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.     

New reaction system for hydrocarbon oxidation by chloroperoxidase

A novel reaction system was developed to maximize the catalytic efficiency of chloroperoxidase (CPO, from Caldariomyces fumago) toward the oxidation of hydrocarbons. The reaction system consisted of an organic/aqueous emulsion comprising pure substrate and aqueous buffer supplemented with the surfactant dioctyl sulfosuccinate. The emulsion system attenuated not only the destabilizing effects of the substrate and product on the enzyme by emulsifying the compounds, but also oxidant toxicity (oxidative stress) by increasing substrate availability. As a result, CPO exhibited total turnover numbers (TTNs, defined as the amount of product produced over the catalytic lifetime of the enzyme) of ca. 20,000 mol product/mol enzyme for the oxidation of styrene, toluene, and o-, m-, p-xylenes. The TTNs are over 10-fold higher than those previously reported for the oxidation of benzylic hydrocarbons by CPO. This study represents a significant step toward the development of CPO as a practical catalyst for large-scale organic syntheses.     

Chloroperoxidase-catalyzed Epoxidation of Styrene in Aqueous and Nonaqueous Media

To overcome poor product yields and stability in aqueous solution, we have examined the chloroperoxidase (CPO from Caldariomyces fumago ) catalyzed oxidation of styrene in organic media using tert -butyl hydroperoxide as external oxidant. CPO's intrinsic catalytic activity in tert -butanol , as reflected in its k cat value, was ca. one-fourth of that in aqueous buffer, indicating that the enzyme remains highly active in the organic solvent. Styrene epoxidation reactions were modeled in both aqueous and nonaqueous media to provide global kinetic information, which dominates non-initial rate conditions and is heavily influenced by continuous deactivation of the CPO. Deactivation studies revealed that the enzyme is deactivated quickly by the combination of the tert -butyl hydroperoxide and styrene, possibly due to the styrenic free radicals generated during the enzymatic reaction. These results may enable catalyst-engineering strategies to be initiated to improve the prospects of using CPO in nonaqueous media for large-scale epoxidation reactions.     

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.     

Chloroperoxidase-catalyzed Epoxidation of Styrene in Aqueous and Nonaqueous Media

To overcome poor product yields and stability in aqueous solution, we have examined the chloroperoxidase (CPO from Caldariomyces fumago ) catalyzed oxidation of styrene in organic media using tert -butyl hydroperoxide as external oxidant. CPO's intrinsic catalytic activity in tert -butanol, as reflected in its k cat value, was ca. one-fourth of that in aqueous buffer, indicating that the enzyme remains highly active in the organic solvent. Styrene epoxidation reactions were modeled in both aqueous and nonaqueous media to provide global kinetic information, which dominates non-initial rate conditions and is heavily influenced by continuous deactivation of the CPO. Deactivation studies revealed that the enzyme is deactivated quickly by the combination of the tert -butyl hydroperoxide and styrene, possibly due to the styrenic free radicals generated during the enzymatic reaction. These results may enable catalyst-engineering strategies to be initiated to improve the prospects of using CPO in nonaqueous media for large-scale epoxidation reactions.     

Chloroperoxidase-catalysed oxidation of alcohols to aldehydes

Chloroperoxidase (CPO) catalyses the oxidation of primary alcohols (hexan-1-ol, hexen-1-ols, epoxyhexan-1-ols and 3-phenylglycidol) selectively to the aldehyde in biphasic systems of hexane or ethyl acetate and a buffer (pH 5.0). The cis to trans isomerization in the case of cis-2-hexenal can be avoided by working at low water contents or in organic solvents saturated with water. In the case of epoxyalcohols, oxidation to the aldehyde proceeds enantioselectively. Hydrogen peroxide and tert-butyl hydroperoxide have been used as an oxidant.     

