KR101325622B1 - A method for preparing an enantiopure epoxide using sphingopyxis sp. microorganism having enantioselective epoxide hydrolase activity - Google Patents

Abstract

The present invention relates to marine microorganisms having enantioselective epoxide hydrolase activity against a racemic epoxide substrate and a method for preparing enantiopure epoxide having enantioselective epoxide hydrolase activity from the racemic epoxide substrate using the microorganisms.

Description

A method for preparing an enantiopure epoxide using Sphingopyxis sp. microorganism having enantioselective epoxide hydrolase activity}

The present invention is directed to selected marine microorganisms having enantioselective hydrolase activity against racemic epoxide substrates, and to methods for producing optical purity epoxides having high optical activity from racemic epoxide substrates using the same. It is about.

Many bioactive materials, including pharmaceuticals, contain several types of optical isomers, and only certain optical isomers show the correct activity, and other isomers often cause serious side effects. As such, there is a great deal of research on the synthesis of pure optically active materials as there is a difference in the biological activity of the optical isomers. Optically active chiral epoxides, which are representative synthetic intermediates that can be used for the synthesis of optically active substances, are highly reactive and can induce a variety of reactions and are widely used as intermediates for synthesizing optically active pharmaceuticals, pesticides and functional foods (Archelas and Furstoss, Annu Rev Microbiol 51: 491-525, 1997; Besse and Veschambre, Tetrahedron 50: 8885-8892, 1994; Grogan et al., FEMS Microbiol. Lett., 141: 239-243, 1996).

Optically active epoxides can be prepared using a variety of chiral chemical and biocatalysts, in particular epoxide hydrolyse (EHase; EC) for each optical isomer of a racemic epoxide substrate. Optoselective kinetic hydrolysis techniques that produce only a single optical isomer using the differential resolution of 3.3.2.3) are of interest. This method is a highly commercialized technique that can produce high value-added optically active epoxides from low-cost racemic substrates using low-cost biocatalysts. Epoxide hydrolase removes only one of the (R) or (S) -isomers from the racemic epoxide substrate by optically hydrolyzing with diol optically and leaving only the remaining isomers to produce optically pure epoxides. Enzymes (eg, FIG. 1). In addition, the optical selectivity of the epoxide hydrolase for the (R)-or (S) -isomer is determined depending on the type of microorganism and the substrate structure.

Epoxide hydrolyse (EHase; EC 3.3.2.3) is known as an ubiquitous enzyme isolated from bacteria, yeast, fungi, insects, plants and mammals (Weijers, et al., J. Mol. Catal. B Enzym., 6: 199-214, 1999 and Archelas & Furstoss, Curr. Opin. Chem. Biol., 5: 112-119, 2001). The biocatalyst of epoxide hydrolase does not require the recycling of cofactors, and can be used as a biocatalyst (whole-cell biocatalyst). It is becoming. In addition, optically active diols, which are reaction by-products, also have the added advantage of being very useful, high value-added synthetic intermediates. Therefore, it is meaningful to develop an epoxide hydrolase biocatalyst having various substrate specificities. However, since the number of optically selective epoxide hydrolase is very limited in the pharmaceutical industry, there is an urgent need for a new optically selective epoxide hydrolase capable of producing optically pure epoxide.

The present inventors selected strains showing high optical selective hydrolytic activity against epoxide substrate styrene oxide and its derivatives in tidal flats in Yeosu, Byeonsan, Gunsan and Taean, which have been contaminated with oil in the past. As a result of analyzing the optical selective hydrolytic activity of, it was confirmed that showed higher optical purity and yield in a shorter reaction time than previously reported wild strains. Therefore, the present invention was completed by confirming that the selected strain can be used as a new optical selective epoxide hydrolase.

SUMMARY OF THE INVENTION An object of the present invention is to select selected marine strains having optical selective hydrolase activity against racemic epoxide substrates, biocatalyst compositions for preparing optical purity epoxides comprising the strains, and preparation of optical purity epoxides using the strains. To provide a way.

More specifically, one object of the present invention is to provide a Sphingopyxis sp. BSNA05 strain (Accession No. KCTC 12262BP) having optically selective epoxide hydrolysis activity.

