroxide. These enzymes usually contain the FeHeme moiety or vanadium as an essential constituent at their active site, however, a few Haloperoxidases which lack a metal cofactor are known. This review will examine (1) the reactivity of the vanadium Haloperoxidases, particularly the mechanism of halide oxidation by hydrogen peroxide, and the mechanism of halogenation and sulfoxidation, including the newly reported regioselectivity and enantioselectivity of the vanadium Haloperoxidases; (2) the X-ray structure of vanadium chloroperoxidase, the vanadium(V) active site and the role of critical amino acid side chains for catalysis and (3) functional biomimetic systems, with specific relevance to the mechanism of the vanadium Haloperoxidase enzymes.
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vanadium bromoperoxidase and functional mimics
1997Co-Authors: Alison Butler, Anne H BaldwinAbstract:Haloperoxidases are enzymes which catalyze the oxidation of halides (iodide, bromide and chloride) by hydrogen peroxide, resulting in the halogenation of appropriate organic substrates or the halide-assisted disproportionation of hydrogen peroxide forming dioxygen. Two classes of vanadium Haloperoxidases are known: vanadium bromoperoxidase, isolated mainly from marine algae, and vanadium chloroperoxidase from terrestrial fungi. A review of the structure, reactivity and enzyme kinetics of these enzymes is presented. Kinetic and mechanistic studies of several functional mimics of the vanadium Haloperoxidases are also presented, including peroxo-complexes of vanadium(V) and other transition metal ions. These catalysts have proved useful in testing hypotheses concerning the role of vanadium in the Haloperoxidase enzymes.
Ron Wever - One of the best experts on this subject based on the ideXlab platform.
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Marine Vanadium-Dependent Haloperoxidases, Their Isolation, Characterization, and Application.
Methods in enzymology, 2018Co-Authors: Ron Wever, Bea E. Krenn, Rokus RenirieAbstract:Vanadium-dependent Haloperoxidases in seaweeds, cyanobacteria, fungi, and possibly phytoplankton play an important role in the release of halogenated volatile compounds in the environment. These halocarbons have effects on atmospheric chemistry since they cause ozone depletion. In this chapter, a survey is given of the different sources of these enzymes, some of their properties, the various methods to isolate them, and the bottlenecks in purification. The assays to detect and quantify Haloperoxidase activity are described as well as their kinetic properties. Several practical tips and pitfalls are given which have not yet been published explicitly. Recent developments in research on structure and function of these enzymes are reviewed. Finally, the application of vanadium-dependent Haloperoxidases in the biosynthesis of brominated and other compounds is discussed.
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51V solid-state NMR spectroscopy of vanadium Haloperoxidases and bioinorganic Haloperoxidase mimics
2007Co-Authors: Tatyana Polenova, Rokus Renirie, Dieter Rehder, Neela Pooransingh Margolis, Ron WeverAbstract:We present 51 V solid-state magic angle spinning NMR spectroscopy as a probe of geometric and electronic environments in vanadium Haloperoxidases and in oxovanadium (V) complexes mimicking the active site of these enzymes. In the bioinorganic complexes, 51 V MAS spectra are sensitive reporters of the coordination environment and coordination geometry. In vanadium chloro- and bromoperoxidases, the spectra reveal unique electronic environments of the vanadate cofactor in each species. The experimental NMR observables and DFT calculations of the NMR parameters yield the most likely protonation states of the individual oxygen ligands in vanadium chloroperoxidase. A combination of experimental solid-state NMR and quantum mechanical calculations thus offers a powerful strategy for analysis of diamagnetic spectroscopically silent vanadium (V) states in inorganic and biological systems.
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bromoperoxidase activity of vanadate substituted acid phosphatases from shigella flexneri and salmonella enterica ser typhimurium
FEBS Journal, 2002Co-Authors: Naoko Tanaka, Qianning Liao, Valerie Dumay, Alex J. Lange, Ron WeverAbstract:Vanadium Haloperoxidases and the bacterial class A nonspecific acid phosphatases have a conserved active site. It is shown that vanadate-substituted recombinant acid phosphatase from Shigella flexneri (PhoN-Sf) and Salmonella enterica ser. typhimurium (PhoN-Se) in the presence of H2O2 are able to oxidize bromide to hypobromous acid. Vanadate is essential for this activity. The kinetic parameters for the artificial bromoperoxidases have been determined. The Km value for H2O2 is about the same as that for the vanadium bromoperoxidases from the seaweed Ascophyllum nodosum. However, the Km value for Br– is about 10–20 times higher, and the turnover values of about 3.4 min−1 and 33 min−1 for PhoN-Sf and PhoN-Se, respectively, are much slower, than those of the native bromoperoxidase. Thus, despite the striking similarity in the active-site structures of the vanadium Haloperoxidases and the acid phophatase, the turnover frequency is low, and clearly the active site of acid phosphatases is not optimized for Haloperoxidase activity. Like the native vanadium bromoperoxidase, the vanadate-substituted PhoN-Sf and PhoN-Se catalyse the enantioselective sulfoxidation of thioanisole.
