Effect of mass transfer limitations on the enzymatic kinetic resolution of epoxides in a two-liquid-phase system

Optically active epoxides can be obtained by kinetic resolution of racemic mixtures using enantioselective epoxide hydrolases. To increase the productivity of the conversion of sparingly aqueous soluble epoxides, we investigated the use of a two-phase aqueous/organic system. A kinetic model which takes into account interphase mass transfer, enzymatic reaction, and enzyme inactivation was developed to describe epoxide conversion in the system by the epoxide hydrolase from Agrobacterium radiobacter. A Lewis cell was used to determine model parameters and results from resolutions carried out in the Lewis cell were compared to model predictions to validate the model. It was found that n-octane is a biocompatible immiscible solvent suitable for use as the organic phase. Good agreement between the model predictions and experimental data was found when the enzyme inactivation rate was fitted. Simulations showed that mass transfer limitations have to be avoided in order to maximize the yield of enantiomerically pure epoxide. Resolution of a 39 g/L solution of racemic styrene oxide in octane was successfully carried out in an emulsion batch reactor to obtain (S)-styrene oxide in high enantiomeric excess (>95% e.e.) with a yield of 30%.     

Interfacial area effects of a biphasic aqueous/organic system on growth kinetic of xenobiotic-degrading microorganisms

The activity of xenobiotic-degrading microorganisms is generally high in a biphasic aqueous/organic system. Therefore, the influence of interfacial area variation on kinetic parameters of Candida sp. growing on ethyl butyrate was evaluated. Interfacial areas of both aseptic and cultured biphasic systems were utilized. Substrate transport measurements in aseptic system (where the interface varied with the organic-phase fraction and agitator speed) showed that the substrate concentration in the aqueous phase was constant at different agitation speeds and decreased as the organic phase increased. Kinetic measurements of the cultured system showed that kinetic parameters vary as functions of their respective aseptic interfacial areas. Higher µ_max and K _i and lower K _s values were obtained with larger interfacial areas. Measurements of the cultured system showed that the interfacial area increased as the biomass increased, and that about 50% of the biomass was attached to the interface as an interfacial biofilm at the end of the culture. Results suggest that the growth and selection of xenobiotic-degrading microorganisms in a biphasic aqueous/organic system should be evaluated mainly on the basis of the activity of adhering biomass (forming a biofilm) at the interfacial area rather than on substrate transport to the aqueous phase     

Structure of Aspergillus niger epoxide hydrolase at 1.8 A resolution: implications for the structure and function of the mammalian microsomal class of epoxide hydrolases

Background: Epoxide hydrolases have important roles in the defense of cells against potentially harmful epoxides. Conversion of epoxides into less toxic and more easily excreted diols is a universally successful strategy. A number of microorganisms employ the same chemistry to process epoxides for use as carbon sources. Results: The X-ray structure of the epoxide hydrolase from Aspergillus niger was determined at 3.5 A resolution using the multiwavelength anomalous dispersion (MAD) method, and then refined at 1.8 A resolution. There is a dimer consisting of two 44 kDa subunits in the asymmetric unit. Each subunit consists of an alpha/beta hydrolase fold, and a primarily helical lid over the active site. The dimer interface includes lid-lid interactions as well as contributions from an N-terminal meander. The active site contains a classical catalytic triad, and two tyrosines and a glutamic acid residue that are likely to assist in catalysis. Conclusions: The Aspergillus enzyme provides the first structure of an epoxide hydrolase with strong relationships to the most important enzyme of human epoxide metabolism, the microsomal epoxide hydrolase. Differences in active-site residues, especially in components that assist in epoxide ring opening and hydrolysis of the enzyme-substrate intermediate, might explain why the fungal enzyme attains the greater speeds necessary for an effective metabolic enzyme. The N-terminal domain that is characteristic of microsomal epoxide hydrolases corresponds to a meander that is critical for dimer formation in the Aspergillus enzyme.     