It is another object of the present invention to provide a method for preparing an optical purity epoxide, comprising reacting the strain with a racemic epoxide substrate.

It is another object of the present invention to provide a biocatalyst composition for preparing an optical purity epoxide from a racemic epoxide substrate comprising the strain.

As one aspect for achieving the above object, the present invention relates to the sphinxis BSNA05 strain (Accession No. KCTC 12262BP) having optically selective epoxide hydrolysis activity.

In another aspect, the present invention provides a method for preparing an optically pure epoxide, comprising reacting a Sphinxis BSNA05 strain (Accession No. KCTC 12262BP) having an optical selective epoxide hydrolysis activity with a racemic epoxide substrate. It is about a method.

In another aspect, the present invention provides a biocatalyst composition for preparing an optical purity epoxide from a racemic epoxide substrate comprising a Sphinxis BSNA05 strain (Accession No. KCTC 12262BP) having optically selective epoxide hydrolysis activity. It is about.

Hereinafter, the present invention will be described in more detail.

As used herein, the term "epoxide" refers to an organic compound having a tricyclic ring molecule composed of one oxygen atom and two carbon atoms, and is generally used in the art, and is also referred to as "epoxide". Preferably, the epoxide in the present invention includes styrene oxide and its derivatives.

As used herein, the term "racemic epoxide" means a mixture of the (R)-and (S) -isomers of the epoxide.

As used herein, the term “optical purity epoxide” refers to an optically pure epoxide that remains unreacted in the course of hydrolysis of the racemic epoxide substrate by an epoxide hydrolase, (S) -Optical purity epoxide or (R)-optical purity epoxide. The term can be expressed by substituting "optically active epoxide" or "chiral epoxide".

As used herein, the term "enantioselective" means removing only one of the (R)-or (S) -isomers of the racemic epoxide substrate or leaving only one.

As used herein, the term "optical selective epoxide hydrolase" means that only one optical isomer of either the (R)-or (S) -isomer is removed from the racemic epoxide substrate by optically hydrolyzing with diol and the remaining isomers. It refers to enzymes that can be used to prepare optical purity epoxides.

The present invention is characterized by providing a BSNA05 strain of Sphinxis genus (Accession No. KCTC 12262BP) having optically selective epoxide hydrolysis activity.

In the present invention, since the strain has optical selective epoxide hydrolase activity, it has been found that the whole cell itself can be used as a microbial biocatalyst.

More specifically, the present inventors, in order to select a new epoxide hydrolytic activity strain having optical selective hydrolase activity against a variety of epoxide substrates, Yeosu, Byeonsan, Gunsan and Taean, etc. Tidal flat samples were isolated using polycyclic aromatic hydrocarbon (PAH) and thickening culture method. In addition, excellent strains showing epoxide hydrolysis activity in racemic styrene oxide were selected through gas chromatography (GC) analysis.

As a result of analyzing the strain according to the present invention through the 16S rDNA sequence, it could be identified as a strain of new genetic properties having the 16S rDNA sequence of SEQ ID NO: 1, the strain was Sphingopyxis sp. It was named BSNA05 and was deposited on August 14, 2012 to KCTC (Korean Colletion for Type Cultures, 125, Gwahakro, Yuseong-gu, Daejeon, Korea).

The strains selected in the present invention can be used to prepare optical purity epoxides by reacting with epoxide substrates.

In the present invention, the epoxide substrate may be used as the substrate without limitation as long as it is a racemic epoxide present in (R) and (S) forms, and preferably styrene oxide (SO) and its derivatives may be used. have. More preferably, it is composed of styrene oxide, 2-chlorostyrene oxide (2CSO), 3-chlorostyrene oxide (3CSO) and 4-chlorostyrene oxide (4CSO). Epoxides selected from the group can be used.

In a preferred embodiment, (S) type optical purity epoxides can be prepared by reacting sphinxis BSNA05 strain (Accession No. KCTC 12262BP) with optical selective epoxide hydrolysis activity with racemic styrene oxide or derivatives thereof.

