Substrate Report for: o-cresyl-glycidyl-ether

In order to provide more alternative epoxide hydrolases for industrial production, a novel cDNA gene Rpeh-encoding epoxide hydrolase (RpEH) of Rhodotorula paludigena JNU001 identified by 26S rDNA sequence analysis was amplified by RT-PCR. The open-reading frame (ORF) of Rpeh was 1236 bp encoding RpEH of 411 amino acids and was heterologously expressed in Escherichia coli BL21(DE3). The substrate spectrum of expressed RpEH showed that the transformant E. coli/Rpeh had excellent enantioselectivity to 2a, 3a, and 5a-10a, among which E. coli/Rpeh had the highest activity (2473 U/g wet cells) and wonderful enantioselectivity (E = 101) for 8a, and its regioselectivity coefficients, alphaR and betaS, toward (R)- and (S)-8a were 99.7 and 83.2%, respectively. Using only 10 mg wet cells/mL of E. coli/Rpeh, the near-perfect kinetic resolution of rac-8a at a high concentration (1000 mM) was achieved within 2.5 h, giving (R)-8a with more than 99% enantiomeric excess (ees) and 46.7% yield and producing (S)-8b with 93.2% eep and 51.4% yield with high space-time yield (STY) for (R)-8a and (S)-8b were 30.6 and 37.3 g/L/h.

To provide more options for the stereoselective hydrolysis of epoxides, an epoxide hydrolase (VrEH3) gene from Vigna radiata was cloned and expressed in Escherichia coli. Recombinant VrEH3 displayed the maximum activity at pH 7.0 and 45 degrees C and high stability at pH 4.5-7.5 and 55 degrees C. Notably, reVrEH3 exhibited high and complementary regioselectivity toward styrene oxides 1a-3a and high enantioselectivity (E = 48.7) toward o-cresyl glycidyl ether 9a. To elucidate these interesting phenomena, the interactions of the three-dimensional structure between VrEH3 and enantiomers of 1a and 9a were analyzed by molecular docking simulation. Using E. coli/vreh3 whole cells, gram-scale preparations of (R)-1b and (R)-9a were performed by enantioconvergent hydrolysis of 100 mM rac-1a and kinetic resolution of 200 mM rac-9a in the buffer-free water system at 25 degrees C. These afforded (R)-1b with >99% eep and 78.7% overall yield after recrystallization and (R)-9a with >99% ees, 38.7% overall yield, and 12.7 g/L/h space-time yield.

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84-99% enantiomeric excess and with 21-73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.

In order to provide more alternative epoxide hydrolases for industrial production, a novel cDNA gene Rpeh-encoding epoxide hydrolase (RpEH) of Rhodotorula paludigena JNU001 identified by 26S rDNA sequence analysis was amplified by RT-PCR. The open-reading frame (ORF) of Rpeh was 1236 bp encoding RpEH of 411 amino acids and was heterologously expressed in Escherichia coli BL21(DE3). The substrate spectrum of expressed RpEH showed that the transformant E. coli/Rpeh had excellent enantioselectivity to 2a, 3a, and 5a-10a, among which E. coli/Rpeh had the highest activity (2473 U/g wet cells) and wonderful enantioselectivity (E = 101) for 8a, and its regioselectivity coefficients, alphaR and betaS, toward (R)- and (S)-8a were 99.7 and 83.2%, respectively. Using only 10 mg wet cells/mL of E. coli/Rpeh, the near-perfect kinetic resolution of rac-8a at a high concentration (1000 mM) was achieved within 2.5 h, giving (R)-8a with more than 99% enantiomeric excess (ees) and 46.7% yield and producing (S)-8b with 93.2% eep and 51.4% yield with high space-time yield (STY) for (R)-8a and (S)-8b were 30.6 and 37.3 g/L/h.

To provide more options for the stereoselective hydrolysis of epoxides, an epoxide hydrolase (VrEH3) gene from Vigna radiata was cloned and expressed in Escherichia coli. Recombinant VrEH3 displayed the maximum activity at pH 7.0 and 45 degrees C and high stability at pH 4.5-7.5 and 55 degrees C. Notably, reVrEH3 exhibited high and complementary regioselectivity toward styrene oxides 1a-3a and high enantioselectivity (E = 48.7) toward o-cresyl glycidyl ether 9a. To elucidate these interesting phenomena, the interactions of the three-dimensional structure between VrEH3 and enantiomers of 1a and 9a were analyzed by molecular docking simulation. Using E. coli/vreh3 whole cells, gram-scale preparations of (R)-1b and (R)-9a were performed by enantioconvergent hydrolysis of 100 mM rac-1a and kinetic resolution of 200 mM rac-9a in the buffer-free water system at 25 degrees C. These afforded (R)-1b with >99% eep and 78.7% overall yield after recrystallization and (R)-9a with >99% ees, 38.7% overall yield, and 12.7 g/L/h space-time yield.

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84-99% enantiomeric excess and with 21-73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.

An open reading frame (ORF) encoding a putative epoxide hydrolase (EHase) was identified by analyzing the genome sequence of Sphingophyxis alaskensis. The EHase gene (seh) was cloned and expressed in E. coli. To facilitate purification, the gene was fused in-frame to 6x histidine at the C-terminus. The recombinant EHase (rSEH) was highly soluble and could be purified to apparent homogeneity by one step of metal affinity chromatography. The purified SEH displayed hydrolyzing activities toward various epoxides such as styrene oxide, glycidyl phenyl ether, epoxyhexane, epoxybutane, epichlorohydrin, and epifluorohydrin. The optimum activity toward styrene oxide was observed at pH 6.5 and 35 degrees . The purified SEH showed a cold-adapted property, displaying more than 40% of activity at low temperature of 10 degrees compared with the optimum activity. Despite the catalytic efficiency, the purified SEH did not hydrolyze various epoxides enantioselectively. Km and kcat of SEH toward (R)-styrene oxide were calculated as 4+/-0.3 mM and 7.42 s(-1), respectively, whereas Km and kcat of SEH toward (S)-styrene oxide were 5.25+/-0.3 mM and 10.08 s(-1), respectively.