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Vanadium Haloperoxidases from brown algae of the Laminariaceae family.
Phytochemistry, 2001Co-Authors: Mércia Melo De Almeida, S. Filipe, Madalena Humanes, M.f. Maia, Ricardo Melo, N. Severino, J. Da Silva, J.j.r.fraústo Da Silva, Ron WeverAbstract:Vanadium Haloperoxidases were extracted, purified and characterized from three different species of Laminariaceae — Laminaria saccharina (Linne´ ) Lamouroux, Laminaria hyperborea (Gunner) Foslie and Laminaria ochroleuca de la Pylaie. Two different forms of the vanadium Haloperoxidases were purified from L. saccharina and L. hyperborea and one form from L. ochroleuca species. Reconstitution experiments in the presence of several metal ions showed that only vanadium(V) completely restored the enzymes activity. The stability of some enzymes in mixtures of buffer solution and several organic solvents such as acetone, ethanol, methanol and 1-propanol was noteworthy; for instance, after 30 days at least 40% of the initial activity for some isoforms remained in mixtures of 3:1 buffer solution/organic solvent. The enzymes were also moderately thermostable, keeping full activity up to 40 � C. Some preliminary steady-state kinetic studies were performed and apparent Michaelis–Menten kinetic parameters were determined for the substrates iodide and hydrogen peroxide. Histochemical studies were also performed in fresh tissue sections from stipe and blade of L. hyperborea and L. saccharina, showing that Haloperoxidase activity was concentrated in the external cortex near the cuticle, although some activity was also observed in the inner cortical region. # 2001 Elsevier Science Ltd. All rights reserved.
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peroxidase and phosphatase activity of active site mutants of vanadium chloroperoxidase from the fungus curvularia inaequalis implications for the catalytic mechanisms
Journal of Biological Chemistry, 2000Co-Authors: Rokus Renirie, Wieger Hemrika, Ron WeverAbstract:Mutation studies were performed on active-site residues of vanadium chloroperoxidase from the fungus Curvularia inaequalis, an enzyme which exhibits both Haloperoxidase and phosphatase activity and is related to glucose-6-phosphatase. The effects of mutation to alanine on Haloperoxidase activity were studied for the proposed catalytic residue His-404 and for residue Asp-292, which is located close to the vanadate cofactor. The mutants were strongly impaired in their ability to oxidize chloride but still oxidized bromide, although they inactivate during turnover. The effects on the optical absorption spectrum of vanadium chloroperoxidase indicate that mutant H404A has a reduced affinity for the cofactor, whereas this affinity is unchanged in mutant D292A. The effect on the phosphatase activity of the apoenzyme was investigated for six mutants of putative catalytic residues. Effects of mutation of His-496, Arg-490, Arg-360, Lys-353, and His-404 to alanine are in line with their proposed roles in nucleophilic attack, transition-state stabilization, and leaving-group protonation. Asp-292 is excluded as the group that protonates the leaving group. A model based on the mutagenesis studies is presented and may serve as a template for glucose-6-phosphatase and other related phosphatases. Hydrolysis of a phospho-histidine intermediate is the rate-determining step in the phosphatase activity of apochloroperoxidase, as shown by burst kinetics.
Bradley S. Moore - One of the best experts on this subject based on the ideXlab platform.
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a unifying paradigm for naphthoquinone based meroterpenoid bio synthesis
Nature Chemistry, 2017Co-Authors: Zachary D Miles, Bradley S. Moore, Stefan Diethelm, Henry P Pepper, David M Huang, Jonathan H GeorgeAbstract:Bacterial naphthoquinone meroterpenoid natural products defy biosynthetic logic via classical biochemical paradigms. Now, an enzyme promoted α-hydroxyketone rearrangement catalysed by vanadium-dependent Haloperoxidases reveals a conserved biosynthetic reaction in this molecular class that further has inspired a concise biomimetic synthesis of naphthomevalin, a prominent member of the napyradiomycin meroterpenes.
