Biotechnological production of enantiopure epoxides by enzymatic kinetic resolution

Enantiopure epoxides are high value-added synthons for the production of pharmaceuticals, agrochemicals, as well as versatile fine chemicals and have broad scope of market demand for their applications. A major challenge in conventional organic synthesis is to generate such compounds in high enantiopurity with reasonable yield. Among possible chemical and biological technologies for enantiopure epoxide preparation, enzymatic kinetic resolution has been paid much attention with respect to its high enantioselectivity. Epoxide hydrolase (EH) has shown promising characteristics for the preparation of enantiopure epoxides and vicinal diols during enantioselective hydrolysis of racemic epoxides. EH is readily available from microbial resources thus it is being employed for biohydrolysis of a variety of epoxides. Recent technical progress in EH-catalyzed enantioselective hydrolysis is summarized in terms of exploration of novel EH, its functional improvement, high throughput assay, and preparative scale resolution process.     

One-pot biotransformation of racemic styrene oxide into (R)-1,2-phenylethandiol by two recombinant microbial epoxide hydrolases

An enantioconvergent biotransformation of racemic styrene oxide by using two recombinant microbial epoxide hydrolases (EHs) in one pot has been investigated to prepare enantiopure vicinal diols. The recombinant whole cell possessing EH gene from Aspergillus niger LK or Rhodotorula glutinis exhibited a complementary enantioselectivity and regioselectivity, compared to the recombinant cell containing Caulobacter crescentus EH gene. When two recombinant microbial EHs were used in combination, 1.3 g of enantiopure (R)-1,2-phenylethandiol with more than 90% enantiopurity and 95% overall yield was obtained from 1.2 g of racemic styrene oxide in a preparative-scale batch enantioconvergent biotransformation.     

Production of chiral epoxides: Epoxide hydrolase-catalyzed enantioselective hydrolysis

Chiral epoxides are highly valuable intermediates, used for the synthesis of pharmaceutical drugs and agrochemicals. They have broad scope of market demand because of their applications. A major challenge in modern organic chemistry is to generate such compounds in high yields, with high stereo- and regio-selectivities. Epoxide hydrolases (EH) are promising biocatalysts for the preparation of chiral epoxides and vicinal diols. They exhibit high enantioselectivity for their substrates, and can be effectively used in the resolution of racemic epoxides through enantioselective hydrolysis. The selective hydrolysis of a racemic epoxide can produce both the corresponding diols and the unreacted epoxides and vicinal diol has prompted researchers to explore their use in the synthesis of epoxides and diols with high ee values.     

Epoxide hydrolase-mediated enantioconvergent bioconversions to prepare chiral epoxides and alcohols

A number of epoxide hydrolase (EH)-mediated bioconversions have been developed to prepare single enantiomeric product from racemic substrates with a yield greater than 50%. Enantioconvergent hydrolysis using single or two EHs possessing complementary enantio- and regio-selectivity, EH-based chemoenzymatic reactions, and EH-triggered cascade-reactions have been developed for the preparation of chiral epoxides, epoxyalcohols, tetrahydrofuran derivatives and vicinal diols. All these bioconversions are based on stereochemical flexibilities of various EHs and can be used in total synthesis of biologically active compounds without the formation of unwanted enantiomers.     

环氧水解酶的结构基础及新酶开发

环氧水解酶能应用于外消旋环氧化物的动力学拆分或对映归一性水解制备光学纯的环氧或邻位二醇,具有广阔的应用前景。近年来,多个环氧水解酶晶体结构的报道使人们对它的结构基础有了更深入的理解。随着基因信息的增长,分子生物学和蛋白质工程技术的发展大大简化了大量克隆表达多样性环氧水解酶的过程,降低了环氧水解酶分子改造的难度,为新型具有工业应用潜力的环氧水解酶的开发提供了技术支持。本文综述了环氧水解酶的结构与机制以及近年来环氧水解酶重组表达及分子改造的研究进展。     

