WO2007043777A1 - Enantioselective epoxide hydlrolase and method for preparing an enantiopure epoxide using the same - Google Patents
Enantioselective epoxide hydlrolase and method for preparing an enantiopure epoxide using the same Download PDFInfo
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- WO2007043777A1 WO2007043777A1 PCT/KR2006/004003 KR2006004003W WO2007043777A1 WO 2007043777 A1 WO2007043777 A1 WO 2007043777A1 KR 2006004003 W KR2006004003 W KR 2006004003W WO 2007043777 A1 WO2007043777 A1 WO 2007043777A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
- C12P41/004—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of alcohol- or thiol groups in the enantiomers or the inverse reaction
Definitions
- the present invention relates to an enantioslective epoxide hydrolase and a method of epoxide having enantiopure activity on various epoxide substrates by using the enantioselective epoxide hydrolase.
- bioactive materials such as medicines are in a form of various enantiomer, and only a specific enantiomer has the desired efficacy, and the remnant enantionmers causes serious undesirable effect.
- enantiomer only single enantiomer must be produced, and thus many researches on the synthesis of enantio-pure bioactive material can be processed.
- Enantiopure epoxides and vicinal diols are versatile synthetic intermediates for the preparation of enantiopure bioactive compounds such as pharmaceutical compounds, peticides, and functional foods (Grogan, et al., FEMS Microbiol. Lett., 141 :239-243, 1996 ; Arahira, et al., Eur. J. Biochem., 267:2649-2657, 2000), because the compounds has excellent reactivity and can induce the various reactions.
- the enantiopure epoxides can be prepared by using the chiral chemical catalysts and enzymes, and only single enantiomer is prepared by performing the selective hydrolysis with epoxide hydrolase to each enantiomer in racemic epoxide substrates.
- the method can be used commercially in the near future, because it can change inexpensive racemic substrate to enantiopure epoxide having higher added value.
- the epoxide hydrolase hydrolyzes only (R) or (S)- enantiomer among racemic epoxide substrate with enantio-selectivity to diol and leave the other type of enantiomer, so as to produce enantiopure epoxide.
- the enantioselectivity of epoxide hydrolase to (R) or (S)-enantiomer depends on microorganisms and substrate structure.
- Epoxide hydrolases (EHase; EC 3.3.2.3) are ubiquitous enzymes that have been isolated from a wide variety of sources such bacteria, yeast, fungi, insect, plant and mammalian (Weijers, et al., J. MoI. Catal. B Enzym., 6:199-214, 1999; Archelas, & Furstoss, Curr. Opin. Chem. Biol., 5:112-119, 2001). Due to the potential application in the production of enantiopure epoxides by kinetic resolution of enantioselective EHase, several EHases have been developed (Tokunaga, et al., Science, 277:936-938, 1997).
- EHases are members of the ⁇ / ⁇ hydrolase family which includes proteases, lipases, esterases, dehalogenases, and peroxidases (Nardini, & Dijkstra, Curr. Opin. Struct. Biol, 9:732-737, 1999; Rick, et al,. J. Am. Chem. Soc, 121 :7417-7418, 1999).
- ⁇ / ⁇ domains consist of a central, parallel or mixed ⁇ sheet surrounded by ⁇ helices.
- the conserved catalytic triad of ⁇ / ⁇ hydrolase fold enzymes consists of a nucleophilic residue (Asp or Ser), an acidic residue (Asp or GIu) and a conserved histidine residue.
- Another conserved amino acid sequence is the HGXP motif containing the oxyanion hole of the enzyme (Ollis, et al., Protein Eng., 5:197- 211, 1992).
- the present inventor found the epoxide hydrolase having high enantioselective hydrolyzing activity by screening Erythrobacter sp., Sphingopyxis sp., Novosphingobium sp. and Rhodobacterium sp. from various marine environments, analying the ORF sequence in their genome to determine a candidate gene, and expressing the candidate gene.
- the object of the present invention is to provide enantioselective epoxide hydrolase proteins which produce high enantiopure epoxide and are isolated from marine environments.
- the further object of the present invention is to provide a method of preparing enantiopure epoxide by using the epoxide hydrolase proteins having high enantio-selectivity to various epoxide substrates.
- Another object of the present invention is to provide Erythrobacter sp.,
- Sphingophyxis sp. Novosphingobium sp.