Microbial Stereoselective Oxidation of 2-methyl-1,3-propanediol to (R)-β-hydroxyisobutyric Acid in Aqueous/organic Biphasic Systems

The aim of this work was to evaluate (R)-g-hydroxyisobutyric acid (HIBA) production by microbial stereoselective oxidation of 2-methyl-1,3 propanediol under different conditions, and to compare the performance of this bioconversion in traditional aqueous media and aqueous/organic biphasic media. The oxidation is a two-step reaction with hydroxyisobutanal as an intermediate. Among the operational factors tested, pH and aeration were those, which most significantly affected the biocatalytic activity. Enantiomeric excesses higher than 95% were consistently obtained. For substrate concentrations above 50 mg ml^-1 a slight substrate inhibition was observed. Product inhibition was much stronger, and together with the decrease of the pH during the bioconversion was the most important limiting factor in long-term bioconversions. Kinetic parameters were determined for different pH values. A compromise pH value of 4 was determined to be the optimum for HIBA production and simultaneous extraction with an organic phase of trioctyl phosphine oxide (TOPO) in isooctane.     

Biocatalytic oxidation of S-alkylcysteine derivatives by chloroperoxidase and Beauveria species

Treatment of N-methoxycarbonyl C-carboxylate ester derivatives of S-methyl- -cysteine by chloroperoxidase (CPO)/hydrogen peroxide resulted in oxidation at sulfur to produce the (R_S) sulfoxide in moderate to high diastereomeric excess (DE). The (S_S) natural product sulfoxide chondrine was obtained via biotransformation of the N-t.boc derivative of -4-S-morpholine-2-carboxylic acid using Beauveria bassiana or Beauveria caledonica.     

Reaction of manganese-dependent peroxidase from Bjerkandera adusta in aqueous organic media

The oxidation of various phenolics and aromatic amines by manganese-dependent peroxidase (MnP) of Bjerkandera adusta was examined in aqueous organic media. MnP retained its activities in several 70% (v/v) aqueous solutions of water-miscible organic solvents including ethylene glycol, diethylene glycol, acetone and acetonitrile. The absorption spectra of MnP in these aqueous organic media were similar to that observed in the reaction without solvent addition, indicating that the heme of MnP was little affected by the addition of these water-miscible organic solvents. MnP was also found to oxidize Mn(II) to Mn(III) in these 70% (v/v) aqueous organic media. The oxidation of Mn(II) by MnP was correlated with the Dimroth–Reichardt parameter, E_T(30), of the solvents. Furthermore, MnP catalyzed the oxidation of anisidines, aminophenols, phenylenediamines and phenolics in aqueous 70% (v/v) acetone, acetonitrile and diethylene glycol media. Aromatic amines that have high hydrophobicity were shown to be suitable for the reaction of MnP in aqueous water-miscible organic media.     

Immobilization of chloroperoxidase on silica-based materials for 4,6-dimethyl dibenzothiophene oxidation

Silica-based materials have been used as effective supports for the immobilization of enzymes. Moreover, the understanding on the oxidation of sulfur compounds by immobilized chloroperoxidase represents a step further in the development of a biocatalytic desulfurization process of fossil fuels. Here, chloroperoxidase from Caldariomyces fumago was immobilized on amorphous and structured silica-based materials either physically or covalently using an organosilane derivative for the oxidation of a recalcitrant organosulfur compound currently found in gas oil and diesel, such as 4,6-dimethyldibenzothiophene (4,6-DMDBT). Such materials were characterized by FTIR, N₂-adsorption, XRD, SEM and TEM. We have found that the chemical differences on the silanol/siloxane groups of SG/67 and SBA15 mesoporous materials deeply modify the enzymatic load, activity, thermal stability and reusability. The physical immobilization of CPO was characterized by a high adsorption capacity (q_m) and affinity constants (K_m) when compared to the covalent approach, but it resulted more sensitive to temperature than free, the silanized and covalently immobilized enzyme. The thermal residual activity as well as reusability of CPO were first improved by silanization, then by covalent immobilization in a support with a large pore size and high silanol/siloxane ratio.     

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%。     

Continuous production of isovaleraldehyde through extractive bioconversion in a hollow-fiber membrane bioreactor

A membrane bioreactor was developed to perform an extractive bioconversion aimed at the production of isovaleraldehyde by isoamyl alcohol oxidation with whole cells of Gluconobacter oxydans. A liquid/liquid extractive system using isooctane as extractant and assisted by a hollow-fiber hydrophobic membrane was chosen to recover the product. The aqueous bioconversion phase and the organic phase were maintained apart with the aid of the membrane. The extraction of alcohol and aldehyde was evaluated by performing equilibrium and mass transfer kinetic studies. The bioprocess was then performed in a continuous mode with addition of the substrate to the aqueous phase. Fresh solvent was added to the organic phase and exhausted solvent was removed at the same flow rate. The extractive system enabled a fast and selective in situ removal of the aldehyde from the water to the organic phase. High conversions (72–90%) and overall productivity (2.0–3.0 g l⁻¹ h⁻¹) were obtained in continuous experiments performed with different rates of alcohol addition (1.5–3.5 g l⁻¹ h⁻¹). Cell deactivation was observed after 10–12 h of operation.     