Sequence and structure of epoxide hydrolases: a systematic analysis

Epoxide hydrolases (EC 3.3.2.3) are ubiquitous enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. More than 100 epoxide hydrolases (EH) have been identified or predicted, and 3 structures are available. Although they catalyze the same chemical reaction, sequence similarity is low. To identify conserved regions, all EHs were aligned. Phylogenetic analysis identified 12 homologous families, which were grouped into 2 major superfamilies: the microsomal EH superfamily, which includes the homologous families of Mammalian, Insect, Fungal, and Bacterial EHs, and the cytosolic EH superfamily, which includes Mammalian, Plant, and Bacterial EHs. Bacterial EHs show a high sequence diversity. Based on structure comparison of three known structures from Agrobacterium radiobacter AD1 (cytosolic EH), Aspergillus niger (microsomal EH), Mus musculus (cytosolic EH), and multisequence alignment and phylogenetic analysis of 95 EHs, the modular architecture of this enzyme family was analyzed. Although core and cap domain are highly conserved, the structural differences between the EHs are restricted to only two loops: the NC-loop connecting the core and the cap and the cap-loop, which is inserted into the cap domain. EHs were assigned to either of three clusters based on loop length. By using this classification, core and cap region of all EHs, NC-loops and cap-loops of 78% and 89% of all EHs, respectively, could be modeled. Representative models are available from the Lipase Engineering Database, http://www.led.uni-stuttgart.de.     

Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase

Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr(465) and Tyr(381) act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr(465) and Tyr(381) are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr(465) and Tyr(381) decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr(465) is conserved in microsomal EHs. The substitution of analogous Tyr(374) with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.     

Substrate and inhibitor selectivity,and biological activity of an epoxide hydrolase from Trichoderma reesei

Molecular Biology Reports - Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EH are involved in the...     

An Overview on the Enhancement of Enantioselectivity and Stability of Microbial Epoxide Hydrolases

Epoxide hydrolases (EHs; 3.3.2.x) catalyze the enantioselective ring opening of racemic epoxides to the corresponding enantiopure vicinal diols and remaining equivalent unreacted epoxides. These epoxides and diols are used for the synthesis of chiral drug intermediates. With an upsurge in the methods for identification of novel microbial EHs, a lot of EHs have been discovered and utilized for kinetic resolution of racemic epoxides. However, there is still a constraint on the account of limited EHs being successfully applied on the preparative scale for industrial biotransformations. This limitation has to be overcome before application of identified functional EHs on large scale. Many strategies such as optimizing reaction media, immobilizing EHs and laboratory-scale directed evolution of EHs have been adopted for enhancing the industrial potential of EHs. In this review, these approaches have been highlighted which can serve as a pathway for the enrichment of already identified EHs for their application on an industrial scale in future studies.     

Role of epoxide hydrolases in lipid metabolism

Epoxide hydrolases (EH), enzymes present in all living organisms, transform epoxide-containing lipids to 1,2-diols by the addition of a molecule of water. Many of these oxygenated lipid substrates have potent biological activities: host defense, control of development, regulation of blood pressure, inflammation, and pain. In general, the bioactivity of these natural epoxides is significantly reduced upon metabolism to diols. Thus, through the regulation of the titer of lipid epoxides, EHs have important and diverse biological roles with profound effects on the physiological state of the host organism. This review will discuss the biological activity of key lipid epoxides in mammals. In addition, the use of EH specific inhibitors will be highlighted as possible therapeutic disease interventions.     

Mutation of an atypical oxirane oxyanion hole improves regioselectivity of the α/β-fold epoxide hydrolase Alp1U

Epoxide hydrolases (EHs) have been characterized and engineered as biocatalysts that convert epoxides to valuable chiral vicinal diol precursors of drugs and bioactive compounds. Nonetheless, the regioselectivity control of the epoxide ring opening by EHs remains challenging. Alp1U is an α/β-fold EH that exhibits poor regioselectivity in the epoxide hydrolysis of fluostatin C (compound 1) and produces a pair of stereoisomers. Herein, we established the absolute configuration of the two stereoisomeric products and determined the crystal structure of Alp1U. A Trp-186/Trp-187/Tyr-247 oxirane oxygen hole was identified in Alp1U that replaced the canonical Tyr/Tyr pair in α/β-EHs. Mutation of residues in the atypical oxirane oxygen hole of Alp1U improved the regioselectivity for epoxide hydrolysis on 1. The single site Y247F mutation led to highly regioselective (98%) attack at C-3 of 1, whereas the double mutation W187F/Y247F resulted in regioselective (94%) nucleophilic attack at C-2. Furthermore, single-crystal X-ray structures of the two regioselective Alp1U variants in complex with 1 were determined. These findings allowed insights into the reaction details of Alp1U and provided a new approach for engineering regioselective epoxide hydrolases.     

Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in ~ 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of ~ 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application.     

Clues for the existence of two different epoxide hydrolase activities in the fungus Beauveria bassiana

Epoxide hydrolase activity was produced during the exponential and stationary growth phases of the fungus Beauveria bassiana ATCC 7159. It was completely cell-associated. After cell disruption epoxide hydrolase activity was recovered in both the cell debris (EH "A") and the soluble fraction (EH "B"), but not in the membrane fraction. Activity assays of these fractions with two different substrates indicated that their substrate specificity, as well as the corresponding E value and, to a lesser extent, their regioselectivity, were different. Also, we could observe that the absolute configuration of the residual epoxide was opposite. This indicates that these two epoxide hydrolase activities are substantially different and are, therefore, interestingly complementary biocatalysts for the preparation of the corresponding epoxides and/or vicinal diols in nearly enantiopure form.     

Effect of carbon and nitrogen sources on the production of a highly enantioselective epoxide hydrolase from Aspergillus niger

The highly enantioselective epoxide hydrolase from Aspergillus niger is well utilized as biocatalysts for the preparation of enantiopure chiral epoxides and diols. Both growth of the fungus and EH activity production were found greatly affected by changing the carbon or the nitrogen source with fructose and corn steep liquor being the best. Their concentrations were optimized (10 g.l–1 of fructose and 15 g.l–1 of corn steep) which resulted in an increase of both the biomass produced (31%) and the epoxide hydrolase specific activity (38%). The results obtained suggested a complex regulation of the EH production. On the whole, a two times increase of the total EH activity was obtained. © Rapid Science Ltd. 1998     

Microbial epoxide hydrolases for preparative biotransformations

Epoxide hydrolases from microbial sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which furnishes the corresponding vicinal diol and remaining non-hydrolysed epoxide in nonracemic form, enantioconvergent processes are possible: these are highly attractive as they lead to the formation of a single enantiomeric diol from a racemic oxirane. The data accumulated over recent years reveal a common picture of the substrate structure selectivity pattern of microbial epoxide hydrolases and indicate that substrates of various structural types can be selectively hydrolysed with enzymes from certain microbial sources.     

Structure–function relationships of epoxide hydrolases and their potential use in biocatalysis

Background

Chiral epoxides and diols are important synthons for manufacturing fine chemicals and pharmaceuticals. The epoxide hydrolases (EC 3.3.2.-) catalyze the hydrolytic ring opening of epoxides producing the corresponding vicinal diol. Several isoenzymes display catalytic properties that position them as promising biocatalytic tools for the generation of enantiopure epoxides and diols.

Scope of review

This review focuses on the present data on enzyme structure and function in connection to biocatalytic applications. Available data on biocatalysis employed for purposes of stereospecific ring opening, to produce chiral vicinal diols, and kinetic resolution regimes, to achieve enantiopure epoxides, are discussed and related to results gained from structure–activity studies on the enzyme catalysts. More recent examples of the concept of directed evolution of enzyme function are also presented.

Major conclusions

The present understanding of structure–activity relationships in epoxide hydrolases regarding chemical catalysis is strong. With the ongoing research, a more detailed view of the factors that influence substrate specificities and stereospecificities is expected to arise. The already present use of epoxide hydrolases in synthetic applications is expected to expand as new enzymes are being isolated and characterized. Refined methodologies for directed evolution of desired catalytic and physicochemical properties may further boost the development of novel and useful biocatalysts.

General significance

The catalytic power of enzymes provides new possibilities for efficient, specific and sustainable technologies to be developed for production of useful chemicals.     

