Substrate Report for: 1,2-epoxybutane

The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities. The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases (EHs) reveals its resourceful microscopic machinery. The whole-genome sequence of Streptomyces sp. CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, was explored by genome mining, and a putative alpha/beta-epoxide hydrolase named B1EPH2 and encoded by 344 amino acids was selected for functional and structural studies. The crystal structure of B1EPH2 was obtained at a resolution of 2.2 and it was found to have a similar fold to other EHs, despite its hexameric quaternary structure, which contrasts with previously solved dimeric and monomeric EH structures. While B1EPH2 has a high sequence similarity to EHB from Mycobacterium tuberculosis, its cavity is similar to that of human EH. A group of 12 aromatic and aliphatic racemic epoxides were assayed to determine the activity of B1EPH2; remarkably, this enzyme was able to hydrolyse all the epoxides to the respective 1,2-diols, indicating a wide-range substrate scope acceptance. Moreover, the (R)- and (S)-enantiomers of styrene oxide, epichlorohydrin and 1,2-epoxybutane were used to monitor enantiopreference. Taken together, the functional and structural analyses indicate that this enzyme is an attractive biocatalyst for future biotechnological applications.

The white-rot basidiomycetes Phanerochaete chrysosporium is a model fungus used to investigate the secondary metabolism and lignin degradation. Genomic sequencing reveals the presence of at least 18 genes encoding putative epoxide hydrolases (EHs). One cDNA encoding EH (designated as PchEHA) was cloned and expressed in Escherichia coli. Transcriptional analysis demonstrated that the transcripts of PchEHA could be detected under the ligninolytic and nonligninolytic conditions as well as amended with anthracene. The recombinant enzyme exhibits broad hydrolytic activity toward several racemic epoxides including styrene oxide, epichlorohydrin, and 1,2-epoxybutane, but with different specificity. Using racemic styrene oxide as the substrate, the optimal pH and temperature are pH 9.0 and 40 degrees C, respectively. The enzyme is not sensitive to EDTA, and is inhibited by H2O2, and several metal ions including Zn(2+), Cd(2+), and Hg(2+) at various extents. Several organic cosolvents including acetone, dimethylsulfoxide, formamide, glycerol and ethanol at 10% (v/v) cause slight or no inhibition of the hydrolytic reaction. More importantly, the recombinant enzyme displays distinct enantioselective preference to several chiral epoxides. The enzyme showed good enantioselectivity toward chiral styrene oxide with preferential hydrolysis of (R)-enantiomer. PchEHA is likely a novel soluble EH based on the sequence analysis and catalytic properties, and is a great potential biocatalyst for the preparation of enantiopure styrene oxide in racemic kinetic resolution.

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.

The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities. The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases (EHs) reveals its resourceful microscopic machinery. The whole-genome sequence of Streptomyces sp. CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, was explored by genome mining, and a putative alpha/beta-epoxide hydrolase named B1EPH2 and encoded by 344 amino acids was selected for functional and structural studies. The crystal structure of B1EPH2 was obtained at a resolution of 2.2 and it was found to have a similar fold to other EHs, despite its hexameric quaternary structure, which contrasts with previously solved dimeric and monomeric EH structures. While B1EPH2 has a high sequence similarity to EHB from Mycobacterium tuberculosis, its cavity is similar to that of human EH. A group of 12 aromatic and aliphatic racemic epoxides were assayed to determine the activity of B1EPH2; remarkably, this enzyme was able to hydrolyse all the epoxides to the respective 1,2-diols, indicating a wide-range substrate scope acceptance. Moreover, the (R)- and (S)-enantiomers of styrene oxide, epichlorohydrin and 1,2-epoxybutane were used to monitor enantiopreference. Taken together, the functional and structural analyses indicate that this enzyme is an attractive biocatalyst for future biotechnological applications.

The white-rot basidiomycetes Phanerochaete chrysosporium is a model fungus used to investigate the secondary metabolism and lignin degradation. Genomic sequencing reveals the presence of at least 18 genes encoding putative epoxide hydrolases (EHs). One cDNA encoding EH (designated as PchEHA) was cloned and expressed in Escherichia coli. Transcriptional analysis demonstrated that the transcripts of PchEHA could be detected under the ligninolytic and nonligninolytic conditions as well as amended with anthracene. The recombinant enzyme exhibits broad hydrolytic activity toward several racemic epoxides including styrene oxide, epichlorohydrin, and 1,2-epoxybutane, but with different specificity. Using racemic styrene oxide as the substrate, the optimal pH and temperature are pH 9.0 and 40 degrees C, respectively. The enzyme is not sensitive to EDTA, and is inhibited by H2O2, and several metal ions including Zn(2+), Cd(2+), and Hg(2+) at various extents. Several organic cosolvents including acetone, dimethylsulfoxide, formamide, glycerol and ethanol at 10% (v/v) cause slight or no inhibition of the hydrolytic reaction. More importantly, the recombinant enzyme displays distinct enantioselective preference to several chiral epoxides. The enzyme showed good enantioselectivity toward chiral styrene oxide with preferential hydrolysis of (R)-enantiomer. PchEHA is likely a novel soluble EH based on the sequence analysis and catalytic properties, and is a great potential biocatalyst for the preparation of enantiopure styrene oxide in racemic kinetic resolution.

A gene encoding a putative epoxide hydrolase (EHase) was identified by analyzing an open reading frame of the genome sequence of Novosphingobium aromaticivorans, retaining the conserved catalytic residues such as the catalytic triad (Asp177, Glu328, and His355) and the oxyanion hole. The enantioselective EHase gene (neh) was cloned, and the recombinant EHase could be purified to apparent homogeneity by one step of metal affinity chromatography and further characterized. The purified N. aromaticivorans enantioselective epoxide hydrolase (NEH) showed enantioselective hydrolysis toward styrene oxide, glycidyl phenyl ether, epoxybutane, and epichlorohydrin. The optimal EHase activity toward styrene oxide occurred at pH 6.5 and 45 degrees C. The purified NEH could preferentially hydrolyze (R)-styrene oxide with enantiomeric excess of more than 99% and 11.7% yield after 20-min incubation at an optimal condition. The enantioselective hydrolysis of styrene oxide was also confirmed by the analysis of the vicinal diol, 1-phenyl-1,2-ethanediol. The hydrolyzing rates of the purified NEH toward epoxide substrates were not affected by as high as 100 mM racemic styrene oxide.

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.