- Rhodobacterium sp. with enantioselective hydrolase activity from the various marine environments and a method of screening them.
- the present invention provide an enantioselective epoxide hydrolase protein which is isolated from Erythrobacter litoralis, and has the following characteristics: 1) a molecular weight of 30 to 45kDa as measured by SDS-
- the protein comprises an amino acid sequence as shown in SEQ ID NO: 1
- amino acid sequence as shown in SEQ ID NO: 13 is coded by a nucleotide sequence as shown in SEQ ID NO:
- amino acid sequence as shown in SEQ ID NO: 15 is coded by a nucleotide sequence as shown in SEQ ID NO: 16
- amino acid sequence as shown in SEQ ID NO: 17 is coded by a nucleotide sequence as shown in SEQ ID NO: 18.
- the present invention an enantioselective epoxide hydrolase protein which is isolated from Sphingophyxis alaskensis, and has the following characteristics: 1) a molecular weight of 45 to 50 kDa as measured by SDS-PAGE method; 2) an optimum pH 7.0 to 8.0; and 3) an optimum temperature of 30 to 40 ° C.
- the protein has an amino acid sequence as shown in SEQ ID NO: 28, and more preferably, is coded by a nucleotide sequence as shown in SEQ ID NO: 29.
- the present invention an enantioselective epoxide hydrolase protein which is isolated from Novosphingobium aromaticivorans, and has the following characteristics: 1) a molecular weight of 40 to 45 kDa measured by SDS-PAGE method; 2) an optimum pH 7.0 to 8.0; and 3) an optimum temperature of 30 to 40 °C .
- the protein has an amino acid sequence as shown in SEQ ID NO: 30, and more preferably, is coded by a nucleotide sequence as shown in SEQ ID NO: 31.
- the present invention provides an enantioselective epoxide hydrolase protein which is isolated from Rhodobacterium sp. HTCC2654, and has the following characteristics: 1) a molecular weight of 35 to 40 kDa measured by SDS-PAGE method; 2) an optimum pH 7.0 to 8.0; and 3) an optimum temperature of 30 to 40 °C .
- the protein has an amino acid sequence as shown in SEQ ID NO: 32, and more preferably is coded by a nucleotide sequence as shown in SEQ ID NO:
- the present invention provides a method of preparing enantiopure epoxide by using enantioselective epoxide hydrolase protein.
- Fig. IA shows the type of various epoxide substrates, wherein A, B, C and D indicate styrene oxide(SO), glycidyl phenyl ether(GPE), 1 ,2-epoxyhexane(EX) and 1 ,2-epoxybutane(EB), respectively.
- Fig. IB shows the hydrolyzing activity of Erythrobacter spp. JCS358 toward the racemic SO substrate with gas chromatographty(GC), wherein A and B indicate (S)-styrene oxide and (R)-styrene oxide, respectively.
- Fig. 2 shows the kinetic resolution of Erythrobacter spp. JCS358 toward the racemic SO substrate, wherein o, • and D indicate (R)-styrene oxide area, (S)- styrene oxide area and enantiomeric excess(%), respectively.
- Fig. 3A to 3C show alignment between amino acid sequence of purified
- Fig. 4A and 4B show alignment between amino acid sequence purified from EEHl and EEH2 to that of known protein.
- Fig. 5 shows a phylogenetic analysis of epoxide hydrolase(EHase).
- Fig. 6 shows the purity of three EHases isolated from Erythrobacter litoralis
- Fig.A indicates purified EEHl
- B indicates purified EEH2 and EEH3, respectively.
- Fig. 7A and 7B show the effects of pH and temperature on the activity of the purified rEEHl, rEEH2 and rEEH3, wherein O , • and T indicate EEHl, EEH2 and EEH3,, respectively.
- Fig. 8 shows the hydrolyzing activities of enantioselective EHases(EEHl, EEH2 and EEH3) toward the racemic SO substrate with gas chromatography.
- Fig. 9 shows the purity of EHase isolated from Novosphingobium aromativorans with SDS-PAGE.
- the present invention provides purified enantioselective epoxide hydrolase proteins from Erythrobacter litoralis, Sphingophyxis alaskensis, Novosphingobium aromaticivorans and Rhodobacterium.
- Rhodobacterium sp. are selected by screening, and then ORF sequence in their genome are aligned to select a candidate gene.