Enantioselective hydrolysis of p-nitrostyrene oxide by an epoxide hydrolase preparation from Aspergillus niger

The epoxide hydrolase activity of Aspergillus niger was synthesized during growth of the fungus and was shown to be associated with the soluble cell fraction. An enzyme preparation was worked out which could be used in place of the whole mycelium as biocatalyst for the hydrolysis of epoxides. The effect of four different cosolvents on enzyme activity was investigated. Consequently, dimethylsulfoxide (DMSO) was selected for epoxide solubilization. The effect of temperature on both reaction rate and enzyme stability was studied in the presence of DMSO (0.2 volume ratio). A temperature of 25 degrees C was selected for the reaction of bioconversion. With a substrate concentration of 4.5 mM a batch reactor showed that the enzyme preparation hydrolyzed para-nitrostyrene oxide with very high enantioselectivity. The (S) enantiomer of the epoxide remained in the reaction mixture and showed an enantiomeric excess higher than 99%. The substrate concentration could be increased to 20 mM without affecting the enantiomeric excess and degree of conversion. Therefore, the method is potentially useful for the preparative resolution of epoxides. Application are in the field of chiral synthons which are important building blocks in organic synthesis. (c) 1996 John Wiley & Sons, Inc.     

Cytosolic epoxide hydrolase

Epoxide hydrolase activity is recovered in the high-speed supernatant fraction from the liver of all mammals so far examined, including man. For some as yet unexplained reason, the rat has a very low level of this activity, so that cytosolic epoxide hydrolase is generally studied in mice. This enzyme selectively hydrolyzes trans epoxides, thereby complementing the activity of microsomal epoxide hydrolase, for which cis epoxides are better substrates. Cytosolic epoxide hydrolase has been purified to homogeneity from the livers of mice, rabbits and humans. Certain of the physicochemical and enzymatic properties of the mouse enzyme have been thoroughly characterized. Neither the primary amino acid, cDNA nor gene sequences for this protein are yet known, but such characterization is presently in progress. Unlike microsomal epoxide hydrolase and most other enzymes involved in xenobiotic metabolism, cytosolic epoxide hydrolase is not induced by treatment of rodents with substances such as phenobarbital, 2-acetylaminofluorene, trans-stilbene oxide, or butylated hydroxyanisole. The only xenobiotics presently known to induce cytosolic epoxide hydrolase are substances which also cause peroxisome proliferation, e.g., clofibrate, nafenopin and phthalate esters. These and other observations indicate that this enzyme may actually be localized in peroxisomes in vivo and is recovered in the high-speed supernatant because of fragmentation of these fragile organelles during homogenization, i.e., recovery of this enzyme in the cytosolic fraction is an artefact. The functional significance of cytosolic epoxide hydrolase is still largely unknown. In addition to deactivating xenobiotic epoxides to which the organism is exposed directly or which are produced during xenobiotic metabolism, primarily by the cytochrome P-450 system, this enzyme may be involved in cellular defenses against oxidative stress.     

Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis

Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative alpha/beta-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.     

Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis

Major characteristics, substrate specificities and enantioselectivities of epoxide hydrolases from various sources are described. Epoxide hydrolase activity in yeasts is discussed in more detail and is compared with activities in other microorganisms. Constitutively produced bacterial epoxide hydrolases are highly enantioselective in the hydrolysis of 2,2- and 2,3-disubstituted epoxides. A novel bacterial limonene-1,2-epoxide hydrolase, induced by growth on monoterpenes, showed high activities and selectivities in the hydrolysis of several substituted alicyclic epoxides. Constitutively produced epoxide hydrolases are found in eukaryotic microorganisms. Enzymes from filamentous fungi are useful biocatalysts in the resolution of aryl- and substituted alicyclic epoxides. Yeast epoxide hydrolase activity has been demonstrated for the enantioselective hydrolysis of various aryl-, alicyclic- and aliphatic epoxides by a strain of Rhodotorula glutinis. The yeast enzyme, moreover, is capable of asymmetric hydrolysis of meso epoxides and performs highly enantioselective resolution of unbranched aliphatic 1,2-epoxides. Screening for other yeast epoxide hydrolases shows that high enantioselectivity is restricted to a few basidiomycetes genera only. Resolution of very high substrate concentrations is possible by using selected basidiomycetes yeast strains.     