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Exploring the chemistry and biology of vanadium-dependent Haloperoxidases.
The Journal of biological chemistry, 2009Co-Authors: Jaclyn M. Winter, Bradley S. MooreAbstract:Nature has developed an exquisite array of methods to introduce halogen atoms into organic compounds. Most of these enzymes are oxidative and require either hydrogen peroxide or molecular oxygen as a cosubstrate to generate a reactive halogen atom for catalysis. Vanadium-dependent Haloperoxidases contain a vanadate prosthetic group and utilize hydrogen peroxide to oxidize a halide ion into a reactive electrophilic intermediate. These metalloenzymes have a large distribution in nature, where they are present in macroalgae, fungi, and bacteria, but have been exclusively characterized in eukaryotes. In this minireview, we highlight the chemistry and biology of vanadium-dependent Haloperoxidases from fungi and marine algae and the emergence of new bacterial members that extend the biological function of these poorly understood halogenating enzymes.
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molecular basis for chloronium mediated meroterpene cyclization cloning sequencing and heterologous expression of the napyradiomycin biosynthetic gene cluster
Journal of Biological Chemistry, 2007Co-Authors: Jaclyn M. Winter, Michelle C Moffitt, Emmanuel Zazopoulos, James B Mcalpine, Pieter C Dorrestein, Bradley S. MooreAbstract:Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent Haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent Haloperoxidases.
Eva Holm Hansen - One of the best experts on this subject based on the ideXlab platform.
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Effect of environmental and physiological factors on the antibacterial activity of Curvularia Haloperoxidase system against Escherichia coli.
Journal of Applied Microbiology, 2005Co-Authors: Eva Holm Hansen, Soren Molin, Thomas Schäfer, Lone GramAbstract:E.H. HANSEN, T. SCHAFER, S. MOLIN AND L. GRAM. 2004. Aims: The aim of this study was to investigate the influence of environmental and physiological factors on the susceptibility of Escherichia coli to the Curvularia Haloperoxidase system. Methods and Results: The Curvularia Haloperoxidase system is a novel enzyme system that produces reactive oxygen species which have an antimicrobial effect. Escherichia coli MG1655 was exposed to the Curvularia Haloperoxidase system under different temperatures and NaCl concentrations and after exposure to different stress factors. Temperature clearly affected enzymatic activity with increasing antibacterial effect at increasing temperature. The presence of NaCl interfered with the enzyme system and in the presence of 1% NaCl, no antibacterial effect could be observed at pH 7. Cells grown at pH 8AE0 were in one experiment more resistant than cells grown at pH 6AE5, whereas cells grown in the presence of 2% NaCl were more susceptible to the Curvularia Haloperoxidase system. Conclusions: Environmental and physiological factors can affect the antibacterial activity of the Curvularia Haloperoxidase system. Significance and Impact of the Study: The study demonstrates a systematic approach in assessing the effect of environmental and physiological factors on microbial susceptibility to biocides. Such information is crucial for prediction of application as well as potential side-effects.
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Elucidation of the Antibacterial Mechanism of the Curvularia Haloperoxidase System by DNA Microarray Profiling
Applied and environmental microbiology, 2004Co-Authors: Eva Holm Hansen, Soren Molin, Thomas Schäfer, Mark A. Schembri, Per Klemm, Lone GramAbstract:A novel antimicrobial enzyme system, the Curvularia Haloperoxidase system, was examined with the aim of elucidating its mechanism of antibacterial action. Escherichia coli strain MG1655 was stressed with sublethal concentrations of the enzyme system, causing a temporary arrest of growth. The expression of genes altered upon exposure to the Curvularia Haloperoxidase system was analyzed by using DNA microarrays. Only a limited number of genes were involved in the response to the Curvularia Haloperoxidase system. Among the induced genes were the ibpA and ibpB genes encoding small beat shock proteins, a gene cluster of six genes (b0301-b0306) of unknown function, and finally, cpxP, a member of the Cpx pathway. Knockout mutants were constructed with deletions in b0301-b0306, cpxP, and cpxARP, respectively. Only the mutant lacking cpxARP was significantly more sensitive to the enzyme system than was the wild type. Our results demonstrate that DNA microarray technology cannot be used as the only technique to investigate the mechanisms of action of new antimicrobial compounds. However, by combining DNA microarray analysis with the subsequent creation of knockout mutants, we were able to pinpoint one of the specific responses of E. coli-namely, the Cpx pathway, which is important for managing the stress response from the Curvularia Haloperoxidase system.