Since the strains selected in the present invention exhibited higher optical purity and yield even in a shorter reaction time than previously reported wild strains, they can be usefully used to prepare optical purity epoxides.

In a specific embodiment of the present invention, the remaining epoxide substrate in the aqueous solution of the aqueous phase is extracted with an organic solvent and then subjected to gas chromatography (GC) equipped with a chiral column to obtain optical purity and chirality of the product. The epoxide hydrolase activity of the strain was measured by the method of analysis.

As a result, the strain of the present invention exhibited epoxide hydrolase activity against racemic styrene oxide and its derivative substrate (see Tables 1 to 4 and 3 to 8 of Examples). The strain of the present invention showed optical purity of 100% by optically decomposing (R) -form on various epoxide substrates, and the optical purity, yield and E value for each substrate were also reported in previously reported wild strains. In particular, the yield and E value for 2CSO were very good. In particular, it has been shown that the activity against 2CSO is much better than the strains or enzymes reported so far, and in particular, there was no report on the strain having better activity against 2CSO than other derivatives. It is expected that the strains of the present invention will be enzymes that fully follow existing degradation mechanisms. In addition, the reaction time was much shorter than the conventional reported strains, and showed superior optical selective epoxide hydrolysis activity on various substrates. Therefore, the strains of the present invention are expected to be useful because they exhibit optical selective hydrolytic activity against various epoxide substrates.

In the present invention, the reaction temperature or the reaction pH is not particularly limited because it changes depending on the activity temperature or the active pH of the selected strain, but the preferred reaction temperature is suitable 20 to 40 ℃ in the nature of the general biocatalyst, pH at the reaction The range of pH 6-9 is suitable. In addition, the ratio of the biocatalyst and the substrate of the present invention during the hydrolysis reaction may be 1:10 to 10: 1, preferably 1: 6 to 10: 1.2.

The reaction process can be carried out by appropriately selecting methods known in the art. More specifically, the optical splitting reaction may be performed on an aqueous solution or on an organic solvent, and a two-phase system of an aqueous solution and an organic solvent may be used. In the case of using a two-phase system of an aqueous solution and an organic solvent, since the triangular ring structure of the epoxide is easily decomposed by water, an organic solvent is used for the substrate epoxide, and the biocatalyst is in contact with the organic solvent. Since it tends to be inactivated, the separation efficiency can be improved by using an aqueous solution for the strain. In addition, while the diol produced after the reaction is mostly water-soluble, epoxide is easy to dissolve in the organic solvent can be used two-phase system in the separation and purification process. In this case, the optically active diol, which is a product, may be present in an aqueous solution layer and separated from the biocatalyst by centrifugal separation. can do.

The recovered strain biocatalyst can be synthesized by repeating the chiral epoxide or chiral 1,2-diol by adding new racemic epoxide to the reactor and adding water slowly to the reactor without repeated catalyst regeneration. .

For the commercialization of optically active epoxides, which are widely used in the synthesis of optically active pharmaceuticals, it is very important to secure new microorganisms having epoxide hydrolase with excellent optical selective hydrolytic catalytic activity on various epoxide substrates. In the present invention, as a result of investigating these new microorganisms by GC analysis, using a whole cell as a biocatalyst, a strain showing optical selective hydrolytic activity against various epoxide substrates was obtained. Among the obtained strains, the BSNA05 strain of Sphinxis (Accession No. KCTC 12262BP) showed very good optical selective epoxide hydrolysis activity against all derivative substrates including racemic styrene oxide. The results shown in the present invention can be a good model for the investigation of optical selective epoxide hydrolase activity, and it is expected to be a high value-added organic intermediate material which is widely used in the synthesis of optically active pharmaceuticals that can increase the industrial applicability. do.

Since the selected strain of the present invention has optical selective epoxide hydrolysis activity, it can be usefully used for biosynthesis of optical purity epoxide with high efficiency. In addition, it can be a good model for the investigation of optical selective epoxide hydrolase activity in the future and can be used as a high value-added organic intermediate material widely used in the synthesis of optically active pharmaceuticals that can increase the industrial applicability.