Epoxide hydrolases: biochemistry and molecular biology

Epoxides are organic three-membered oxygen compounds that arise from oxidative metabolism of endogenous, as well as xenobiotic compounds via chemical and enzymatic oxidation processes, including the cytochrome P450 monooxygenase system. The resultant epoxides are typically unstable in aqueous environments and chemically reactive. In the case of xenobiotics and certain endogenous substances, epoxide intermediates have been implicated as ultimate mutagenic and carcinogenic initiators Adams et al. (Chem. Biol. Interact. 95 (1995) 57-77) Guengrich (Properties and Metabolic roles 4 (1982) 5-30) Sayer et al. (J. Biol. Chem. 260 (1985) 1630-1640). Therefore, it is of vital importance for the biological organism to regulate levels of these reactive species. The epoxide hydrolases (E.C. 3.3.2. 3) belong to a sub-category of a broad group of hydrolytic enzymes that include esterases, proteases, dehalogenases, and lipases Beetham et al. (DNA Cell Biol. 14 (1995) 61-71). In particular, the epoxide hydrolases are a class of proteins that catalyze the hydration of chemically reactive epoxides to their corresponding dihydrodiol products. Simple epoxides are hydrated to their corresponding vicinal dihydrodiols, and arene oxides to trans-dihydrodiols. In general, this hydration leads to more stable and less reactive intermediates, however exceptions do exist. In mammalian species, there are at least five epoxide hydrolase forms, microsomal cholesterol 5,6-oxide hydrolase, hepoxilin A(3) hydrolase, leukotriene A(4) hydrolase, soluble, and microsomal epoxide hydrolase. Each of these enzymes is distinct chemically and immunologically. Table 1 illustrates some general properties for each of these classes of hydrolases. Fig. 1 provides an overview of selected model substrates for each class of epoxide hydrolase.     

Hydroxynitrile lyase at the diisopropyl ether/water interface: evidence for interfacial enzyme activity.

A novel recycle reactor has been designed to determine the interfacial activity of hydroxynitrile lyase in a diisopropyl ether (DIPE)/water two-phase system. The reactor provides a known interfacial area. Enzyme activity toward mandelonitrile cleavage is continuously measured in the reactor by following benzaldehyde product formation in the DIPE organic phase with an optical flow cell. For the first time, we establish that this enzymatic reaction is carried out by the hydroxynitrile lyase residing at the organic solvent/water interface and not in the aqueous bulk phase. Hydroxynitrile lyase adsorbs at the interface and exhibits extraordinary stability. Denaturation does not occur over several hours, although the surface pressure increases under the same conditions over this time span. Increases in surface pressure indicate enzyme penetration through the interface although no loss of enzyme activity is observed. Adsorption of p-Hnl at the interface is fit by the Langmuir equilibrium adsorption model with an adsorption equilibrium constant of 0.032 L mg(-1). For the mandelonitrile-cleavage reaction at ambient temperature, p-Hnl follows Michaelis-Menten kinetics at the interface with a Michaelis constant of 14.4 mM and a specific activity close that for the bulk aqueous phase.     

Interfacial versus homogeneous enzymatic cleavage of mandelonitrile by hydroxynitrile lyase in a biphasic system

The question of an interfacial versus a homogeneous reaction is carefully addressed for the enzymatic biphasic cleavage of mandelonitrile to benzaldehyde by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) (Hickel et al. [1999] Biotechnol Bioeng 36:425-436). Experimental evidence, including 1) the reaction ceases when the interface is populated by previously adsorbed denatured pa-Hnl, 2) the reaction continues even after washout of the bulk enzyme from the aqueous phase, 3) highly nonpolar organic solvents initially promote fast reaction kinetics that relatively quickly decay to zero product production, and 4) the reaction rate is nonlinear in the bulk enzyme concentration, provide robust grounds for an interfacial reaction. We also model enzymatic mandelonitrile cleavage assuming a homogeneous aqueous-phase reaction. The homogeneous reaction scheme does not simultaneously account for the experimental observations of a linear dependence of the reaction rate on organic/water interfacial area, no dependence on the aqueous-phase volume, and a nonlinear dependence on pa-Hnl aqueous concentration. Further, simple calculations demonstrate that the homogeneous reaction rate is at least three orders of magnitude slower than those observed by Hickel et al. (1999). We again conclude that enzyme adsorbed at the organic solvent/water interface primarily catalyzes the biphasic mandelonitrile cleavage reaction.     