A novel enantioselective epoxide hydrolase from Agromyces mediolanus ZJB120203: Cloning,characterization and application

A new strain Agromyces mediolanus ZJB120203, capable of enantioselective epoxide hydrolase (EH) activity was isolated employing a newly established colorimetric screening and chiral GC analysis method. The partial nucleotide sequence of an epoxide hydrolase (AmEH) gene from A. mediolanus ZJB120203 was obtained by PCR using degenerate primers designed based on the conserved domains of EHs. Subsequently, an open reading frame containing 1167 bp and encoding 388 amino acids polypeptide were identified. Expression of AmEH was carried out in Escherichia coli and purification was performed by Nickel-affinity chromatography. The purified AmEH had a molecular weight of 43 kDa and showed its optimum pH and temperature at 8.0 and 35 °C, respectively. Moreover, this AmEH showed broad substrates specificity toward epoxides. In this study, it is demonstrated that the AmEH could unusually catalyze the hydrolysis of (R)-ECH to produce enantiopure (S)-ECH. Enantiopure (S)-ECH could be obtained with enantiomeric excess (ee) of >99% and yield of 21.5% from 64 mM (R,S)-ECH. It is indicated that AmEH from A. mediolanus is an attractive biocatalyst for the efficient preparation of optically active ECH.     

Biosynthesis of (R)-phenyl-1,2-ethanediol from racemic styrene oxide by using bacterial and marine fish epoxide hydrolases

Enantio-convergent hydrolysis of racemic styrene oxides was achieved to prepare enantiopure (R)-phenyl-1,2-ethanediol by using two recombinant epoxide hydrolases (EHs) of a bacterium, Caulobacter crescentus, and a marine fish, Mugil cephalus. The recombinant C. crescentus EH primarily attacked the benzylic carbon of (S)-styrene oxide, while the M. cephalus EH preferentially attacked the terminal carbon of (R)-styrene oxide, thus leading to the formation of (R)-phenyl-1,2-ethanediol as the main product. (R)-Phenyl-1,2-ethanediol was obtained with 90% enantiomeric excess and yield as high as 94% from 50 mM racemic styrene oxides in a one-pot process.     

Biocatalytic conversion of epoxides

Epoxides are attractive intermediates for producing chiral compounds. Important biocatalytic reactions involving epoxides include epoxide hydrolase mediated kinetic resolution, leading to the formation of diols and enantiopure remaining substrates, and enantioconvergent enzymatic hydrolysis, which gives high yields of a single enantiomer from racemic mixtures. Epoxides can also be converted by non-hydrolytic enantioselective ring opening, using alternative anionic nucleophiles; these reactions can be catalysed by haloalcohol dehalogenases. The differences in scope of these enzymatic conversions is related to their different catalytic mechanisms, which involve, respectively, covalent catalysis with an aspartate carboxylate as the nucleophile and non-covalent catalysis with a tyrosine that acts as a general acid-base. The emerging new possibilities for enantioselective biocatalytic conversion of epoxides suggests that their importance in green chemistry will grow.     

The rabbit liver microsomal biotransformation of 1,1-dialkylethylenes: Enantioface selection of epoxidation and enantioselectivity of epoxide hydrolysis

The rabbit liver microsomal biotransformation of α-methylstyrene ( 1a ), 2-methyl-1-hexene ( 1b ), 2,4,4-trimethyl-1-pentene ( 1c ), and 2,3,3-trimethyl-1-butene ( 1d ) has been investigated with the aim at establishing the enantioface selection of the cytochrome P-450-promoted epoxidation of the double bond and the enantioselectivity of microsomal epoxide hydrolase (mEH)-catalyzed hydrolysis of the resulting epoxides. GLC on a Chiraldex G-TA (ASTEC) column was used to determine the enantiomeric composition of the products. The epoxides 2 first produced in incubations carried out in the presence of an NADPH regenerating system were not detected, being rapidly hydrolyzed by mEH to diols 3 . The enantiomeric composition of the latter showed that no enantioface selection occurred in the epoxidation of 1c and 1d , and a very low (8%) ee of the (R)-epoxide was formed from 1b . Incubation of racemic epoxides 2b–d with the microsomal fraction showed that the mEH-catalyzed hydrolysis of 2c and 2d was practically nonenantioselective, while that of 2b exhibited a selectivity E = 4.9 favoring the hydrolysis of the (S)-enantiomer. A comparison of these results with those previously obtained for linear and branched chain alkyl monosubstituted oxiranes shows that the introduction of the second alkyl substituent suppresses the selectivity of the mEH reaction of the latter and reverses that of the former substrates. © 1994 Wiley-Liss, Inc.