KR101325623B1 - Marine microorganisms having epoxide hydrolase activity and use thereof - Google Patents

Abstract

The present invention relates to sorted marine microorganisms with hydrolytic activities on racemic epoxide substrates and a method for hydrolyzing the racemic epoxide substrates using the same. According to the present invention, chiral epoxide or chiral diol generated by hydrolysis of the racemic epoxide is used as a high added-value synthetic intermediate in various industries such as pharmaceutical products, agrochemicals, and foods.

Description

Marine microorganisms having epoxide hydrolase activity and use

The present invention relates to selected marine microorganisms having hydrolase activity against racemic epoxide substrates, and methods of hydrolyzing racemic epoxide substrates using the same. Chiral epoxides or chiral diols produced as a result of hydrolysis of racemic epoxides according to the present invention can be usefully used in various industries such as pharmaceuticals, pesticides and foods as high value synthetic intermediates.

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 and chiral diols, 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. Widely used as synthetic intermediates (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).

Epoxides are highly reactive because they contain a thermodynamically unstable triangular ring structure and polar oxygen atoms. In nature, racemic epoxides are a mixture of (R) -isomers and (S) -isomers. Exist). In addition, racemic epoxides are hydrolyzed by various kinds of chiral chemical catalysts and biocatalysts acting as substrates, and chiral epoxides and chiral diols may be generated through ring-opening reactions.

An exemplary catalyst used to hydrolyze racemic epoxides is epoxide hydrolyse (EHase). Epoxide hydrolase is a ubiquitous enzyme isolated from bacteria, yeast, fungi, insects, plants, and mammals. It can use the enzyme itself as a biocatalyst (whole-cell biocatalyst) and has a stable structure, which is highly commercially available. It is evaluated as a biocatalyst. In particular, optical selective kinetic hydrolysis techniques that produce only a single optical isomer from racemic epoxide substrates using the selective resolution differences of epoxide hydrolase, employ high cost value from low cost racemic substrates using low cost biocatalysts. Optically active epoxides can be prepared, and thus, they have been attracting attention as a technology having high commercial potential. 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 epoxide hydrolases in the pharmaceutical industry is very limited, there is an urgent need for a new epoxide hydrolase that can hydrolyze racemic epoxides to produce intermediates useful for the industry.

The present inventors selected three strains showing high epoxide hydrolysis activity against styrene oxide and its derivatives, which are epoxide substrates in the tidal flats in Yeosu, Byeonsan, Gunsan and Taean, which were previously contaminated with oil. As a result of analyzing the epoxide hydrolysis activity of the selected strains, it was confirmed that the epoxide degradation activity was excellent in a short reaction time than the previously reported wild strains. Therefore, the present invention was completed by confirming that the selected strains could be used as a new epoxide hydrolase.

An object of the present invention is a selected marine strain having hydrolytic activity against racemic epoxide substrate, a biocatalyst composition for preparing chiral epoxides or diols comprising the strains, and a method for producing chiral epoxides or diols using the strains To provide.

More specifically, one object of the present invention in Lactococcus also having optical selective epoxide hydrolysis activity (Rhodococcus sp.) YSMI04 strain (accession No. KCTC 12263BP), Rhodococcus genus (Rhodococcus sp.) YSNA32 strain (accession number KCTC 12260BP), and Roseobacter genus sp.) TSBP12 strain (Accession No. KCTC 12261BP).

Another object of the present invention is to provide a method for preparing a chiral epoxide or diol, comprising reacting the strain with a racemic epoxide substrate.

It is another object of the present invention to provide a biocatalyst composition for preparing chiral epoxides or diols from racemic epoxide substrates comprising said strains.

As one embodiment for achieving the above object, the present invention relates to a strain of Rhodococcus YSMI04 (Accession No. KCTC 12263BP) having optically selective epoxide hydrolysis activity.

As another aspect, the present invention relates to a strain of Rhodococcus YSNA32 (Accession No. KCTC 12260BP) having optically selective epoxide hydrolysis activity.

As another aspect, the present invention relates to a genus TSBP12 strain (Accession No. KCTC 12261BP) having optically selective epoxide hydrolysis activity.

As another embodiment, the present invention, YSMI04 strain (Accession No. KCTC 12263BP), Rhodococcus YSNA32 strain (Accession No. KCTC 12260BP), or Rose B. genus TSBP12 strain (Accession No.), having epoxide hydrolysis activity KCTC 12261BP) is reacted with a racemic epoxide substrate, to a method for preparing a chiral epoxide or diol.

