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The vast scope of the biotechnological aspects of chloroperoxidase from Caldariomyces fumago, as highlighted in this report, is limited due to imposed quantity restraints in contrast to the amount of information on chloroperoxidase freely available, however a broad range of biotechnological aspects are represented.
Chloroperoxidase is a famous and studied heam-thiolate containing bioinorganic molecule of the haloperoxidase class of enzymes which in addition to its namesake, halogenation processes, carries out the functions of other biotechnologically and biochemically important enzyme classes namely catalase, cytochrome p-450, monoxygenase and peroxygenase.
Chloroperoxidase catalyses a wide range of biotechnologically important oxidations and molecular conversions for example the conversion of indole to oxindole, a molecule with great capacity in the pharmaceutical sector.
Halogenation by chloroperoxidase, albeit somewhat restricted, has profound applications in synthetic organic chemistry and in biotechnology as it is not limited to the utilization of one halogen. Chloroperoxidase oxidation mechanisms are highly desirable in a multitude of topical applications from the development and large-scale manufacture of pharmaceuticals to industrial wastewater treatment. Chloroperoxidase has shown great stability in the biotechnological sector and is commercially available at economic rates.
The stability of chloroperoxidase, and its immobilization, has led to the design and manufacture of an innovative, controlled, bactericidal and sporicidal surface cover which has great applicability to domestic, industrial and commercial settings.
This report also illustrates the function of CPO in the synthesis of chloromethyloxirane which is a well-known and economically important precursor monomer constituent in the production of many industrial polymers, in addition to having niche applications of its own in various areas of industry.
The peroxidase family are an enzymatic family which typically act as catalysts for peroxide reactions when peroxides act as electron donors in acidic environments which cleaves the oxygen-oxygen bond to yield two hydroxyl groups. This is expressed in the stoichiometric equation:
ROOR' + 2 e- + 2 H+ â†’ ROH + R'OH
An example of this would be the conversion of two hydrogen peroxide molecules to two water molecules and two oxygen molecules by the enzyme catalase. (Chelikani P., Fita I., Loewen P.C. 2004)
2 H2O2 + 2 e- + 2 H+ â†’ 2 H2O + 2 O2
A substantial amount of peroxidases use hydrogen peroxide as an ideal substrate, an example of this would be the haloperoxidases which are a class of peroxidase which function to oxidize halides by means of hydrogen peroxide. (Butler A., 1998)
Amongst these are the oxidoreductases, an enzyme class that catalyse electrons transfer from the reductant to the oxidant. This group of enzymes is important for metabolic purposes as it generally utilizes NADP or NAD+ as cofactors. (Berg J., Tymoczko J., Stryer L. 2012)
From a purely thermodynamic point of view the Nernst equation, this is described as the potential of the cell under non-standard conditions (V) is equal to the potential of the cell minus the product of the gas constant multiplied by temperature divided by the product of the number of moles multiplied by Faraday's constant, which is then multiplied by the natural log of the product of the activities of the reduced and oxidized ionic species respectively. (Saroff H.A., 2007) This is expressed in the equation:
Ecell = Eo - (RT/nF)ln([Red]/[Ox])
The Nernst equation shows that hydrogen peroxide can oxidize chloride, yielding 1.36 V thermodynamically under standard conditions, for example by reacting with chloroperoxidase. (Clark J., 2002)
For the purposes of clarity this report will focus on the enzyme chloroperoxidase, a 42 kDa oxidoreductase, which is produced from the ascomycetous fungus Caldariomyces fumago.