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curvularia Haloperoxidase antimicrobial activity and potential application as a surface disinfectant
Applied and Environmental Microbiology, 2003Co-Authors: Eva Holm Hansen, Soren Molin, Thomas Schäfer, Line Albertsen, Charlotte Johansen, Jens Christian Frisvad, Lone GramAbstract:A presumed antimicrobial enzyme system, the Curvularia Haloperoxidase system, was examined with the aim of evaluating its potential as a sanitizing agent. In the presence of hydrogen peroxide, Curvularia Haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds. The Curvularia Haloperoxidase system caused several-log-unit reductions in counts of bacteria (Pseudomonas spp., Escherichia coli, Serratia marcescens, Aeromonas salmonicida, Shewanella putrefaciens, Staphylococcus epidermidis, and Listeria monocytogenes), yeasts (Candida sp. and Rhodotorula sp.), and filamentous fungi (Aspergillus niger, Aspergillus tubigensis, Aspergillus versicolor, Fusarium oxysporum, Penicillium chrysogenum, and Penicillium paxilli) cultured in suspension. Also, bacteria adhering to the surfaces of contact lenses were killed. The numbers of S. marcescens and S. epidermidis cells adhering to contact lenses were reduced from 4.0 and 4.9 log CFU to 1.2 and 2.7 log CFU, respectively, after treatment with the Curvularia Haloperoxidase system. The killing effect of the Curvularia Haloperoxidase system was rapid, and 106 CFU of E. coli cells/ml were eliminated within 10 min of treatment. Furthermore, the antimicrobial effect was short lived, causing no antibacterial effect against E. coli 10 min after the system was mixed. Bovine serum albumin (1%) and alginate (1%) inhibited the antimicrobial activity of the Curvularia Haloperoxidase system, whereas glucose and Tween 20 did not affect its activity. In conclusion, the Curvularia Haloperoxidase system is an effective sanitizing system and has the potential for a vast range of applications, for instance, for disinfection of contact lenses or medical devices.
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curvularia Haloperoxidase antimicrobial activity and potential application as a surface disinfectant
Applied and Environmental Microbiology, 2003Co-Authors: Eva Holm Hansen, Soren Molin, Thomas Schäfer, Line Albertsen, Charlotte Johansen, Jens Christian Frisvad, Lone GramAbstract:Bacteria and other microorganisms attach readily to surfaces in both natural and man-made ecosystems. If they are left undisturbed, and if nutrients are supplied, biofilms may form (9, 10). Microorganisms attached as single cells or in monolayers and more mature microbial biofilms are the cause of problems in several areas, for example, in the food industry, where bacteria colonize processing equipment and subsequently contaminate (or recontaminate) the products (22). There are reports of clones of pathogenic bacteria, such as Listeria monocytogenes, which have persisted in food-processing environments for several years (12, 27). Even more severe problems are seen in the medical area, where surface-adhered microorganisms cause infections from contaminated contact lenses (42) or medical devices, such as catheters or ear tubing (4, 28, 34). Effective cleaning and surface disinfection is essential to control these hazards. A vast range of agents or compounds that can inactivate microorganisms are therefore used across many sectors, from the pharmaceutical industry to the food industry to water distribution lines. Such sanitizers are used both for surface disinfection and for water purification (15, 21, 25). Due to the environmental impact, there is a growing concern about the disposal of sanitizers and chemically synthesized disinfectants. This has led to increased research efforts to identify and evaluate natural antimicrobial compounds as novel sanitizers (14, 30). Natural antimicrobial systems are widespread, occurring in mammals, cold-blooded animals, plants, and microorganisms (3). Examples of such compounds are iron-chelating substances, such as lactoferrin (41), small basic peptides (16, 17, 20), and enzymes with antimicrobial activity. Examples of the last are lactoperoxidase, secreted from various mammalian glands (14, 43), and lysozyme, found in body fluids, such as tears, saliva, and human milk (26). Peroxidases other than lactoperoxidase may also exhibit antimicrobial activity. Thus, preliminary studies have demonstrated that a Haloperoxidase isolated from the filamentous fungus Curvularia verruculosa has antimicrobial activity (18). Curvularia Haloperoxidase (Novozymes A/S, Bagsvaerd, Denmark), examined in this study, is not commercially available but is produced by recombinant technology that allows production on an industrial scale. The enzyme oxidizes halides, such as bromide, chloride, and iodide, in the presence of hydrogen peroxide, and it is believed, albeit not experimentally verified, that reactive oxygen species with antimicrobial effects are produced. Commercial sanitizers based on, for example, hydrogen peroxide alone have several side effects, such as corrosion. The Curvularia Haloperoxidase system uses 100-fold-lower concentrations of H2O2 and would therefore be expected to be less corrosive. The present study was undertaken with the aim of evaluating the Curvularia Haloperoxidase system as a sanitizing agent. The stability of the system, as well as effects of potential interfering agents, were studied to evaluate potential areas of application. Finally, the system was tested on bacteria adhering to contact lenses to determine its potential as a surface disinfectant. (This work was carried out as part of an industrial Ph.D. study by Eva Holm Hansen.)
