Engineering chloroperoxidase for activity and stability

Random mutagenesis of Caldariomyces fumago was carried out to produce a chloroperoxidase (CPO) with enhanced activity in mixtures of aqueous buffers and organic cosolvents. A mutant CPO with a 3.4-fold increased activity in 40% aqueous tert-butyl alcohol was obtained.

A CPO-surfactant conjugate, prepared by colyophilisation, mediated the oxidation of thioanisole to its (R)-sulfoxide by TBHP in water-saturated isooctane. Copolymerisation of CPO and a toluenediisocyanate prepolymer resulted in a stable preparation that mediated the oxidation of indole to 2-oxindole in a range of organic solvents. The highest yield was obtained in 1-octanol.     

Stabilization of chloroperoxidase by polyethylene glycols in aqueous media: kinetic studies and synthetic applications

Chloroperoxidase (CPO) is one of the most versatile of the heme peroxidase enzymes for synthetic applications. Despite the potential use of CPO, commercial processes have not been developed because of the low water solubility of many organic substrates of synthetic interest and the limited stability due to inactivation by H(2)O(2). CPO catalytic properties have been studied in aqueous solutions in the presence of short-chain poly(ethylene glycol)s (PEGs), and the sulfoxidation of thioanisole, as model substrate, has been investigated. The addition of PEGs allows a better substrate solubilization in the reaction mixture and the enzyme to retain more of its initial activity, with respect to pure buffer. Kinetic studies were performed to optimize the experimental conditions, and complete enantioselective conversion to the (R)-sulfoxide (ee = 99%) was observed in the presence of PEG 200 and tri(ethylene glycol). The relevant stabilization of chloroperoxidase due to the presence of PEGs allows the enzyme to convert the substrate with significant product yields even after 10 days, with a consequent increase in enzyme productivity. This is a promising result in view of industrial application of the enzyme.     

Selective oxidations with molecular oxygen,catalyzed by chloroperoxidase in the presence of a reductant

Chloroperoxidase (CPO) catalyzes the oxidation of various substrates with molecular oxygen as the primary oxidant, in the presence of dihydroxyfumaric acid (DHF) as a sacrificial reductant. For example, indole is oxidized to 2-oxindole with up to 77% selectivity and thioanisole to the corresponding R-sulfoxide (e.e. >99%). To our knowledge, these are the first examples of (enantio)selective aerobic oxidations catalyzed by peroxidases. A mechanism is proposed which involves initial formation of hydrogen peroxide via autoxidation of DHF. CPO subsequently uses the hydrogen peroxide for the selective oxidation of the substrate, via an oxygen transfer mechanism. In contrast, horseradish proxidase (HRP) primarily catalyzes the oxidation of DHF via a classical peroxidase mechanism and oxidations of added substrates are aselective.     

Reduced‐oxidized difference spectral analysis and chemiluminescence‐based Scatchard analysis demonstrate selective binding of myeloperoxidase to microbes

Myeloperoxidase (MPO), a microbicidal haloperoxidase of neutrophil leukocytes, was observed to selectively bind to bacteria. Binding was quantified by dithionite‐reduced minus oxidized (R? O) difference spectral analysis. Escherichia coli and Pseudomonas aeruginosa showed large MPO binding by R? O difference spectral analysis, whereas Streptococcus sanguinis did not. For increased sensitivity, free and microbe‐bound MPO and chloroperoxidase (CPO) activities were quantified by acid‐optimum haloperoxidase‐dependent chemiluminescence (CL) measurements, and these data were used for Scatchard analysis. The MPO bound/free (B/F) CL ratio was 49.5 for P. aeruginosa, 14.6 for Staphylococcus aureus, 2.8 for E. coli, 0.7 for Candida albicans and 0.4 for S. sanguinis. By comparison, the CPO B/F CL ratio was 0.03 for P. aeruginosa, 0.09 for S. aureus, 0.31 for E. coli, 0.18 for C. albicans and 0.16 for S. sanguinis. As a member of the lactic acid family of bacteria and a viridans streptococcus, S. sanguinis does not synthesize cytochromes and is catalase‐negative. The metabolic products of S. sanguinis, i.e. lactic acid and hydrogen peroxide, provide optimal acidity and substrate for MPO oxidation of chloride to hypochlorite. Hypochlorite can react with organic substrates to yield dehydrogenated or chlorinated products, but when peroxide is not limiting, hypochlorite reacts with peroxide yielding singlet oxygen. The reactivity of hypochlorite is dependent on substrate availability. The microsecond half‐life of electronically excited singlet oxygen restricts reactivity to within a radius of <0.25 µm; i.e. the reactivity of singlet oxygen is both substrate and half‐life dependent. Poor MPO binding provides protection and possibly competitive advantage to viridans streptococci. Copyright © 2010 John Wiley & Sons, Ltd.     