As another embodiment, the present invention, YSMI04 strain (Accession No. KCTC 12263BP), Rhodococcus YSNA32 strain (Accession No. KCTC 12260BP), or Rose B. genus TSBP12 strain (Accession No.), having epoxide hydrolysis activity KCTC 12261BP), to a biocatalyst composition for preparing chiral epoxides or diols from racemic epoxide substrates.

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 "epoxide hydrolytic activity" refers to the activity of producing chiral epoxides or diols through hydrolysis reactions from racemic epoxide substrates. In the present invention, the epoxide hydrolysis activity refers to an activity of hydrolyzing both (R)-and (S) -isomers of racemic epoxide to generate diol, and (R)-or (from racemic epoxide substrate) Only one optical isomer of any one of the S) -isomers is optically selectively hydrolyzed to diol to remove, leaving only the remaining isomers to include both chiral epoxide and chiral diol.

As used herein, the term “chiral epoxide” or “optically active 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) chiral epoxide or (R) 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 "diol" refers to a reaction by-product generated through a ring-opening reaction of an epoxide in the course of hydrolyzing a racemic epoxide substrate by an epoxide hydrolase. Preferably, the diol may be a chiral diol, more preferably a chiral 1,2-diol compound.

The present invention provides YSMI04 strain (Accession No. KCTC 12263BP), Rhodococcus YSNA32 strain (Accession No. KCTC 12260BP), and Roseobacter genus TSBP12 strain (Accession No. KCTC 12261BP), having epoxide hydrolysis activity. It features.

Preferably, the three strains provided in the present invention may have optically selective epoxide hydrolysis activity, the optical selectivity is determined according to the type and substrate structure of the strain.

In the present invention, since the three strains listed above have 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 a hydrolase activity against a variety of epoxide substrates, tidal flats such as Yeosu, Byeonsan, Gunsan and Taean, which were previously contaminated with oil Samples were isolated using polycyclic aromatic hydrocarbons (PAH) and thickening. Finally, three excellent strains showing epoxide hydrolysis activity on 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 rRNA sequence, Rhodococcus YSMI04 strain (Accession No. KCTC 12263BP) is SEQ ID NO: 1, Rhodococcus YSNA32 strain (Accession No. KCTC 12260BP) is SEQ ID NO: 2, and Rose Obacter genus TSBP12 strain (Accession No. KCTC 12261BP) could be identified as a strain of new genetic properties having the 16S rDNA sequence of SEQ ID NO: 3, three of these strains as of August 14, 2012 Korean Colletion for Type Cultures, 125, Gwahak-ro, Yuseong-gu, Daejeon, Korea.

Three strains selected in the present invention can be used to prepare chiral epoxides or diols by reacting with epoxide substrates. Preferably, the diol may be a chiral diol, more preferably a chiral 1,2-diol compound.

In the present invention, the epoxide substrate may be used without limitation as long as it is a racemic epoxide having a (R) type and a (S) type, and preferably styrene oxide (SO) and its derivatives may be used, and more preferably. Preferably, in the group consisting of styrene oxide, 2-chlorostyrene oxide (2CSO), 3-chlorostyrene oxide (3CSO) and 4-chlorostyrene oxide (4CSO). Epoxides of choice can be used.

Since the strains selected in the present invention exhibited superior epoxide degradation activity even in a shorter reaction time than previously reported wild strains, they can be usefully used to prepare chiral epoxides and diols.

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 strains of the present invention exhibited epoxide hydrolase activity against racemic styrene oxide and its derivative substrates (see Table 1 and Table 2 of Examples, FIGS. 3 to 9). Specifically, for styrene oxide (SO), Rhodococcus YSMI04 strain (Accession No. KCTC 12263BP) showed (S) -form, and YSNA32 strain (Accession No. KCTC 12260BP) showed optical selectivity for (R) -form. For the 3-chlorostyrene oxide (3CSO) substrate both strains showed optical selectivity for (S) -form, and for the 4-chlorostyrene oxide (4CSO) substrate both strains for (R) -form Optical selectivity was shown. For 2-chlorostyrene oxide (2CSO) both strains were shown to degrade both racemic 2CSO (R)-and (S)-. Of particular note, the TSBP12 strain genus Roseobacter (Accession No. KCTC 12261BP) hydrolyzed both (R)-and (S)-without optical selectivity for SO and 2CSO, but optically selective hydrolytic activity for 3CSO and 4CSO. The optical selectivity of 3CSO and 4CSO was different from each other, indicating that 3CSO showed optical selectivity for (R) -form and 4CSO for (S) -form. In addition, the present invention is also the first to identify that the strain of genus Roseobacter has optical selective epoxide hydrolysis activity.