- the enantioselective hydrolase proteins having high enantioselectivity to the various substrates are separated and purified from the expressed products from the candidate genes. More specifically, the enantioselective hydrolase having high enantio-selectivity are separated by using the following screening method from the microorganisms including Erythrobacter sp.,
- the screening method includes the steps of:
- the sample of interest in step 1 of the screening method is not limited particularly, but can be marine sediment, sponge and algae which are collected from various marine environments such as Hoogin in Gangwon-Do, Ulleungdo(Island), Dokdo(Island), Taejongdae in Busan, and Sihwa in Gyeonggi-do in Republic of Korea, and Kagoshima in Japan.
- the strains can be isolated from the collected marine sediment directly, or after culturing the marine sediment in enriched medium.
- the enriched medium step 2 in the screening method is not limited particularly, preferably 1 wt% of styrene oxide (SO) or alkan mixture (nC8, ClO, nC12, nC13, nC14, nC15, nC16, C 17, nC18, and cyclohexan(Sigma, MO, USA) are mixed in 1 L of mineral salt medium (MM 2) of sea water.
- the bacteria in step 3) is selected from Erythrobacter litoralis, Erythrobacter sp.
- the microorganism can be Erythrobacter sp. selected from the group consisting of Erythrobacter litoralis HTCC2594, Erythrobacter sp. AKS329, Erythrobacter sp. aquimaris JCS325, Erythrobacter gaetbuli JCS340 JCS325, Erythrobacter sp. JCS340, Erythrobacter sp.
- JCS350 Erythrobacter sp. JCS358, Alterierythrobacter sp. JCS350, Erythrobacter aquimaris sp. JCS360, Erythrobacter aquimaris sp. JCS364, Erythrobacter luteolus sp. JCS368, Erythrobacter sp. HJ239, Erythrobacter longus sp. DokDo 15, Erythrobacter litoralis DMS8509 and Erythrobacter geatbuli KCTC12227(Table 2), but not limited thereto.
- the open reading frame analysis can be carried out by ProteinFinder produced by Ensoltek (www.ensoltek.com) and BLAST program, but not limited thereto.
- analyzing method of amino acid sequence of conventional epoxide hydrolase, and the new epoxide hydrolases of the present invention can be preformed by using CLUSTAL W program(Thompson, et al., Nucleic. Acids. 22:4673-4680, 1994), but not limited thereto.
- the candidate genes in step 3) include EEHl gene represented by SEQ ID NO: 13, EEH2 gene represented by SEQ ID NO: 16 and EEH3 gene represented by
- SEQ ID NO: 29 from Sphingophyxis alaskensis, nEEH gene represented by SEQ ID NO:
- SEQ ID NO: 33 from Rhodobacterium, but not limited thereto.
- the candidate gene can be entire open reading frame or its fragment derived from the genes.
- the expression vector can be any expression vector used in the prior art, for example pET-24a(+).
- step 4 analysis of the hydrolyzing activity to the various epoxide substrates was performed by a spectrophotometric assay based on the epoxide extracted from the reaction mixture and spectrophotometric quantification of the non-extracted diol or gas chromatography.
- epoxide hydrolase proteins having high enantioselective hydrolyzing activity to various epoxide substrates are separated and purified from the candidate strains.
- the epoxide hydrolase are separated and purified by general separation and purification method used generally in this field. For example, after performing seed culture in LB medium enriched with Kanamycin (50 ug/ml), 1% of the cultured strain is added to the main medium, cultured for 3 hours, and added with IPTG to ImM of the final concentration. To purify the expressed gene product, His-Tag is added to the candidate gene, and then is cleaved by Talon resin (Clontech, Co.).
- 1.122bp gene (EEHl, SEQ ID NO: 14), 870bp gene (EEH2, SEQ ID NO: 16) and 888bp gene (EEH3, SEQ ID NO: 18) are isolated from Erythrobacter litoralis HTCC2594.
- sEEH gene(SEQ ID NO: 29) of Sphingophyxis alaskensis, nEEH gene (SEQ ID NO: 31) of Novosphingobium aromaticivorans, and rEEH gene (SEQ ID NO: 33) of Rhodobacterium sp. HTCC2654 are separated respectively.
- each gene is introduced into expression vector, pET-24a (+), transformed to BL21-CodonPlus (DE3)-RP (Novagen), and then performed by SDS-PAGE electrophoresis.