Lone Gram - One of the best experts on this subject based on the ideXlab platform.
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Effect of environmental and physiological factors on the antibacterial activity of Curvularia Haloperoxidase system against Escherichia coli.
Journal of Applied Microbiology, 2005Co-Authors: Eva Holm Hansen, Soren Molin, Thomas Schäfer, Lone GramAbstract:E.H. HANSEN, T. SCHAFER, S. MOLIN AND L. GRAM. 2004. Aims: The aim of this study was to investigate the influence of environmental and physiological factors on the susceptibility of Escherichia coli to the Curvularia Haloperoxidase system. Methods and Results: The Curvularia Haloperoxidase system is a novel enzyme system that produces reactive oxygen species which have an antimicrobial effect. Escherichia coli MG1655 was exposed to the Curvularia Haloperoxidase system under different temperatures and NaCl concentrations and after exposure to different stress factors. Temperature clearly affected enzymatic activity with increasing antibacterial effect at increasing temperature. The presence of NaCl interfered with the enzyme system and in the presence of 1% NaCl, no antibacterial effect could be observed at pH 7. Cells grown at pH 8AE0 were in one experiment more resistant than cells grown at pH 6AE5, whereas cells grown in the presence of 2% NaCl were more susceptible to the Curvularia Haloperoxidase system. Conclusions: Environmental and physiological factors can affect the antibacterial activity of the Curvularia Haloperoxidase system. Significance and Impact of the Study: The study demonstrates a systematic approach in assessing the effect of environmental and physiological factors on microbial susceptibility to biocides. Such information is crucial for prediction of application as well as potential side-effects.
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Elucidation of the Antibacterial Mechanism of the Curvularia Haloperoxidase System by DNA Microarray Profiling
Applied and environmental microbiology, 2004Co-Authors: Eva Holm Hansen, Soren Molin, Thomas Schäfer, Mark A. Schembri, Per Klemm, Lone GramAbstract:A novel antimicrobial enzyme system, the Curvularia Haloperoxidase system, was examined with the aim of elucidating its mechanism of antibacterial action. Escherichia coli strain MG1655 was stressed with sublethal concentrations of the enzyme system, causing a temporary arrest of growth. The expression of genes altered upon exposure to the Curvularia Haloperoxidase system was analyzed by using DNA microarrays. Only a limited number of genes were involved in the response to the Curvularia Haloperoxidase system. Among the induced genes were the ibpA and ibpB genes encoding small beat shock proteins, a gene cluster of six genes (b0301-b0306) of unknown function, and finally, cpxP, a member of the Cpx pathway. Knockout mutants were constructed with deletions in b0301-b0306, cpxP, and cpxARP, respectively. Only the mutant lacking cpxARP was significantly more sensitive to the enzyme system than was the wild type. Our results demonstrate that DNA microarray technology cannot be used as the only technique to investigate the mechanisms of action of new antimicrobial compounds. However, by combining DNA microarray analysis with the subsequent creation of knockout mutants, we were able to pinpoint one of the specific responses of E. coli-namely, the Cpx pathway, which is important for managing the stress response from the Curvularia Haloperoxidase system.