Effect of organic solvents on a chloroperoxidase biotransformation

The effect of organic solvents on the chlorination activity of chloroperoxidase (CPO) was identified for use in biotransformations with CPO. CPO was found to chlorinate monochlorodimedon (MCD) in the presence of organic solvents with log P values less than 0. The relative rates of chlorination with chloride ion in the presence of H₂O₂, buffer and 2.5–20% of either dimethyl sulfoxide, N,N-dimethyl formamide, methanol or acetonitrile, were in the range of 10–58% of that in buffer (pH 2.8) at the same reactant concentrations. The presence of such organic solvents was found to alter CPO catalysis by altering the protein conformation and the local environment at the active site. CPO did not display chlorination activity in the presence of organic solvents which had log P values greater than 0.     

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 of a mixture of 2-(R) and 2-(S)-heptanol to 2-heptanone by Saccharomyces cerevisiae in a biphasic system

The ability of dehydrated baker's yeast (Sigma, type II) to carry out oxidation reactions was investigated using a mixture of (S)- and (R)-enantiomers of 2-heptanol operated in a biphasic system with hexadecane as the organic layer. The commercial material could be used without preliminary growth provided the external trehalose was removed by centrifugation. It afforded a non enantiospecific biocatalyst with high activity, and 2-heptanone could be obtained in up to 10 g L^-1 after 30 h reaction with a molar yield close to 100% with this material. Yeast cells harvested in the stationary phase of aerobic growth exhibited only a (S)-oxidation activity, which gave a process for the resolution of (R)-enantiomers of secondary alcohols. These results led to the assumption that at least two enzymes were acting in this process, one of them probably being the yeast alcohol dehydrogenase (YADH), which is known to exhibit a (S)-enantioselectivity in Saccharomyces cerevisiae.     

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.     

Whole-cell bioconversion of β-sitosterol in aqueous–organic two-phase systems

The selective cleavage of the β-sitosterol side-chain by free Mycobacterium sp. NRRL B-3805 cells was used as a model system for the study of solvent effects in a whole-cell bioconversion in two phase aqueous–organic media. This multi-step degradation pathway leads to the production of 4-androstene-4,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) as a minor product. In an attempt to correlate the substrate and cell partition effects and solvent hydrophobicity (log P) with biocatalytic activity, 15 carboxylic acid esters with log P values between 3 and 10 were screened. The results indicated that the toxicity of the tested solvents in this system could not be correlated to their log P, but seemed to depend on their ability to accumulate in the cells, as these showed a strong affinity towards the organic phase. Different solvent/aqueous ratios and hydrodynamic conditions were further tested in the solvent systems (phthalates) showing significant biodegradation activity. The bioconversion rate was generally not much affected by the stirring speed in the employed range (150–300 rpm) but was strongly influenced by the aqueous/organic phase ratio. Results suggest that the bioconversion takes place at the interphase, its rate being possibly limited by mass transport inside the organic phase.     

Detoxification of sulfur mustard by enzyme-catalyzed oxidation using chloroperoxidase

One of the most interesting methods for the detoxification of sulfur mustard is enzyme-catalyzed oxidation. This study examined the oxidative destruction of a sulfur mustard by the enzyme chloroperoxidase (EC 1.11.1.10). Chloroperoxidase (CPO) belongs to a group of enzymes that catalyze the oxidation of various organic compounds by peroxide in the presence of a halide ion. The enzymatic oxidation reaction is affected by several factors: pH, presence or absence of chloride ion, temperature, the concentrations of hydrogen peroxide and enzyme and aqueous solubility of the substrate. The optimum reaction conditions were determined by analyzing the effects of all factors, and the following conditions were selected: solvent, Britton–Robinson buffer (pH = 3) with tert-butanol (70:30 v/v); CPO concentration, 16 U/mL; hydrogen peroxide concentration, 40 mmol/L; sodium chloride concentration, 20 mmol/L. Under these reaction conditions, the rate constant for the reaction is 0.006 s⁻¹. The Michaelis constant, a measure of the affinity of an enzyme for a particular substrate, is 1.87 × 10⁻³ M for this system. The Michaelis constant for enzymes with a high affinity for their substrate is in the range of 10⁻⁵ to 10⁻⁴ M, so this value indicates that CPO does not have a very high affinity for sulfur mustard.     