Selection of Xenobiotic-Degrading Microorganisms in a Biphasic Aqueous-Organic System

Microbial selection on mixtures of chlorinated and nonchlorinated compounds that are poorly soluble in water and/or toxic to growing microbial cells was examined in both biphasic aqueous-organic and monophasic aqueous systems. A biphasic system in which silicone oil was used as the organic phase permitted the acceleration of acclimation, leading to rapid selection and to an increase in xenobiotic compound degradation. In contrast, acclimation, selection, and degradation were very slow in the monophasic aqueous system. The variation in microbial growth rate with the degree of dispersion (i.e., dispersion at different silicone oil concentrations and agitation rates), and cell adhesion to the silicone oil indicate that the performance of the biphasic aqueous-organic system is dependent on the interfacial area between the two phases and that microbial activity is important at this interface. Therefore, the biphasic water-silicone oil system could be used for microbial selection in the presence of xenobiotic compounds that are toxic and have low water solubility.     

Resolution of 1,2-epoxyhexane by Rhodotorula glutinis using a two-phase membrane bioreactor

Large-scale resolution of epoxides by the yeast Rhodotorula glutinis was demonstrated in an aqueous/organic two-phase cascade membrane bioreactor. Due to the chemical instability and low solubility of epoxides in aqueous phases, an organic solvent was introduced into the reaction mixture in order to enhance the resolution of epoxide. A cascade hollow-fiber membrane bioreactor was used (1) to minimize the toxicity of organic solvents towards the epoxide hydrolase of R. glutinis, and (2) to remove inhibitory amounts of formed diol from the yeast cell containing aqueous phase. Dodecane was selected as a suitable solvent and 1,2-epoxyhexane as a model substrate. By use of this membrane bioreactor, highly concentrated (0.9 M in dodecane) enantiopure (> 98% ee) (S)-1,2-epoxyhexane (6.5 g, 30% yield) was obtained from the racemic mixture. Received: 28 June 1999 / Received revision: 26 August 1999 / Accepted: 3 September 1999     

Catalytic properties and inhibition of hepatic cholesterol-epoxide hydrolase

Hepatic cholesterol-epoxide hydrolase is a microsomal enzyme which appears to be catalytically distinct from the epoxide hydrolase responsible for the catabolism of a wide variety of aromatic and aliphatic epoxides. The diastereomeric forms of cholesterol epoxide, cholesterol 5 alpha,6 alpha-, and cholesterol 5 beta,6 beta-epoxides are converted to cholestane-3 beta,5 alpha,6 beta-triol with equal facility. Kinetic analysis of cholesterol-epoxide hydrolase demonstrated that both diastereomers bind to a common catalytic site. Apparent Km values of 3.69 and 4.42 microM were derived for cholesterol 5 alpha,6 alpha- and cholesterol 5 beta,6 beta-epoxide, respectively. In addition, enzyme activity with both diastereomers was product-inhibited by cholestanetriol through a competitive mechanism with the apparent Ki for cholestanetriol being 10.8 and 6.8 microM against cholesterol alpha- and beta-epoxides, respectively. This inhibitory effect of cholestanetriol may account for the difference observed in the hydration rates for the cholesterol epoxide isomers when they are incubated together in the presence of liver microsomes. Inhibitors of epoxide hydrolase were studied, and three oxidation products were found to be particularly effective against cholesterol-epoxide hydrolase while producing no significant inhibition of styrene-epoxide hydrolase. These inhibitors were 7-ketocholesterol, 6-ketocholestanol, and 7-ketocholestanol, the latter displaying an apparent Ki lower than the Km for either cholesterol epoxide isomer. None of the xenobiotic epoxide hydrolase inhibitors or activators studied affected cholesterol-epoxide hydrolase activity.     

The Isolation and Characterisation of a Carbocyclic Epoxide-Degrading Corynebacterium sp

A two-phase aqueous/organic isolation system was developed for the isolation of epoxide-degrading bacteria. The potential of this system, for the isolation of cyclohexene oxide-degrading bacteria, was assessed by comparison to an analogous system lacking co-solvent. Using the biphasic isolation strategy, an epoxide-degrading Corynebacterium sp. designated C12, was isolated and was shown to grow on cyclohexene oxide as sole source of carbon and energy. Epoxide degradation appeared to proceed via a diol intermediate implicating the involvement of an epoxide hydrolase. The epoxide hydrolase of Corynebacterium sp. C12 was shown to have activity towards a range of terminal, sub-terminal and cyclic substrates. The enantioselectivity of the hydrolysis reaction was largely dependent on the nature of the substrate. In a series of biotransformations allowed to proceed to 50% substrate conversion, the remaining epoxide ranged from low (5% ee) to moderate (60% ee) optical purity.     