Chloroperoxidase (EC 184.108.40.206) from C. fumago contain a heam, a porhyrin ring-iron ligand, whereas chloroperoxidases from other sources utilize vanadium as a cofactor. (Hofrichter M., Ullrich R., 2006)
C. fumago chloroperoxidase (CPO) is utilized as an enzymatic catalyst for the reversible halogenation, in particular chlorination, of a range of organic molecules in biochemical reactions, forming stable carbon-halide bonds, generically expressed as, where X is a halide:
2 RH + 2 X- + H2O2 \rightleftharpoons 2 RX + 2 H2O
In detail, when used in chlorination, hypochlorous acid (HOCl) production is catalysed by the transferring one oxygen atom from hydrogen peroxide to a chloride atom. At a different enzyme site, activated aliphatic and aromatic organic substrates are chlorinated catalytically using HOCl and other derived species of chloride. (Griffin B.W., 1983)
When halides are not present in the reaction it displays peroxygenase and peroxidase activities; during which an oxygen atom from hydrogen peroxide on to, for example alkene side chains, which results in side chain epoxidation, or methyl side chains where it results in hydroxylation. (Shaik S. et al, 2002)
In addition to acting as a halogenation catalyst exhibiting peroxidase and peroxygenase activities, CPO exhibits cytochrome P450 activities, therefore acting as a catalyst of a large number of organic substances. (Sundaramoorthy M., Terner J, Poulos T.L. 1995)
For example, the metabolically important monooxygenase reaction, where one oxygen atom from a dioxygen molecule is inserted into an organic substrate while the other oxygen atom is reduced to a water molecule:
RH + O2 + NADPH + H+ â†’ ROH + H2O + NADP+
Structurally, CPO is quite unique amongst other heam enzymes, as it folds into a tertiary structure including eight Î±-helices. (Sundaramoorthy M., Terner J, Poulos T.L. 1995)
Image: Haem chloroperoxidase
Source: Protein Data Bank (Google Images)
Biotechnological applications in halogenation:
In addition to peroxidation reactions CPO can perform halogenation reactions, where the donors involved are chloride and bromide ions. As stated previously, CPO mirrors the functions of a select assortment of non-peroxidase haem-containing enzymes, such as cytochrome P-450.
CPO reactions show to be either dependent on, whether it be total dependence or merely to accelerate the reaction, on halide ions, the mode of dependency depends on the circmstances of the reaction required. CPO has the capacity to perform the functions of the enzyme catalase; thereby it has the capacity to oxidise alcohols and formate. (Thomas, J.A., Morris, D.R., and Hager, L.P. 1970)
CPO is a cataylist the reactions which oxidise certain primary alcohols to their corresponding aldehydes in two phase systems of a pH 5.0 buffer and ethyl acetate or hexane respectively.
(Kiljunen E., Kanerva L.T. 2000) A functionally similar enzyme, myeloperoxidase, shows that a free hypohalite intermediates performs the actual molecular chlorination duties required for successful chlorination, in this case HOCl. (Masuda M. et al 2001)
With respect to the biotechnological sector, by understanding the exact mechanism of CPO it is possible to understand the mechanics of other heam containing enzymes and their relationships; therefore the oxidation of halide ions is key to fully comprehending the functionality of CPO and its related enzymes.
However, a downside to CPO practicality exists due to the tendency of CPO to dismutate components of hydrogen peroxide added which results in the formation of the highly reactive oxygen species, superoxide (O2-) which can result in haem bleaching and in turn possible enzyme inactivation. To overcome this experimental protective antioxidants have been added to immobilize CPO on various chemical carrier molecules. Due to successful attempts there are plans to introduce CPO into highly specific synthesis in the future. (Hofrichter M., 2010)
Biotechnological applications for industrial waste water treatment:
In the presence of hydrogen peroxide, CPO catalyses oxidation of a multitude of chlorinated phenols which are normally present as by-products in industrial waste waters.
Chlorinated phenols are considered environmental pollutants due to their high pH and biological toxicity. (Carmichael et al. 1985) These compounds are usually found in wastewaters released from industrial plants such as those specialising in petrochemical and pharmaceutical production and from agricultural run-off which would contain pesticides. (Exon J.H., 1984) Conventionally oxidation, degradation by microbial processes and extraction of solvents from effluents are used as phenol removal techniques. (Rivas et al. 1999) Oxidizing inorganic biomolecules, such as hydrogen peroxide, are widely used in industrial waste water treatment due to their effectiveness and economic value. Hydrogen peroxide can also make toxic and biologically inhibiting organic molecules more susceptible to microbial degradation by oxidising the compounds as a pre-treatment measure.
