Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two alpha/beta hydrolase superfamily fold enzymes were cloned, over-expressed in Escherichia coli, purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of cis-2,3-epoxybutane producing the (2R,3R)-diol product with ee exceeding 99%. Interestingly these enzymes also hydrolyse (4R)-limonene-1,2-epoxide with Sibe-EH being specific for the trans isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85 degrees C retaining 80% of its initial activity after 3 h thermal treatment at 70 degrees C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 A, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs.
        
Title: Enantioselective Hydrolysis of Racemic and Meso-Epoxides with Recombinant Escherichia coli Expressing Epoxide Hydrolase from Sphingomonas sp. HXN-200: Preparation of Epoxides and Vicinal Diols in High ee and High Concentration Wu S, Li A, Chin YS, Li Z Ref: ACS Catal, 3:752, 2013 : PubMed
A unique epoxide hydrolase (SpEH) from Sphingomonas sp. HXN-200 was identified and cloned based on genome sequencing and expressed in Escherichia coli. The engineered E. coli (SpEH) showed the same selectivity and substrate specificity as the wild type strain and 172 times higher activity than Sphingomonas sp. HXN-200 for the hydrolysis of styrene oxide 1. Hydrolysis of racemic styrene oxide 1, substituted styrene oxides 3, 57, and N-phenoxycarbonyl-3,4-epoxypiperidine 8 (200100 mM) with resting cells of E. coli (SpEH) gave (S)-epoxides 1, 3, 57 and (-)-8 in 98.099.5% enantiomeric excess (ee) and 37.646.5% yield. Hydrolysis of cyclopentene oxide 9, cyclohexene oxide 10, and N-benzyloxycarbonyl-3,4-epoxypyrrolidine 11 (100 mM) afforded the corresponding (R, R)-vicinal trans-diols 1214 in 8693% ee and 9099% yield. The ee of (1R, 2R)-cyclohexane-1,2-diol 13 was improved to 99% by simple crystallization. These biotransformations showed high specific activity (0.284.3 U/mg cdw), product concentration, product/cells ratio, and cell-based productivity. Hydrolysis at even higher substrate concentration was also achieved: (S)-1 was obtained in 430 mM (51 g/Lorg) and 43% yield; (1R, 2R)-13 was obtained in 500 mM (58 g/L) and >99% yield. Gram-scale preparation of epoxides (S)-1, (S)-3, (S)-6 and diols (1R, 2R)-12, (1R, 2R)-13, (3R, 4R)-14 were also demonstrated. E. coli (SpEH) cells showed the highest enantioselectivity to produce (S)-1 (E of 39) among all known EHs in the form of whole cells or free enzymes and the highest enantioselectivities to produce (S)-3, 5, 6, 7, (-)-8, and (R, R)-14 (E of 36, 35, 28, 57, 22, and 28) among all known EHs. The easily available and highly active E. coli (SpEH) cells are the best biocatalysts known thus far for the practical preparation of these useful and valuable enantiopure epoxides and vicinal diols via hydrolysis.