- the hydrolases are 41kDa-sized protein (rEEHl, SEQ ID NO: 13) isolated from Erythrobacter litoralis HTCC2594, 33.4kDa-sized protein (rEEH2, SEQ ID NO: 15) and 34.5 kDa-sized protein (rEEH3, SEQ ID NO: 17), 49kDa-sized protein (sEEH, SEQ ID NO: 28) isolated from Sphingophyxis alaskensis, 43kDa-sized protein (nEEH, SEQ ID NO: 30) isolated from Novosphingobium aromaticivorans, and 36kDa43kDa-sized isolated from Sphingophyxis alaskensis (rEEH, SEQ ID NO: 32) isolated from Rhodobacterium sp.
- HTCC2594
- the present invention provides an enantioselective epoxide hydrolase protein which is isolated from Erythrobacter litoralis HTCC2594, and has the following characteristics:
- the rEEHl, rEEH2 and rEEH3 hydrolase isolated from Erythrobacter litoralis has a molecular weight of 4IkDa (polypeptide as shown in SEQ ID NO: 13), 33.4kDa(polypeptide as shown in SEQ ID NO: 15) and 34.5kDa(polypeptide as shown in SEQ ID NO: 17) (Fig. 6A and Fig. 6B), optimum pH of 6.5(rEEHl), 7.5(rEEH2) and 8.0(rEEH3)(Fig. 7A), and optimum temperature of 50 ° C (rEEHl), 55 ° C(rEEH2) and 45 °C (rEEH3)(Fig. 7B), respectively.
- the present invention provides an enantioselective epoxide hydrolase protein which is isolated from Sphingophyxis alaskemis, and has the following characteristics: 1) a molecular weight of 45 to 50 kDa measured by SDS-PAGE method;
- the rEEH hydrolase has a molecular weight of 49kDa (polypeptide as shown in SEQ ID NO: 28) (see Fig.9), optimum pH of about 7 and optimum temperature of 30-40 °C .
- the present invention provides an enantioselective epoxide hydrolase protein which is isolated from Novosphingobium aromaticivorans, and has the following characteristics:
- the rEEH hydrolase has a molecular weight of 43kDa (polypeptide as shown in SEQ ID NO: 30)(see Fig.9), optimum pH of 7.0-8.0 and optimum temperature of 30-40 ° C .
- the present invention provides an enantioselective epoxide hydrolase protein which is isolated from Rhodobacterium sp. HTCC2654, and has the following characteristics:
- the rEEH hydrolase has a molecular weight of 36kDa
- polypeptide as shown in SEQ ID NO: 32 (polypeptide as shown in SEQ ID NO: 32) (see Fig. 9), an optimum pH 7.0 to 8.0; and an optimum temperature of 30 to 40 ° C .
- a method of preparing epoxides with high enantiopure by using the enantioselective epoxide hydrolase protein having a high enantioselectivity on various epoxide substrates is also provided.
- 2-100 mM racemic styrene oxide can be reacted with purified enzymes such as EEHl, EEH2, EEH3, sEEH, nEEH, and rEEH, recombinant E.coli or wild type strain in each optimum condition(as confirmed by Gas Chromatography), and then the produced epoxide are used.
- purified enzymes such as EEHl, EEH2, EEH3, sEEH, nEEH, and rEEH, recombinant E.coli or wild type strain in each optimum condition(as confirmed by Gas Chromatography), and then the produced epoxide are used.
- the substrates of enantioselective epoxide hydrolase can not be limited particularly, and the examples are styrene oxide (SO), glycidyl phenyl ether (GPE), epichlorohydrin (ECH), epifluorohydrin (EF), 1,2-epoxybutane (EB) and 1,2-epoxyhexane ( EX).
- SO styrene oxide
- GPE glycidyl phenyl ether
- EH epichlorohydrin
- EF epifluorohydrin
- EB 1,2-epoxybutane
- EX 1,2-epoxyhexane
- the hydrolase has an amino acid sequence as shown in SEQ ID NO: 13, an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence as shown in SEQ ID NO: 17.
- the preparation method can be performed at pH 6.5 to 8.0 and temperature of 40 to 60 ° C .
- the hydrolase has an amino acid sequence as shown in SEQ ID NO: 28, an amino acid sequence as shown in SEQ ID NO: 30, or an amino acid sequence as shown in SEQ ID NO: 32.
- the preparation method can be performed at pH 7.0 to 8.0 and temperature of 30 to 40 0 C .