Hydrogen peroxide utilization as a catalyst as biological reactions as a peroxidase substrate suffers limitations due to inhibitions when presented in concentrations above 25 mM and inhibitory consequences when exposed to low concentrations for extended periods of time. (Nicell J., Wright H., 1997). In situ electrogeneration can provide a solution for this inhibitory circumstance, by means of controlling the introduction of very low concentrations of hydrogen peroxide supplements thus achieving an effective basal oxidation level, and preventing inhibition. This method was applied to some economically important biocatalytical reactions such as CPO oxidation of thioanisole. The electrogeneration method yields a much lower and a much more easily controlled formation rate of hydrogen peroxide than any other method reported. (Lutz S. et al. 2004). This illustrates the economic and ecological, by virtue of biotechnological, value of CPO in the industrial sector.
A biotechnological application in the potential treatment of HIV:
CPO shows a great affinity for the oxidation of the amine group (N-oxidation) of arylamines. Nitroso compound synthesis from arylamines shows to be very efficient in the presence of CPO. (Corbett M.D. et al., 1978)
Studies show that rapid oxidation of indole in the presence of chloroperoxidase, using hydrogen peroxide as an electron donor, yields the "unusual" metabolite oxindole as the single major product. (Corbett M.D., Chipko B.R., 1979)
The production of oxindole in an industrial setting can be utilized for various pharmaceutical uses. For example, oxindoles are being studied due to their mechanistic properties which allow them to act as non-nucleoside reverse transcriptase inhibitors for the treatment of HIV infections by preventing viral replication; this is particularily successful due to the interaction of oxindole with the enzyme reverse transcriptase. Oxindoles perform this function by binding to the non-nucleoside reverse transcriptase inhibition site of the HIV-1 virion. (Tao J. et al., 2006)
Source: Google Images
Courtesy of www.sigmaaldrich.com
Biotechnological applications for the synthesis of a bactericide and sporicide:
Due to the concern regarding surface hygiene and bacterial surface contamination and their spores and the potential environmental toxicity of conventional disinfectants, for example aldehydes, alcohols and ammonium compounds, halides have been studied due to their antimicrobial effects. Studies have also shown significant activity against antibiotic resistant bacterial strains in addition to virucidal and fungicidal properties. (Taylor G., Butler M. 1982)
High concentrations of halogen, for example iodine and chlorine, which may be required prove to be environmentally unappealing. Both iodine and chlorine are made commercially available and are commonly used as a topical precaution against surface pathogens due to their activity which interferes with metabolism by rapidly oxidising cytosol and membrane proteins of the cell. (Mcdonnell G., Russell A.D. 1999). It has been reported that controlled enzymatically generated chloride containing compounds or molecular iodine provides a solution to problems surrounding toxicity as opposed to directly applying halogens as antimicrobial agents. (Amitai G. et al. 2003) While CPO catalyses the oxidation of chloride to HOCl, an effective biocide, laccase utilizes molecular oxygen as a terminal electron acceptor to catalyse the oxidation of two iodine anions to molecular iodine. (Xu F. 1996)
The activity of laccase can be regulated by small molecules that play an active role in electron transfer between the substrate and enzyme, which yields bactericidal and sporicidal results. (Kulys J, Bratkovskaja I, Vidziunaite R. 2005) The production of a surface coating containing CPO and laccase would provide broad spectrum sterility to work surfaces over extended periods of time. Nanoscale supports obtained by multi-walled carbon nanotubes provided a structure which allowed for the physical immobilization of the enzyme. This immobilization allowed for the introduction into a suitable surface coating. These coatings
are highly bactericidal and sporicidal; which may lead to further advancements in the area of antimicrobial surfaces. (Grover et al. 2012)
Biotechnological applications for the synthesis of industrially important epoxides:
CPO can catalyse an asymmetric epoxidation reaction of 3-chloropropene thus converting it to chloromethyloxirane, utilizing t-butyl hydroperoxide as oxygen donor, and carried out in a phosphate buffer. For optimum yields the process is carried out for 60 minutes at pH 5.5 at room temperature in the presence of the co-solvent 1-ethyl-3-methylimidazolium. (Wu J. et al 2010) The production of chloromethyloxirane in this fashion is mechanically achieved via a biphasic reaction process. The primary phase is hydrochlorination using CPO obtained HOCl which results in a mixture of two distinct alcohol species. (Sienel G., Rieth R., and Rowbottom K.T. 2005)
CH2=CHCH2Cl + HOCl â†’ HOCH2CHClCH2Cl &/or ClCH2CH(OH)CH2Cl
The secondary phase involves in the neutralization treatment with an alkali, yielding an epoxide. (http://2.imimg.com/data2/PF/OB/MY-2946707/epichlorohydrine.pdf)
HOCH2CHClCH2Cl &/or ClCH2CH(OH)CH2Cl + NaOH â†’ CH2CHOCH2Cl + NaCl + H2O
Chloromethyloxirane is industrially converted to yield 2-[[4-[2-[4-(oxiran-2-ylmethoxy)phenyl]propan-2-yl]phenoxy]methyl]oxirane, commercially known as bisphenol A diglycidyl ether (BADGE), from a reaction with bisphenol A; a primary component involved in epoxy resin manufacture. (Srivastava A.K. & Mohan P. 1997).
CPO produced chloromethyloxirane is perceived as a precursor in synthetic organic chemistry. Chloromethyloxirane reacts with alkali nitrate compounds to yield the solvent glycidyl nitrate, which may also be used as an insecticide and propellant. (Oyama S.T. 2008)
The enzyme chloroperoxidase from Caldariomyces fumago is an incredibly potent catalyst for a multitude of reactions in a substantial quantity of biotechnological areas.
Since their discovery substantial progress has been made in the study and application of oxidative fungal peroxidases such as peroxidases, phenol oxidases and peroxygenases, the mechanisms of all can be carried out by CPO.
Hofrichter M. & Ullrich R. (2010) expect a breakthrough regarding biocatalysts such as CPO in the coming decade which further illustrates the potential biotechnological aspects of CPO and its value in industry. Large-scale production of CPO will aid in this by means of genetic engineering of Caldariomyces fumago cells which will allow for the systematic overproduction of CPO.
With the exception of the slight downfall regarding the synthesis of superoxide, which is becoming less troublesome with advancements, the future applications of CPO are potentially limitless.
Currently, certain industries show great dependency on CPO and its applications, one such industry that benefits in this fashion is any plant which requires epoxidation which can form monomers and resultant polymers such as the commercially available epoxy resin.
This thesis is the outcome of great support from many gifted people from a multitude of academic backgrounds during Semester 1 of my penultimate year in Ardmore House, University College Dublin. Firstly I would like to express my gratitude to my research supervisor, Dr. Cormac D. Murphy for his guidance and support which helped guide me in the right direction with this report. I would like to thank the other academic members of staff for the MICR30070 (Skills in Microbiology) module: Professor James P. O'Gara, Dr. Hilary McMahon, Dr. Tadhg O'Croinin, and Dr. Kevin O'Connor respectively for the education they collectively provided in other modules in my penultimate stage and in previous stage, I would particularily like to thank Dr. Patrick Caffrey and Professor Wim G. Meijer for the information provided in the module MICR30030 (Microbial Physiology) which greatly assisted in my comprehension of biochemical processes applicable to this report.
I would like to express my gratitude to Ms. Carmel Norris, Liaison Librarian for the Main Library of University College Dublin for her support with regards to the Harvard referencing and essay writing skills - a meeting arranged by Dr. Jarlath Nally, School of Veterinary Medicine, University College Dublin, who I would also like to thank. I also would like to extend my appreciation to the staff at the University College Dublin Main Library and to the staff at the library at the Institute of Technology, Carlow for their assistance in helping me acquire certain articles and references from the extensive archives at both institutions.