1 is a schematic diagram showing that styrene oxide (SO) is hydrolyzed to (S) -styrene oxide and (R) -1-phenylethane-1,2-diol by the hydrolytic activity of epoxide hydrolase.
Figure 2 shows the chemical structure of the epoxide substrate used in the present invention. A, styrene oxide (SO); B, 2-chlorostyrene oxide (2CSO); C, 3-chlorostyrene oxide (3CSO); D, 4-chlorostyrene oxide (4CSO).
Figure 3 shows the optical resolution (kinetic resolution) of 0.2g 8-NA-5 strain of wet cell weight acts on 4mM of styrene oxide (SO).
Figure 4 shows a phylogenetic tree based on the 16S rRNA sequence of the epoxide hydrolase active strain 8-NA-5. The parenthesis shows the registration number of the GenBank database.
Figure 5 shows the results confirmed by the GC analysis of the activity of strain 8-NA-5 hydrolyzes 4 mM 2CSO, 3CSO, 4CSO.
FIG. 6 shows the optical resolution of strain 8-NA-5 (0.2 g, wet cell weight) on 4 mM 2CSO.
7 shows the optical resolution of strain 8-NA-5 (0.2 g, wet cell weight) on 4 mM 3CSO.
FIG. 8 shows the optical resolution of strain 8-NA-5 (0.2 g, wet cell weight) on 4 mM 4CSO.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Preparation of experimental materials

The basal medium used for strain culture was a product of Difco, USA. Among the various epoxide substrates used in this study, 2-chlorostyrene oxide (2CSO) and 3-chlorostyrene oxide (3CSO) were obtained from Rstec (RStec, Korea), and the remaining styrene oxide (SO) and 4-chlorostyrene oxide. (4CSO) was purchased from Aldrich, USA. All products used in this study were purchased from Aldrich as HPLC Grade. Figure 2 shows the chemical structure of the epoxide substrate used in the present invention.

Example  2. Thickening culture  through Polycyclic Aromatic Hydrocarbons  Consortium ( PAH consortium ) And Single strain  detach

The medium used in this study was MSM (Mineral salt medium, 1 L of DW), NaNO ₃ 4g, KH ₂ PO ₄ 1.5g, FeCl ₃ · 6H ₂ O 0.005g, MgSO ₄ 0.2g, CaCl · 2H ₂ O 0.01g, 0.5g of Na ₂ HPO ₄ was added and pH was used 7.2) using a pH meter. Marine broth medium (DIFCO) was used as a general nutrient medium, and selected strains were separated by polycyclic aromatic hydrocarbons and thickening cultures from the past tidal flat samples such as Yeosu, Byeonsan, Gunsan and Taean, which were previously contaminated with oil. .

0.5 g of tidal-flat samples were added together with polyaromatic hydrocarbons (naphthalene, phenanthrene, pyrene and benzopyrene) in 5 mL MSM medium and incubated at 25 ° C. for 50 days. After 2 weeks of culture in concentrated culture, 1 mL of the supernatant was added to fresh MSM medium, followed by further incubation for 50 days as described above. By this process, a consortium capable of degrading various polycyclic aromatic hydrocarbons was obtained, and pure monopolyaromatic hydrocarbon decomposition strains were isolated from the consortium. Finally, an excellent strain showing epoxide hydrolysis activity in racemic styrene oxide was selected through gas chromatography (GC) analysis.

Example  3. Culture conditions of selected good strains

The superior strains selected in this study were inoculated in marine broth medium and incubated for 3-4 days at agitation speed of 180 rpm and temperature of 25 ℃ using shaking incubator. The cultured cells were washed twice with 50 mM Tris-HCl (pH 8.0) after centrifugation and used as biocatalysts.