- epoxides used in the present invention are indicated in Fig. 1.
- racemic styrene oxide was purchased from Fluka Co.
- pure (R)-styrene oxide, pure (S)-styrene oxide, and all other racemic epoxides were purchased from Aldrich Co, respectively.
- the chiraldex gamma-cyclodextrin trifluoroacetyl(G-TA) capillary GC column was purchased from Astec Co.(Whippany, NJ), and other medium components were purchased Merck and Difco Co.
- Marine sediment, sponge and algae samples were collected from Hujin(depth, -20 m; 37° 51' N, 129° 45' E), Ulleungdo(depth, -758.7 m; 38° 00' N, 131° 27' E), Dokdo(depth, -620 m; 37° 14' N, 131° 45' E), Taejongda(depth, -20 m; 35° 14' N, 129° 45' E), Sihwa(Yellow sea, Korea), and Kagoshima bay(depth, 100-200 m; 31° 90' N, 130° 48' E).
- the sediment samples were collected using various method such as grab, core sampler, and scuba diving. Immediately after sampling, 0.3 g of chilled sediment was ground in a mortar, and incubated in the nutrition abundant culture media. Samples were collected under formal agreement with all legal parties.
- HTCC2594 was cultured in ZoBeIl 2216E broth(Oppenheimer & Zobell, 1952) medium consisting of 0.5% peptone, 0.1% yeast extract, and 75% seawater(pH 7.5) at 30 ° C for 1 day.
- Sphingopyxis alaskensis and Novosphingobium aromaticivorans were cultured in nutrient medium consisting of 0.5% peptone and 0.3% yeast extract at 30 ° C .
- Rhodobacterium sp. HTCC2654 was cultured in marine broth 2216(Difco) medium at 25 ° C .
- E. coli DH5 ⁇ and E. coli BL21-CodonPlus (DE3)-RIL cells(Stratagene, LaJolla, CA) were used for plasmid propagation and gene expression, respectively.
- the cells were cultured in Luria-Bertani(LB) broth medium containing appropriate antibiotics at 37 " C . ⁇ l-5> Identification of an EHase-producing strain
- an embodiment of the present invention isolated total 181 strains from marine environments.
- 31 stains were shown to hydrolyze SO substrate by spectrophotometric measurement(Table 1).
- JCS358 was shown to preferentially hydrolyze (R)-epoxide of SO(TaWe 1 and Fig. IB).
- ⁇ -pro ⁇ -proteobacteria
- b ⁇ -pro ⁇ -proteobacteria
- 0 GP Gram-positive
- d CFB Cytophaga-Flavobacteria-Bacteroides
- An embodiment of the present invention was performed by the sequence analysis of 16S rRNA gene on the genomic DNA sequence of the strain.
- the 16S rRNA gene was amplified from genomic DNA by PCR (Weisburg, et al., J. Bacterioaol, 173: 697-703, 1991) using the SEQ ID NO: 2(5'- AGAGTTTGATCATGGCTCAG-S', 27F) and SEQ ID NO: 3(5'- AAGGAGGTGATCCAGCCGCA-3 1 , 1518R).
- DNA sequencing was performed with the automated sequencer (ABI 3100) using a BigDye terminator kit(PE Applied Biosystems, Foster City, CA).
- JCS 350, JCS 358, JCS 360 and JCS 364) displayed ee value of high enantioselective hydrolyzing activity toward SO substrate.
- the kinetic preference of the EHase of the strains was mostly toward (R)-SO.
- EHase activity of the strains was measured by a spectrophotometric assay based on the epoxide extracted from the reaction mixture and spectrophotometric quantification of the non-extracted diol(Bhatnagar, et al., J. Biochem. Biophys. Methods., 50 :1-13, 2001).
- the isolated strains were shake-cultured in a flask containing 30 ml of ZoBeIl medium at 25 ° C . 24 h after incubation, the supernatant was removed by centrifugation at 4,300 x g for 20 min at 4 ° C .
- the whole cells were washed twice with 10 mM of sodium phosphate buffer(pH 6.8), and 4 mM of SO containing dimethyl formamide(DMF) was mixed with 0.04 g of whole cells which were resuspended in 10 mM of sodium phosphate buffer(pH 6.8), and the mixture was incubated at 30 ° C for 15 min. Then, 40 ul of the NaIO 4 stocked solution(stocked with 200 mM of NaIO 4 in DMF) was added, and immediately vortexed for 2 min. After centrifugation at 16,500 x g for 90 sec, the supernatant was quantified by spectrophotometric measurement at 290 ran.