Peroxidase-catalyzed bromination of tyrosine, thyroglobulin, and bovine serum albumin: comparison of thyroid peroxidase and lactoperoxidase

A recent paper (Buchberger, W., 1988, J. Chromatogr. 432, 57) on lactoperoxidase-catalyzed bromination of tyrosine and thyroglobulin stated, without evidence, that thyroid peroxidase (TPO) is able to use bromide as a substrate. This was in disagreement with unpublished experiments previously performed in this laboratory, and we undertook, therefore, to examine this subject further. Highly purified porcine TPO was compared with lactoperoxidase (LPO) and chloroperoxidase (CPO) for ability to catalyze bromination of tyrosine, thyroglobulin, and bovine serum albumin (BSA). The incubation mixture contained 50-100 nM peroxidase, 10-500 microM 82Br-, tyrosine (150 microM), thyroglobulin (0.3 or 1 microM), or BSA (7.5 microM), and a source of H2O2. The latter was either generated by glucose (1 mg/ml)-glucose oxidase (0.5 or 1 micrograms/ml), or added initially as a bolus (100 microM). With TPO, formation of organically bound 82Br was undetectable under all conditions in the pH range 5.4-7.0. Lactoperoxidase and CPO, on the other hand, displayed considerable brominating activity. Lactoperoxidase was much more active at pH 5.4 than at pH 7.0 and was more active with BSA as acceptor than with tyrosine or thyroglobulin. The distribution of 82Br among the various amino acids in LPO-brominated thyroglobulin and BSA was determined by HPLC. As expected, monobromotyrosine and dibromotyrosine together comprised the greatest part of the bound 82Br. However, a surprisingly high percentage (20-25%) was present as monobromohistidine. Evidence was also obtained for the presence of a small percentage of the bound 82Br as tetrabromothyronine. Peroxidase-catalyzed bromination probably depends on the oxidation of Br- to Br+ by the Compound I form of the enzyme. Since oxidation of Br- to Br+ requires a stronger oxidant than oxidation of I- to I+, our results suggest that Compound I of LPO and of CPO has a higher oxidation potential than Compound I of TPO. In vivo experiments with rats on a low iodine diet injected with 82Br- showed that even under conditions of high stimulation by thyrotropic hormone, there is negligible formation of organic bromine in the thyroid. Measurements of thyroid:serum concentration ratios for 82Br- in similar rats provided no evidence that Br- is a substrate for the iodide transport system of the thyroid.     

Bioremediation of polycyclic aromatic hydrocarbons by fungal laccases engineered by directed evolution

Fungal laccases are useful for several remarkable transformations, such as bioremediation of polycyclic aromatic hydrocarbons (PAHs), synthesis of phenolic-based resins, oxidation of lignin derivatives and others. Most of these substrates are barely water-soluble, and although polar organic co-solvents may be added to enhance their solubility, transformation rates dramatically decrease due to the negative effect of organic solvents on the protein structure. Laccase from Myceliophthora thermophila variant T2 (MtLT2) has been submitted to laboratory evolution in Saccharomyces cerevisiae with the aim of improving activity and stability in organic co-solvents. Some 4500 clones created by random mutagenesis were screened in two rounds of directed evolution. Libraries were explored under increasing concentrations of acetonitrile and ethanol, and several mutants with improved features were purified and further characterised. Turnover rates of MtLT2 in 30% (v/v) acetonitrile and 50% (v/v) ethanol were increased up to 6.5- and 7.5-fold, respectively. The best variants showed similar rates in 20% (v/v) acetonitrile or 30% (v/v) ethanol as the parent type in aqueous media. Mutant laccases were also tested for the oxidation of anthracene in the presence of 20% (v/v) acetonitrile.