Immiscible organic solvent inactivation of urease, chymotrypsin, lipase, and ribonuclease: separation of dissolved solvent and interfacial effects

A new technique with controlled interface generation allows separation and quantitation of enzyme inactivation by both solvent/aqueous interface and dissolved solvent. This has now been used in n-butanol, isopropylether, 2-octanone, n-hexane, n-butylbenzene, and n-tridecane. Ribonuclease was stable with all the solvent/aqueous interfaces studied. Chymotrypsin was mainly inactivated by the more hydrophobic solvent/aqueous interfaces, whereas lipase was only inactivated by the less hydrophobic solvent/aqueous interfaces. Urease was inactivated by some interfaces, but not all, without an obvious trend. Thus, the commonly expected simple relationship with solvent polarity (e.g., log P) does not apply when interfacial inactivation is determined specifically. Greater dissolved solvent inactivation occurred with the more polar solvents, though only a general trend was apparent with log P. A better correlation was noted with the Hilde-brand solubility parameter. Interfacial effects are discussed with reference to enzyme molecular weight, denaturation temperature, hydrophobicity, and adiabatic compressibility. (c) 1994 John Wiley & Sons, Inc.     

Stereoselectivity of microsomal epoxide hydrolase toward diol epoxides and tetrahydroepoxides derived from benz[a]anthracene

We have examined the selectivity of rat liver microsomal epoxide hydrolase (EC 3.3.2.3) toward all of the possible positional isomers of benzo-ring diol epoxides and tetrahydroepoxides of benz[a]anthracene, as well as the 1,2-diol 3,4-epoxides of triphenylene. This set includes compounds with no bay region in the vicinity of the benzo-ring, a bay-region diol group, a bay-region epoxide group, and (for the triphenylene derivatives) both a bay-region diol and a bay-region epoxide. In all cases where both the tetrahydroepoxides and the corresponding diol epoxides were examined, there is a large retarding effect of hydroxyl substitution on the rate of the enzyme-catalyzed hydration. When the tetrahydroepoxides are fair or poor substrates (epoxide group in the 1,2-, 8,9-, or 10,11-position), the additional retardation introduced by adjacent hydroxyl groups causes the enzyme-catalyzed hydrolysis of the corresponding diol epoxides to be insignificantly slow or nonexistent. In contrast, a benz[a]anthracene derivative with an epoxide group in the 3,4-position, (-)-tetrahydrobenz[a]anthracene (3R,4S)-epoxide, has been identified as the best substrate known for epoxide hydrolase, with a Vmax at 37 degrees C and pH 8.4 of 6800 nmol/min/mg of protein, and the two diastereomeric (+/-)-benz[a]anthracene 1,2-diol 3,4-epoxides, unlike all the other diol epoxides examined to date, are moderately good substrates for epoxide hydrolase. This novel observation is accounted for by the fact that the very high reactivity of the tetrahydrobenz[a]anthracene 3,4-epoxide system towards epoxide hydrolase is large enough to overcome a kinetically unfavorable effect of hydroxyl substitution. The enantioselectivity and positional selectivity of the enzyme have been determined for the tetrahydro-1,2- and -3,4-epoxides of benz[a]anthracene as well as the 1,2-diol 3,4-epoxides. When the epoxide is located in the 3,4-position, the benzylic carbon is the preferred site of attack, whereas for the enantiomers of the bay-region tetrahydro-1,2-epoxides, the chemically less reactive non-benzylic carbon is preferred. The regio- and enantioselectivity of epoxide hydrolase are discussed in terms of a possible model for the hydrophobic binding site of this enzyme.     