- GC gas chromatography
- the temperatures of oven, injector and detector in GC analysis for racemic SO were 90 ° C, 220 ° C and 230 ° C, respectively.
- the hydrolysis toward other epoxide substrates depicted in Fig. IA was also analyzed with the method as described above.
- the hydrolyzing rate toward (R)-SO substrate was to be faster than that of (S)-SO substrate, and the enantiopurity of (S)-SO was increased from 0 to 99% after 16 h.
- the fact that it was taken 16 h for kinetic resolution of 2 mM of SO indicates that the endogeneous level of EHase produced by Erythrobacter spp. JCS 358 strain was not be enough to efficient kinetic resolution of racemic SO.
- Fig. 3 A to 3C were aligned amino acid sequence of purified EEHl protein with that of known protein, wherein the protein accession numbers were followed as:
- Ephxl (Rattus norvegivcu), P07687; EPHXl (Homo sapiens), AAH08291 ;
- Ephl Xanthophyllomyces dendrorhous
- AAF 18956 hyll (Aspergillus niger)
- CAB59813 Ephl (Xanthophyllomyces dendrorhous), AAF 18956; hyll (Aspergillus niger), CAB59813 and
- Fig. 4A and 4B were aligned amino acid sequence of purified EEH2 and EEH3 protein with that of known protein, wherein the protein accession numbers were followed as:
- Rattus norvegicus (Ephx2, Rat sEH), CAA46211;
- Solanum tuberosum (pEHSt and potato sEH), AAA81890; Glycine max (sEHGm and soybean sEH), CAA55293;
- ephA Bradyrhizobium japonicum
- eehl gene showed 35% of similarity to human microsomal EHase, and contained GGD 173 WGS motif, catalyst triad(Asp 173 , GIu 324 and His 351 ) and oxyaion hole HGXP (HGW 99 P)(Fig. 3A to 3C).
- eeh2 and eeh3 genes showed similarity to soluble EHase containing Sm-X-Nu-X-Sm-Sm motif(VHD 107 YGV for eeh2 and AHD 106 WGA for eeh3), catalytic triad(Asp 107 , GIu 250 and His 269 for eeh2; Asp 106 , GIu 251 and His 270 for eeh3) and oxyanion hole HGXP(HGY 42 P for eeh2 and HGF 38 P for eeh3) conserved in EHase(Arahira et al., 2000; Kaneko et al., 2002; Knehr et al., 1993; Stapleton et al., 1994 and Strausberg et al., 2002; Fig. 4A and 4B).
- EHase For phylogenetic analysis of EHase, the known EHase sequences received from SwissProt or EMBL protein database were compared with sequences of eehl, eeh2 and eeh3 gene. Phylogenetic distances were calculated with the CLUSTAL W program and phylogenetic trees were drawn with the Molecular Evolutionary
- Fig. 5 was drawn by phylogenetic analysis of EHase, wherein the protein accession numbers were followed as:
- Rhodotorula glutinis (AAF64649)
- Glycine max (CAA55293); Agrobactrium radiobacter sEEH (031243);
- Haloalkane dehalogenase (P22643).
- the underlined sequences in the forward and reverse primer indicate Nhe I and Xho I/Not I site, respectively.
- the reverse primers of eeh IRX, eeh2RX and eeh3RX were also designed as above Table 3.
- the amplified DNA fragment was restricted with restriction enzyme Nhe I and Xho I/Not I, and the fragment was ligated with Nhe I and Xho I/Not I-restricted plasmid pET- 24a (+) vector, and then the vector was transformed into E. coli DH5 ⁇ .
- the recombinant vector was introduced into BL21-CondonPlus(DE3)-RP(Novagen) for expression.
- the transformant was cultured at 37 "C, and was induced by the addition of 1 mM IPTG when the optical density(O.D) reached 0.4 to 0.6 at 600 nm. 3h after induction, the cells were harvested by centrifugation at 5,000 x g for 20 min, resuspended in a buffer[50 mM phosphate (pH 7.0), 0.5 M KCl and 10% glycerol], and then disrupted by sonication. Cell debris was removed by centrifugation at 15,000 x g for 30 min using a His-Bind Purification Kit(Novagen Co.).