Example  4. 16s of Selected Strains rRNA  Sequencing and Taxonomic Positioning

Genomic DNA of excellent strains was isolated using Wizard Genomic DNA Purification Kit (Promega, Madison, Wis.). 16S rRNA is a 16S rRNA primer, 27F (5'-AGA GTT TGA TCM TGG CTC AG-3 '; Escherichia coli nucleotides 8-27) and 1518R (5'-AAG GAG GTG ATC CAN CCR CA-3'; Escherichia coli nucleotides 1541-1522) were used to amplify from genomic DNA by PCR. The reaction mixture for PCR was 50 mM Tris-HCl (pH 9.0), 0.1% TritonX-100, 1.5 mM MgCl 2, 0.2 mM dNTPs, 0.2 μM primer, 2.5 U Taq DNA polymerase (Promega Co., Madison, WI), 2.5 50 μl of 1 × PCR complete solution containing ˜250 ng template was prepared. DNA amplification was performed using a model 2400 thermal cycler (PE, Applied Biosystem) and the reaction conditions were as follows. After the initial denaturation reaction at 94 ° C. for 5 minutes, the reaction was repeated 35 times under conditions of denaturation (94 ° C., 1 minute), annealing (60 ° C., 1 minute), and elongation (72 ° C., 2 minutes), followed by further 7 minutes at 72 ° C. Reacted. PCR products were confirmed that the DNA was amplified by electrophoresis (0.8% agarose). PCR products were isolated using a separation kit (Wizard PCR Preps DNA Purification System, Promega) and then ligation to pGEM-T Easy Vector (Promega, Madison, WI) and transformed into E. coli JM109. 16S rDNA was determined by using an automatic base sequencer (ABI Prism 377 DNA Sequencer, Perkin Elmer). Analysis of the 16S rDNA base sequence was carried out using the SIMILARITY-RANK of the Ribosomal Database Project (RDP) and the Basic Local Alignment Search Tool (BLAST) by National Center Biotechnology Information (NCBI) (Altschul et al., J. Mol Biol. 215, 403-410). , 1990). The Phylogenetic Interference Package (PHYLIP), version 3.57c (Felsenstein, 1993) was used to analyze the sequence data, and the DNADIST program was used to determine the sequence similarity, and the FITCH program was used to generate the schematic.

Example  5. Derived Strain Cells Epoxide  Various using hydrolysis activity Epoxide  Temperamental Optical selective  Hydrolysis reaction

Hydrolysis of the various epoxide racemic substrates was carried out by suspending the selected strain whole cells (0.2 g / ml) and 4 mM racemic epoxide substrate in 50 mM Tris-HCl (pH 8.0), followed by shaking incubator (25 The reaction proceeded while stirring at 200 ℃). For the GC analysis of the epoxide hydrolysis reaction, the epoxides present in the samples obtained at regular intervals were extracted with 2 ml of hexane, and the organic solvent layer was analyzed by chiral GC to obtain optical purity. excess, ee ), conversion, yield, and E value expressing the degree of activity of optical selectivity were determined.

Optical purity (ee) = [ee (%) = {(S) -epoxide-(R) -epoxide} / {(S) -epoxide + (R) -epoxide} X 100

Conversion (c) = [1-{residual (S) -epoxide + residual (R) -epoxide} / {initial (S) -epoxide + residual (R) -epoxide}]

Yield (%) = [{residual (S) -epoxide} / {initial racemic (S, R) -epoxide}] X 100 (maximum theory 50%)

Optical selectivity (E) = In {(1-c) (1-ees)} / In {(1-c) (1 + ees)}

Example  6. Gas Chromatography GC ) analysis

In order to measure the epoxide decomposed by the epoxide hydrolysis activity, the reaction solution was taken, the remaining epoxide was extracted with hexane, and the organic solvent layer was analyzed by GC. FID was used as a detector and silica cyclodextrin beta-DEX 120 (30 m length, 0.25 mm ID, and 0.25 μm film thickness; Supelco, USA) was used as an analytical column. Helium was used as the transfer gas, and the temperature of the injector and detector was 180 ° C and 180 ° C, respectively, and the column temperature was determined by referring to Supelco's guide for each substrate [SO, ( R )- SO, t _r = 10.7 min; ( S ) -SO, t _r = 11.2 min (oven temperature 105 ° C.); 2CSO, ( R ) -2CSO, t _r = 19.6 min; ( S ) -2CSO, t _r = 40 min (oven temperature 105 ° C.); 3CSO, ( R ) -3CSO, t _r = 34.9 min; ( S ) -3CSO, t _r = 35.6 min (oven temperature 100 ° C.); And 4CSO, ( R ) -4CSO, t _r = 24.6 min; ( S ) -4CSO, t _r = 25.4 min (oven temperature 110 ° C.)].