Chloroperoxidase-catalyzed enantioselective oxidations in hydrophobic organic media.

Chloroperoxidase from Caldariomyces fumago, a peroxidase that performs P450-like chemistry, was immobilized via covalent attachment into polyurethane foam as well as conjugated with a surfactant or polymer via colyophilization. The resulting preparations catalyzed enantio- and regioselective oxidations in hydrophobic organic media with tert-butyl hydroperoxide as the oxidant.Dried PUR-foam immobilized CPO mediated the selective oxidation of indole to 2-oxindole (regioselectivity: 99%) in water-saturated isooctane or 1-octanol. Thioanisole was converted into the corresponding (R)-sulfoxide (ee > 99%) in isooctane medium.The complexes of CPO with sodium octadecylsulphate or ethyl cellulose mediated the oxidation of thioanisole in water-immiscible organic media with variable enantioselectivity due to radical side-reactions. In the presence of alpha-tocopherol, acting as radical scavenger, the (R)-sulfoxide was formed with ee > 90%. The effect of the water activity on the catalytic activity of the complexes was investigated.The CPO complexes likewise mediated the regioselective oxidation of indole into 2-oxindole in water-saturated isooctane or 1-octanol and its kinetics were investigated. The reaction suffered from substrate inhibition when carried out in isooctane.     

Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme

The Caldariomyces fumago chloroperoxidase was successfully expressed in Aspergillus niger. The recombinant enzyme was produced in the culture medium as an active protein and could be purified by a three-step purification procedure. The catalytic behavior of recombinant chloroperoxidase (rCPO) was studied and compared with that of native CPO. The specific chlorination activity (47 units/nmol) of rCPO and its pH optimum (pH 2.75) were very similar to those of native CPO. rCPO catalyzes the oxidation of various substrates in comparable yields and selectivities to native CPO. Indole was oxidized to 2-oxindole with 99% selectivity and thioanisole to the corresponding R-sulfoxide (enantiomeric excess >98%). Incorporation of (18)O from labeled H(2)18O(2) into the oxidized products was 100% in both cases.     

Oxygenation of organosulfur compounds by peroxidases: evidence of an electron transfer mechanism for lactoperoxidase

The oxygenation of benzyl methylsulfide, thioanisole, and thiobenzamide to the respective sulfoxides was found to be catalyzed by chloroperoxidase, lactoperoxidase, and horseradish peroxidase. The activities of lactoperoxidase and horseradish peroxidase were similarly low toward benzyl methylsulfide and thioanisole but lactoperoxidase efficiently catalyzed the oxygenation of thiobenzamide while horseradish peroxidase showed low activity. Chloroperoxidase had high reactivity toward all three substrates tested in halide-independent reactions and only small differences in the rates of enzymatic sulfoxidation were observed. The logarithm of lactoperoxidase activity was found to linearly correlate with the voltammetric peak potentials for oxidation of the three substrates tested. The results of this study are consistent with a one-electron transfer mechanism for lactoperoxidase-mediated sulfoxidation.     