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curvularia Haloperoxidase antimicrobial activity and potential application as a surface disinfectant
Applied and Environmental Microbiology, 2003Co-Authors: Eva Holm Hansen, Soren Molin, Thomas Schäfer, Line Albertsen, Charlotte Johansen, Jens Christian Frisvad, Lone GramAbstract:A presumed antimicrobial enzyme system, the Curvularia Haloperoxidase system, was examined with the aim of evaluating its potential as a sanitizing agent. In the presence of hydrogen peroxide, Curvularia Haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds. The Curvularia Haloperoxidase system caused several-log-unit reductions in counts of bacteria (Pseudomonas spp., Escherichia coli, Serratia marcescens, Aeromonas salmonicida, Shewanella putrefaciens, Staphylococcus epidermidis, and Listeria monocytogenes), yeasts (Candida sp. and Rhodotorula sp.), and filamentous fungi (Aspergillus niger, Aspergillus tubigensis, Aspergillus versicolor, Fusarium oxysporum, Penicillium chrysogenum, and Penicillium paxilli) cultured in suspension. Also, bacteria adhering to the surfaces of contact lenses were killed. The numbers of S. marcescens and S. epidermidis cells adhering to contact lenses were reduced from 4.0 and 4.9 log CFU to 1.2 and 2.7 log CFU, respectively, after treatment with the Curvularia Haloperoxidase system. The killing effect of the Curvularia Haloperoxidase system was rapid, and 106 CFU of E. coli cells/ml were eliminated within 10 min of treatment. Furthermore, the antimicrobial effect was short lived, causing no antibacterial effect against E. coli 10 min after the system was mixed. Bovine serum albumin (1%) and alginate (1%) inhibited the antimicrobial activity of the Curvularia Haloperoxidase system, whereas glucose and Tween 20 did not affect its activity. In conclusion, the Curvularia Haloperoxidase system is an effective sanitizing system and has the potential for a vast range of applications, for instance, for disinfection of contact lenses or medical devices.
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curvularia Haloperoxidase antimicrobial activity and potential application as a surface disinfectant
Applied and Environmental Microbiology, 2003Co-Authors: Eva Holm Hansen, Soren Molin, Thomas Schäfer, Line Albertsen, Charlotte Johansen, Jens Christian Frisvad, Lone GramAbstract:Bacteria and other microorganisms attach readily to surfaces in both natural and man-made ecosystems. If they are left undisturbed, and if nutrients are supplied, biofilms may form (9, 10). Microorganisms attached as single cells or in monolayers and more mature microbial biofilms are the cause of problems in several areas, for example, in the food industry, where bacteria colonize processing equipment and subsequently contaminate (or recontaminate) the products (22). There are reports of clones of pathogenic bacteria, such as Listeria monocytogenes, which have persisted in food-processing environments for several years (12, 27). Even more severe problems are seen in the medical area, where surface-adhered microorganisms cause infections from contaminated contact lenses (42) or medical devices, such as catheters or ear tubing (4, 28, 34). Effective cleaning and surface disinfection is essential to control these hazards. A vast range of agents or compounds that can inactivate microorganisms are therefore used across many sectors, from the pharmaceutical industry to the food industry to water distribution lines. Such sanitizers are used both for surface disinfection and for water purification (15, 21, 25). Due to the environmental impact, there is a growing concern about the disposal of sanitizers and chemically synthesized disinfectants. This has led to increased research efforts to identify and evaluate natural antimicrobial compounds as novel sanitizers (14, 30). Natural antimicrobial systems are widespread, occurring in mammals, cold-blooded animals, plants, and microorganisms (3). Examples of such compounds are iron-chelating substances, such as lactoferrin (41), small basic peptides (16, 17, 20), and enzymes with antimicrobial activity. Examples of the last are lactoperoxidase, secreted from various mammalian glands (14, 43), and lysozyme, found in body fluids, such as tears, saliva, and human milk (26). Peroxidases other than lactoperoxidase may also exhibit antimicrobial activity. Thus, preliminary studies have demonstrated that a Haloperoxidase isolated from the filamentous fungus Curvularia verruculosa has antimicrobial activity (18). Curvularia Haloperoxidase (Novozymes A/S, Bagsvaerd, Denmark), examined in this study, is not commercially available but is produced by recombinant technology that allows production on an industrial scale. The enzyme oxidizes halides, such as bromide, chloride, and iodide, in the presence of hydrogen peroxide, and it is believed, albeit not experimentally verified, that reactive oxygen species with antimicrobial effects are produced. Commercial sanitizers based on, for example, hydrogen peroxide alone have several side effects, such as corrosion. The Curvularia Haloperoxidase system uses 100-fold-lower concentrations of H2O2 and would therefore be expected to be less corrosive. The present study was undertaken with the aim of evaluating the Curvularia Haloperoxidase system as a sanitizing agent. The stability of the system, as well as effects of potential interfering agents, were studied to evaluate potential areas of application. Finally, the system was tested on bacteria adhering to contact lenses to determine its potential as a surface disinfectant. (This work was carried out as part of an industrial Ph.D. study by Eva Holm Hansen.)