Experiment result

One. GC Styrene Through Analysis Oxide  About Optical selective Epoxide  Hydrolysis Activity Measurement

Whole cells (0.2 g / ml) and 17 mM racemic styrene oxide in whole isolated cells after polycultured with polycyclic aromatic hydrocarbons, which were contaminated with oil in the past, and polyculture with thickened aromatics. ) Was suspended in 50 mM Tris-HCl (pH 8.0), followed by an optical selective hydrolysis reaction with stirring in a shake incubator (30 ° C., 200 rpm). Chiral epoxide was extracted with hexane, and the organic solvent layer was analyzed by chiral GC column to determine optical purity (enantiomeric excess, ee ). As a result, 8-NA-5 strain having the best optical selective epoxide hydrolysis activity was finally selected. Table 1 shows the epoxide hydrolysis activity of 8-NA-5 strain based on racemic styrene oxide (Table 1).

Sampling area Strain name Phylogenically closest strain 16S rRNA
Homology (%) ee ^a (%) / abs.
Conf. ^b / (R): (S) Hydrolysis rate
(x10 ^-2 ) mg / h SO (R) (S) Oil Pollution Area Sediment 8-NA-5 ^c Sphingopyxis flavimaris 99.53 10.6 / S / (3.0) 1.48 0.50 a, optical purity: (SR) / (S + R) X 100
b, absolute form representing remaining epoxide
c, whole cell (wet. 0.2 g) and reaction time 16 hours

To further investigate the properties of 8-NA-5, which showed optical selective epoxide hydrolysis activity, the optical purity of racemic styrene oxide was investigated by the amount of whole cells and reaction time. The amount of whole cells of 8-NA-5 was 0.1 g and 0.2 g, and the concentration of racemic styrene oxide was 4 mM, and the reaction time was analyzed until the time when the optical purity reached 100%. As a result, when the substrate concentration was 4 mM, the optical purity reached 100% at 7 hours in 0.2 g of whole cells, and 86% at 13 g in 0.1 g of whole cells. (Figure 3 and Table 2). As a result of comprehensive analysis of the optically selective epoxide hydrolysis activity of racemic styrene oxide of 8-NA-5, 8-NA-5 optically decomposes (R) -styrene oxide and has 100% optical purity. Seems. In addition, high optical purity and yield are shown to be excellent in optical selective epoxide hydrolysis activity, which is inferior to previously reported wild strains.

Optically Selective Epoxide Hydrolysis Activity of 8-NA-5 Strains Substrate concentration Time (h) ee (%) c ^a E ^b Yield (%) ^c Abs.
Conf. ^d 4 mM SO ^e 13 86.46 0.8039 3.9 21.8 S 4mM SO ^f 7 99.99 0.7606 12.2 20.6 S a, conversion ( c ) [ c = {1- (ERs + ESs / (ERso + ESso)}], the initial (R) and (S) epoxides are denoted by Eso and the remaining (R) and (S) epoxides Side is represented by Es
b, the optical selectivity (E) is derived from the conversion (c) and the optical purity (ees) of the isomer of the remaining substrate [ E = In {(1- c ) (1-ees)} / In {(1- c ) (1 + ees)}]
c, yield of remaining epoxide
d, an absolute form representing the remaining epoxide
e, amount of whole cells used (wet. 0.2 g)
f, amount of whole cells used (wet. 0.1 g)

2. 16s of Selected Strains rRNA  Sequencing and Taxonomic Positioning

The nucleotide sequence of 16S rRNA clone was analyzed for strain 8-NA-5 showing epoxide hydrolysis activity in racemic styrene oxide. 16S rRNA of 8-NA-5 strain is shown in SEQ ID NO: 1. Figure 4 Phylogenetic figure 8-NA-5 was created based on the 1293 base pairs of the 16S rRNA, system analysis 8-NA-5 shown in has to have high homology with 99.53% Sphingopyxis flavimaris (1444bp, NR025814 ) appear. Based on these results, strain 8-NA-5 was isolated from Sphingopyxis sp. It was named BSNA05 and was deposited on August 14, 2012 to KCTC (Korean Colletion for Type Cultures, 125, Gwahakro, Yuseong-gu, Daejeon, Korea).