Deactivation mechanisms of chloroperoxidase during biotransformations

Inactivation mechanisms of chloroperoxidase (CPO) from Caldariomyces fumago have been investigated with the aim of improving the practical utility of CPO for hydrocarbon oxidation. Deactivation studies in the presence of oxidants (i.e., hydrogen peroxide and t-butyl hydroperoxide) indicated that CPO lost oxidation activity toward hydrocarbon substrates during dismutation of hydrogen peroxide. The loss of enzyme activity was accompanied by the apparent destruction of the heme rather than aggregation or denaturation of the apo-protein. The decrease of enzyme activity was significantly retarded by adding the radical scavenger t-butyl alcohol at pH 4.1, or by optimizing the reaction pH. CPO retained greatest oxidation activity at pH 5-6, which may produce a more favorable ionization state of the key amino acid (Glu-183) and thus reduce radical formation. As a result of higher activity at pH 5-6, the total turnover numbers (TTN, defined as the amount of product produced over the catalytic lifetime of the enzyme) for the oxidation of toluene and o-, m-, p-xylenes in substrate/aqueous emulsion systems ranged from ca. 10% to 110% higher at pH 5.5 (20,000 to 45,000 mol product/mol enzyme) compared to pH 4.1. Furthermore, TTNs of CPO increased with increasing turnover frequencies, indicating that higher activity toward reducing substrates reduces radical formation and stabilizes CPO toward inactivation by H(2)O(2). These findings demonstrate the important relationship between CPO stability and activity, and illustrate that large improvements in CPO activity and stability can be achieved through solvent engineering.     

The improvement of chloroperoxidase activities in the presence of ammonium salts and cationic surfactants

The catalytic activities of chloroperoxidase (CPO) including halogenation, oxidation, and peroxidation were investigated in the presence of ammonium salts: tetramethylammonium bromide (TMABr), tetraethylammonium bromide (TEABr), tetrapropylammonium bromide (TPABr), tetrabutylammonium bromide (TBABr), and cationic surfactants: dodecyltrimethylammonium bromide (DTABr) and cetyltrimethylammonium bromide (CTABr). All the mentioned activities were promoted in most cases. The highest modified activity (ABTS peroxidation) was 18.16 times higher in the presence of TMABr than that in pure buffer. The activity enhancement was strongly dependent on the concentration and the hydrophobic chain length of additives, and the structure of substrates. The kinetic parameters showed that the activation was mainly attributed to an increase in k_cat due probably to a catalytically favorable conformation of CPO induced by the additives. Moreover, a lower K_m and higher ratio of k_cat/K_m (specificity constant) was obtained, indicating that both the affinity and specificity of CPO to substrates were improved in the presence of additives. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Ligand recognition by chloroperoxidase using molecular interaction fields and quantum chemistry calculations

We recently reported that chloroperoxidase (CPO) from Caldariomyces fumago showed atypical kinetic behavior for the oxidation of 4,6 dimethyl dibenzothiophene (DMDBT). In this work, we undertake the theoretical study of DMDBT docking into CPO's active site, in order to clarify its binding capacity and affinity using molecular interaction fields and quantum mechanical procedure. The results revealed that CPO has two substrate binding sites with similar affinities for DMDBT. This finding suggests that the atypical kinetic behavior of CPO for the oxidation of DMDBT might be due to the simultaneous binding of two DMDBT molecules during its reaction cycle. Finally, we extend these results to carbazole, a nitrogen-containing PAH, through experimental and theoretical studies.     

Catalytic performance and thermostability of chloroperoxidase in reverse micelle: achievement of a catalytically favorable enzyme conformation

The catalytic performance of chloroperoxidase (CPO) in peroxidation of 2, 2′-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) and oxidation of indole in a reverse micelle composed of surfactant-water-isooctane-pentanol was investigated and optimized in this work. Some positive results were obtained as follows: the peroxidation activity of CPO was enhanced 248% and 263%, while oxidation activity was enhanced 215% and 222% in cetyltrimethylammonium bromide (CTABr) reverse micelle medium and dodecyltrimethylammonium bromide (DTABr) medium, respectively. Thermostability was also greatly improved in reverse micelle: at 40°C, CPO essentially lost all its activity after 5 h incubation, while 58–76% catalytic activity was retained for both reactions in the two reverse micelle media. At 50°C, about 44–75% catalytic activity remained for both reactions in reverse micelle after 2 h compared with no observed activity in pure buffer under the same conditions. The enhancement of CPO activity was dependent mainly on the surfactant concentration and structure, organic solvent ratio (V _pentanol/V _isooctane), and water content in the reverse micelle. The obtained kinetic parameters showed that the catalytic turnover frequency (k _cat) was increased in reverse micelle. Moreover, the lower K _m and higher k _cat/K _m demonstrated that both the affinity and specificity of CPO to substrates were improved in reverse micelle media. Fluorescence, circular dichroism (CD) and UV–vis spectra assays indicated that a catalytically favorable conformation of enzyme was achieved in reverse micelle, including the strengthening of the protein α-helix structure, and greater exposure of the heme prosthetic group for easy access of the substrate in bulk solution. These results are promising in view of the industrial applications of this versatile biological catalyst.     