Continuous production of enantiopure 1,2-epoxyhexane by yeast epoxide hydrolase in a two-phase membrane bioreactor

A two-phase membrane bioreactor was developed to continuously produce enantiopure epoxides using the epoxide hydrolase activity of Rhodotorula glutinis. An aqueous/organic cascade, hydrophilic, hollow-fiber membrane bioreactor was used: (1) to carry out large-scale resolution of epoxides, (2) to continuously extract residual enantiopure epoxides from the aqueous phase, and (3) to separate inhibitory formed diol from the yeast cells contained in the aqueous phase. Dodecane was employed to dissolve-feed epoxide as well as to extract residual epoxide. 1,2-Epoxyhexane was used as a model substrate. By use of this membrane bioreactor, enantiopure (S)-1,2-epoxyhexane (>98% enantiomeric excess) was obtained with a volumetric productivity of 3.8 g l⁻¹ h⁻¹. The continuous-production system was operated for 12 days and resulted in 38 g enantiopure (S)-1,2-epoxyhexane. Received: 14 February 2000 / Received revision: 15 June 2000 / Accepted: 18 June 2000     

Immobilization of epoxide hydrolase from Aspergillus niger onto DEAE-cellulose: enzymatic properties and application for the enantioselective resolution of a racemic epoxide

Recombinant epoxide hydrolase (EH) from Aspergillus niger can be a very promising tool for the resolution of various racemic epoxides by enantioselective hydrolysis. The enzyme was successfully immobilized by ionic adsorption onto DEAE-cellulose (99% yield, 70% of retention activity). The temperature for maximal activity (40 °C) and the activation energy (38.8 kJ/mol) were similar for both the immobilized and free EHs, whereas the optimal pH was about one unit less for the immobilized enzyme. Thermal stability was also affected by immobilization; the immobilized enzyme appeared to be slightly less stable than the free one. However, a gram-scale resolution of racemic para-chlorostyrene oxide (pCSO) was successfully carried out in a repeated batch reactor, operated for seven cycles. Furthermore, using a very high substrate concentration of 2 M (306 g/L), i.e. biphasic conditions, the resolution of 3 g of pCSO was also achieved in a repeated batch reactor using approximately 300 mg of immobilized EH, corresponding to less than 3 mg of the enzymatic powder.     

Thyroid hormone-induced changes in the hepatic monooxygenase system, heme oxygenase activity and epoxide hydrolase activity in adult male, female and immature rats

In 8-day-old rat pups, pretreatment with a single injection of L-triiodothyronine or L-thyroxine decreased hepatic cytochrome P-450 content, aminopyrine N-demethylase activity and epoxide hydrolase activity but increased hepatic microsomal cytochrome c reductase, 7-ethoxyresorufin O-deethylase and heme oxygenase activities without significantly altering UDP-glucuronosyltransferase activity (towards o-aminophenol) or the microsomal yield.

In adult rats of either sex such single injections of L-triiodothyronine failed to significantly alter these enzyme activities. However, multiple injections evoked changes similar to those observed in the pups, in all these enzyme activities, except that 7-ethoxyresorufin O-deethylase activity was slightly decreased rather than increased.

These findings demonstrate that: (1) The hepatic monooxygenase system in the rat pup is more responsive to thyroid hormones than that in adult. (2) Thyroid hormones can decrease rat liver cytochrome P-450 content and its dependent monooxygenase activity independently of sexual maturity. (3) Thyroid hormones also decrease hepatic epoxide hydrolase activity in both pups and adults. Thus, hyperthyroidism could render the rat pup more susceptible to hepatotoxicity from electrophilic epoxides which utilize microsomal epoxide hydrolase as the major detoxication pathway.     

Improved catalytic performance of Bacillus megaterium epoxide hydrolase in a medium containing Tween-80

A new epoxide hydrolase with high enantioselectivity toward (R)-glycidyl phenyl ether (R-GPE) was partially purified from Bacillus megaterium strain ECU1001. The maximum activity of the isolated enzyme was observed at 30 degrees C and pH 6.5 in a buffer system with 5% (v/v) of DMSO as a cosolvent. The enzyme was quite stable at pH 7.5 and retained full activity after incubation at 40 degrees C for 6 h. Interestingly, when the cosolvent DMSO was replaced by an emulsifier (Tween-80, 0.5% w/v) as an alternative additive to help disperse the water-insoluble substrate, the apparent activity of the epoxide hydrolase significantly increased by about 1.8-fold, while the temperature optimum shifted from 30 to 40 degrees C and the half-life of the enzyme at 50 degrees C increased by 2.5 times. The enzymatic hydrolysis of rac-GPE was highly enantioselective, with an E-value (enantiomeric ratio) of 69.3 in the Tween-80 emulsion system, which is obviously higher than that (41.2) observed in the DMSO-containing system.