3. Styrene of Selected Strains Oxide  For derivative substrates Optical selective Epoxide  Hydrolysis activity

The 8-NA-5 strain was analyzed using GC to confirm that it has optical selective epoxide hydrolysis activity against various epoxide substrates in addition to styrene oxide. The various epoxide substrates used in the experiments are all racemic structures, including derivatives of styrene oxide, including styrene oxide (SO), 2-chlorostyrene oxide (2CSO), 3-chlorostyrene oxide (3CSO), and 4-chloro Styrene oxide (4CSO) (FIG. 2). The GC assay was performed in the same manner as above, and the reaction time of whole cells and substrate was screened according to a single condition (preliminary investigation).

As a result, the response of each substrate to each strain is shown by GC results (FIG. 5) and degradation rate (Table 3). Looking at the chromatogram of GC of Figure 5 The optical purity of 8-NA-5 strain against 2-chlorostyrene oxide (2CSO) was 100% and showed optical selectivity for (S) -2CSO. In addition, it showed optically selective activity against 3-chlorostyrene oxide (3CSO) and 4-chlorostyrene oxide (4CSO) substrates, and showed optical selectivity to (S) -3CSO and (S) -4CSO, respectively. As a result, it was found that 8-NA-5 strain showed excellent optical selectivity to all derivatives of styrene oxide (2CSO, 3CSO, 4CSO) including styrene oxide.

Strain Hydrolysis rate
(x10 ^-2 ) mg / h SO 2CSO 3CSO 4CSO ( R ) ( S ) ( R ) ( S ) ( R ) ( S ) ( R ) ( S ) 8-NA-5
( Sphingopyxis sp. BSNA05;
KCTC 12262BP) 1.48 0.50 1.76 0.51 0.87 0.08 0.97 0.34

4. Styrene of Selected Strains Oxide  For derivative substrates Optical selective Epoxide  Hydrolytic activity

Next, under the same conditions as the above GC analysis, the reaction time of all cells of 8-NA-5 strain and derivatives of styrene oxide (2CSO, 3CSO, 4CSO) substrate were investigated. As a result, the response of each substrate to each strain shows the GC results in FIGS. 6 to 8.

As a result, 8-NA-5 strain showed optical selectivity for 2-chlorostyrene oxide (2CSO) substrate (S) -2CSO was selected and the optical purity was 100% at 100 minutes reaction time (Fig. 6) . In general, the papers reported so far have been known that 2CSO is difficult to have high optical selectivity, it can be seen that the optical selectivity of 2CSO of 8-NA-5 strain is much higher activity than the previously reported strain.

The results for the 3-chlorostyrene oxide (3CSO) substrate showed photoselective activity as well as prior irradiation, and the optical selectivity for (S) -3CSO was confirmed. The time taken for the optical purity to be 100% was 120 minutes, which also showed excellent optical selectivity (FIG. 7).

The results for the 4-chlorostyrene oxide (4CSO) substrate showed photoselective activity as well as prior irradiation, and the optical selectivity for (S) -4CSO was confirmed. The time taken for the optical purity to be 100% was 55 minutes, which also showed excellent optical selectivity (FIG. 8).

5. 8- NA - 5 strains  Styrene Oxide  For derivative substrates Optical selective Epoxide  Hydrolysis activity clearance

As described above, the 8-NA-5 strain was very excellent in the optical selective epoxide hydrolysis activity for all derivatives of styrene oxide (2CSO, 3CSO, 4CSO) including styrene oxide. As a result of analysis, 8-NA-5, including the same concentration of styrene oxide, performs a sufficient catalysis to achieve 100% optical purity for the styrene oxide derivative in a short time, thereby clearly confirming the optical selective epoxide hydrolysis activity. (Table 4). The time for optical purity to reach 100% was the difference in time according to the difference in substrate. In other words, at 4 mM concentration, 8-NA-5 of the same whole cell volume (0.2 g), SO was 420 minutes, 2CSO was 100 minutes, 3CSO was 120 minutes, and 4CSO was 55 minutes. Reached .