- the soluble fraction was loaded in a Ni-nitrilotriacetic(Ni-NTA) column equilibrated with binding buffer[500 mM NaCl, 20 mM phosphate (pH 7.0), and 5 mM imidazole]. After washing with washing buffer[500 mM NaCl, 20 mM phosphate (pH 7.0), and 60 mM imidazole], the bound enzyme was eluted with elution buffer [500 mM NaCl, 20 mM phosphate (pH 7.0), and 1 M imidazole], and then dialyzed with 50 mM of phosphate buffer(pH 7.0).
- the purity of the protein was confirmed by SDS-PAGE under denaturing conditions as described by Laemmli(1970).
- the protein concentration was measured by the Bradford method using the Bio-Rad protein assay kit containing a standard protein BSA(Bradford, 1976).
- the vector was introduced into BL21-CondonPlus(DE3)-RP(Novagen), and then separated by SDS- PAGE.
- the optimum activities of sEEH, nEEH, and rEEH occurred at neutral pH and mesophhilic conditions(30 to 40 ° C ).
- Dotted line RSO incubated with rEEFB.
- rEEHl, rEEH2 and rEEH3 toward various epoxide substrates depicted in Fig. 1 was same to Table 4.
- a , b , and c Enzyme purified from Sphingopyxis alaskensis, Novosphingobium aromaticivorans, and Rhodobacterium sp. HTCC2654, respectively.
- EHases purified from Erythrobacter, Sphingopyxis, Novosphingobium, and Rhodobacterium strains can be applied to bioprocess for production of enantiopure epoxides in the pharmaceutical industry.
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BRPI0616814-0A BRPI0616814A2 (en) | 2005-10-07 | 2006-10-04 | Enantioselective epoxide hydrolase proteins and preparation method of styrene or glycidyl phenyl oxide Enantiopure ether or epoxide |
CN2006800461942A CN101522892B (en) | 2005-10-07 | 2006-10-04 | Enantioselective epoxide hydlrolase and method for preparing an enantiopure epoxide using the same |
US12/089,422 US8030048B2 (en) | 2005-10-07 | 2006-10-04 | Enantioselective epoxide hydlrolase and method for preparing and enantiopure epoxide using the same |
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ITRM20080508A1 (en) * | 2008-09-23 | 2010-03-24 | Fond Parco Tecnologico Padano | RECOMBINANT PROTEINS OF EPOXY HYDROLASIS (EH) DI PESCO. |
CN102242085A (en) * | 2011-04-12 | 2011-11-16 | 华东理工大学 | Epoxy hydrolase, gene thereof and application thereof |
WO2013030851A1 (en) * | 2011-08-26 | 2013-03-07 | Council Of Scientific & Industrail Research | A novel bacterial strain of achromobacter sp. mtcc 5605 and a highly enantioselective epoxide hydrolase isolated therefrom |
CN112730683A (en) * | 2020-12-25 | 2021-04-30 | 石家庄四药有限公司 | Method for detecting epichlorohydrin isomer |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ITRM20080508A1 (en) * | 2008-09-23 | 2010-03-24 | Fond Parco Tecnologico Padano | RECOMBINANT PROTEINS OF EPOXY HYDROLASIS (EH) DI PESCO. |
WO2010035154A1 (en) * | 2008-09-23 | 2010-04-01 | Fondazione Parco Tecnologico Padano | Recombinant proteins of epoxide hydrolase (eh) from peach |
CN102242085A (en) * | 2011-04-12 | 2011-11-16 | 华东理工大学 | Epoxy hydrolase, gene thereof and application thereof |
CN102242085B (en) * | 2011-04-12 | 2013-04-24 | 华东理工大学 | Epoxy hydrolase, gene thereof and application thereof |
WO2013030851A1 (en) * | 2011-08-26 | 2013-03-07 | Council Of Scientific & Industrail Research | A novel bacterial strain of achromobacter sp. mtcc 5605 and a highly enantioselective epoxide hydrolase isolated therefrom |
US9150840B2 (en) | 2011-08-26 | 2015-10-06 | Council Of Scientific & Industrial Research | Isolated bacterial strain of Achromobacter sp. MTCC 5605 and a highly enantioselective epoxide hydrolase isolated therefrom |
CN112730683A (en) * | 2020-12-25 | 2021-04-30 | 石家庄四药有限公司 | Method for detecting epichlorohydrin isomer |
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