Regioselective bromohydroxylation of alkenes catalyzed by chloroperoxidase: Advantages of the immobilization of enzyme on talc

The bromohydroxylation of alkenes catalyzed by chloroperoxidase (CPO) from the mould Caldariomyces fumago adsorbed on different types of talc or in reverse micelles was compared to that of the same reaction catalyzed by the free enzyme in buffer. High reactivity was observed in all media, but the reaction was optimized with an enzyme-talc combination that produced the halohydrin with no oxidative by-products in a Markovnikov-type regioselective addition. The reaction was facilitated by the use of this solid and a recyclable biocatalyst, which gave rise to the halohydrin in a quantitative yield. The protective influence of the talc (hydrophilic or hydrophobic) with respect to hydrogen peroxide enabled use of large amounts of oxidizing agent and substrate, opening perspectives for CPO in the synthesis of fine chemicals.     

The intriguing enhancement of chloroperoxidase mediated one-electron oxidations by azide, a known active-site ligand

Azide is a well-known inhibitor of heme–enzymes. Herein, we report the counter-intuitive observation that at some concentration regimes, incorporation of azide in the reaction medium enhances chloroperoxidase (CPO, a heme–enzyme) mediated one-electron abstractions from several substrates. A diffusible azidyl radical based mechanism is proposed for explaining the phenomenon. Further, it is projected that the finding could have significant impact on routine in situ or in vitro biochemistry studies involving heme–enzyme systems and azide.     

Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance

Heme-thiolate haloperoxidases are undoubtedly the most versatile biocatalysts of the hemeprotein family and share catalytic properties with at least three further classes of heme-containing oxidoreductases, namely, classic plant and fungal peroxidases, cytochrome P450 monooxygenases, and catalases. For a long time, only one enzyme of this type—the chloroperoxidase (CPO) of the ascomycete Caldariomyces fumago—has been known. The enzyme is commercially available as a fine chemical and catalyzes the unspecific chlorination, bromination, and iodation (but no fluorination) of a variety of electrophilic organic substrates via hypohalous acid as actual halogenating agent. In the absence of halide, CPO resembles cytochrome P450s and epoxidizes and hydroxylates activated substrates such as organic sulfides and olefins; aromatic rings, however, are not susceptible to CPO-catalyzed oxygen-transfer. Recently, a second fungal haloperoxidase of the heme-thiolate type has been discovered in the agaric mushroom Agrocybe aegerita. The UV–Vis adsorption spectrum of the isolated enzyme shows little similarity to that of CPO but is almost identical to a resting-state P450. The Agrocybe aegerita peroxidase (AaP) has strong brominating as well as weak chlorinating and iodating activities, and catalyzes both benzylic and aromatic hydroxylations (e.g., of toluene and naphthalene). AaP and related fungal peroxidases could become promising biocatalysts in biotechnological applications because they seemingly fill the gap between CPO and P450 enzymes and act as “self-sufficient” peroxygenases. From the environmental point of view, the existence of a halogenating mushroom enzyme is interesting because it could be linked to the multitude of halogenated compounds known from these organisms.     

Mechanism of enantioselective oxygenation of sulfides catalyzed by chloroperoxidase and horseradish peroxidase. Spectral studies and characterization of enzyme-substrate complexes.