In conclusion, the 8-NA-5 strain of the present invention exhibits optical purity of 100% by optically decomposing (R) -form even in various styrene oxide derivatives including styrene oxide. In addition, the optical purity, yield, and E value of the four substrates are also inferior to previously reported wild strains. In particular, the yield and E value of 2CSO are excellent. Therefore, 8-NA-5 was shown to have a much higher activity against 2CSO than the strains or enzymes reported so far, and in particular, there was no report on the strain having better activity against 2CSO than other derivatives. 8-NA-5 is expected to be an enzyme completely following existing degradation mechanisms. In addition, the reaction time was much shorter than the conventional reported strains, and showed superior optical selective epoxide hydrolysis activity on various substrates. Thus, 8-NA-5 strains are expected to be useful because they exhibit optically selective hydrolytic activity against various epoxide substrates.

Optically Selective Epoxide Hydrolysis Activity of 8-NA-5 Strains Substrate concentration Time (minutes) ee (%) c ^a E ^b Yield (%) ^c Abs.
Conf. ^d 4 mM SO ^e 420 99.99 0.7606 12.2 20.6 S 4mM 2CSO ^e 100 99.99 0.6070 42.1 39.3 S 4mM 3CSO ^e 120 99.99 0.7131 18.8 28.7 S 4mM 4CSO ^e 55 99.99 0.7329 16.8 26.8 S a, conversion ( c ) [ c = {1- (ERs + ESs / (ERso + ESso)}], the initial (R) and (S) epoxides are denoted by Eso and the remaining (R) and (S) epoxides Side is represented by Es
b, the optical selectivity (E) is derived from the conversion (c) and the optical purity (ees) of the isomer of the remaining substrate [ E = In {(1- c ) (1-ees)} / In {(1- c ) (1 + ees)}]
c, yield of remaining epoxide
d, an absolute form representing the remaining epoxide
e, amount of whole cells used (wet. 0.2 g)

Korea Biotechnology Research Institute KCTC12262BP 20120814

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Claims (10)

A method of preparing an optically pure epoxide, comprising reacting a Sphingopyxis sp. BSNA05 strain (Accession No. KCTC 12262BP) with optical selective epoxide hydrolysis activity with a racemic epoxide substrate. The method of claim 1, wherein the epoxide is styrene oxide (SO) or a derivative thereof. The method of claim 2, wherein the styrene oxide derivative is 2-chlorostyrene oxide (2-chlorostyrene oxide, 2CSO), 3-chlorostyrene oxide (3-chlorostyrene oxide, 3CSO) or 4-chlorostyrene oxide (4-chlorostyrene oxide, 4CSO). The method of claim 1, wherein the optical purity epoxide is (S) type optical purity epoxide or (R) type optical purity epoxide. The method according to claim 1, wherein (S) type optical purity epoxide is prepared by reacting sphinxis BSNA05 strain (Accession No. KCTC 12262BP) having optical selective epoxide hydrolysis activity with racemic styrene oxide or a derivative thereof. Way. The method of claim 1, further comprising separating and purifying the optical purity epoxide using gas chromatography. A biocatalyst composition for preparing optical purity epoxides from racemic epoxide substrates comprising a Sphinxis genus BSNA05 strain having accessoselective epoxide hydrolysis activity (Accession No. KCTC 12262BP). 8. The composition of claim 7, wherein the epoxide is styrene oxide or a derivative thereof. The composition of claim 8, wherein the styrene oxide derivative is 2-chlorostyrene oxide, 3-chlorostyrene oxide or 4-chlorostyrene oxide. BSNA05 strain of Sphinxis genus having optical selective epoxide hydrolysis activity (Accession No. KCTC 12262BP).

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