The binding of a series of alkyl aryl sulfides to chloroperoxidase (CPO) and horseradish peroxidase (HRP) has been investigated by optical difference spectroscopy, circular dichroism, paramagnetic NMR spectroscopy, and NMR relaxation measurements. The data are consistent with binding of the sulfides in the distal side of the heme pocket with CPO and near the heme edge with HRP. A linear correlation between the binding constants of para-substituted sulfides to CPO and the Taft sigma I parameter suggests that these substrates act as donors in donor-acceptor complexes involving some residue of the protein chain. Spectral studies during turnover show that high enantioselectivity in the CPO-catalyzed oxidation of sulfides results from a reaction pathway that does not involve the accumulation of compound II enzyme intermediate.     

Chloroperoxidase-catalyzed cyclodimerization of methyl (2E)-2,4-pentadienoate: a [4+2] cycloaddition product

The chloroperoxidase (CPO)-catalyzed oxidation of the methyl (2E)-2,4-pentadienoate gives the terminal double bond epoxide (25%) and a cyclodimerization compound (63%) as the major products.     

Oxidation reactions catalyzed by vanadium chloroperoxidase from Curvularia inaequalis

Vanadium haloperoxidases have been reported to mediate the oxidation of halides to hypohalous acid and the sulfoxidation of organic sulfides to the corresponding sulfoxides in the presence of hydrogen peroxide. However, traditional heme peroxidase substrates were reported not to be oxidized by vanadium haloperoxidases. Surprisingly, we have now found that the recombinant vanadium chloroperoxidase from the fungus Curvularia inaequalis catalyzes the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), a classical chromogenic heme peroxidase substrate. The enzyme mediates the oxidation of ABTS in the presence of hydrogen peroxide with a turnover frequency of 11 s(-1) at its pH optimum of 4.0. The Km of the recombinant enzyme for ABTS was observed to be approximately 35 microM at this pH value. In addition, the bleaching of an industrial sulfonated azo dye, Chicago Sky Blue 6B, catalyzed by the recombinant vanadium chloroperoxidase in the presence of hydrogen peroxide is reported.     

海洋真菌Caldariomyces fumago产氯过氧化物酶的双水相提纯条件

采用聚乙二醇(PEG6000)在(NH4)2SO4高饱和度下沉淀夹带蛋白质富集氯过氧化物酶,再利用磷酸盐溶液复溶解共沉淀物形成的双水相萃取体系高浓度回收酶蛋白,最后再经Sephadex G100柱层析纯化获得高纯度酶试样。结果显示:氯过氧化物酶与PEG共沉淀总活力回收率达85.5%,酶在优化的PEG/磷酸盐双水相系统中上下相分配系数k在0.341以下,酶活力回收率达到69.1%,纯度提高了21.57倍,柱层析可使酶纯度进一步提高到24.79倍,总回收率为37.75%。     

Selective oxygen transfer catalysed by heme peroxidases: synthetic and mechanistic aspects

The synthetic and mechanistic aspects of the use of heme peroxidases as functional mimics of the cytochrome P450 monooxygenases in oxygen-transfer reactions have been described. The chloroperoxidase from Caldariomyces fumago (CPO) is the catalyst of choice in sulfoxidation, hydroxylation and epoxidation on account of its high activity and enantioselectivity. Other heme peroxidases were less active by orders of magnitude; protein engineering has resulted in impressive improvements but even the most active mutant was still at least an order of magnitude less active than CPO. The 'oxygen-rebound' mechanisms of oxygen transfer mediated by heme enzymes - as originally conceived - have proved to be untenable. Dual pathway mechanisms, via oxoferryl species that insert oxygen as well as iron hydroperoxide species that insert OH(+), have been proposed that accommodate all of the known experimental data.     

Over-expression of chloroperoxidase in <Emphasis Type="Italic">Caldariomyces fumago</Emphasis>

The filamentous fungus Caldariomyces fumago secretes a chloroperoxidase (CPO). To increase its production, we integrated a CPO-expression cassette into the non-transcribed spacer regions of the rDNA in C. fumago. One strain was obtained that had twice the CPO activity when grown in shake-flask and bioreactor compared to the wild-type. The highest CPO activity from the bioreactor cultivation was 3,236 U ml⁻¹. This is the highest value reported so far.