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 diol product is present in the aqueous solution layer can be separated from the biocatalyst by centrifugation, etc. Another product chiral epoxide is present in the organic solvent layer can be separated purely through evaporation. .

The recovered strain biocatalyst is added with a new racemic epoxide to the reactor immediately without additional catalyst regeneration, and the water is slowly added to repeat the same reaction to remove chiral epoxide or diol (preferably, chiral 1,2-diol). It can be synthesized repeatedly.

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 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 hydrolytic activity against various epoxide substrates was obtained. Three strains provided by the present invention showed very good epoxide hydrolysis activity against all derivative substrates including racemic styrene oxide. The results shown in the present invention can be a good model of epoxide hydrolase activity investigation in the future and is expected to be a high value-added organic intermediate material widely used in the synthesis of optically active pharmaceuticals that can increase the industrial applicability.

Since the selected strains of the present invention have epoxide hydrolysis activity, they can be usefully used for biosynthesis of high value synthetic intermediates such as chiral epoxide and chiral diol with high efficiency. In addition, it can be a good model of epoxide hydrolase activity investigation 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).
FIG. 3 shows the optical resolution of 11-BPY-1-2 strain of 0.02 g of wet cell weight acting on 17 mM styrene oxide (SO).
Figure 4 shows a phylogenetic tree based on 16S rRNA sequences of epoxide hydrolase active strains 1-MIXS-4 and 1-NA-3-2. The parenthesis shows the registration number of the GenBank database.
Figure 5 shows a phylogenetic tree based on the 16S rRNA sequence of epoxide hydrolase active strain 11-BPY-1-2. The parenthesis shows the registration number of the GenBank database.
FIG. 6 shows that three strains (1-MIXS-4, 1-NA-3-2, and 11-BPY-1-2) hydrolyze 4 mM of 2CSO, 3CSO, and 4CSO by GC analysis. Indicates.
7 shows the optical resolution of three strains (1-MIXS-4, 1-NA-3-2, and 11-BPY-1-2; 0.2 g, wet cell weight) on 4 mM 2CSO. ).
FIG. 8 shows the optical resolution in which three strains (1-MIXS-4, 1-NA-3-2, and 11-BPY-1-2; 0.2 g, wet cell weight) act on 4 mM 3CSO. ).
9 shows the optical resolution of three strains (1-MIXS-4, 1-NA-3-2, and 11-BPY-1-2; 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 Excellent Strains rRNA  Sequencing and Taxonomic Positioning

Genomic DNA of excellent strains was isolated using Wizard Genomic DNA Purification Kit (Promega, Madison, Wis.). 16S rRNA was 27F (5'-AGA GTT TGA TCM TGG CTC AG-3 '; Escherichia , a 16S rRNA primer; 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 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, three strains (1-MIXS-4, 1-NA-3-2, and 11-BPY-1-2) showed epoxide hydrolysis activity against racemic styrene oxide (Table 1). In particular, in the case of the 11-BPY-1-2 strain faster than other strains, all 17 mM racemic styrene oxide was decomposed within 2 hours. As a result of the investigation over time, the degradation activity for racemic styrene oxide was very fast and about 80% was degraded in 1 hour (FIG. 3).

Sampling area Strain name Best-matched neighbor Similarity (%) ee ^a (%) / abs.
Conf. ^b / (R): (S) Hydrolysis rate
(x10 ^-2 ) mg / h SO (R) (S) Oil Pollution Area Sediment 1-MIXS-4 ^c Rhodococcus sp. M1 5-2 99.93 5.66 / S / (1.6) 1.22 0.77 1-NA-3-2 ^c Rhodococcus sp. M1 5-2 99.77 5.91 / R / (1.7) 1.03 1.77 11-BPY-1-2 ^d Roseobacter sp.204Z-13
Celeribacter sp.L-6 99.92 0.01 / NE ^e / (1) 51.0 51.0 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
d, whole cell (wet. 0.2 g) and reaction time 2 hours
e, no optical selectivity

2. Select 16 strains s rRNA  Sequencing and Taxonomic Positioning

Sequences for 16S rRNA clones for three strains (1-MIXS-4, 1-NA-3-2, and 11-BPY-1-2) showing epoxide hydrolysis activity in racemic styrene oxide the results, 1-MIXS-4, 1 -NA-3-2, and 11-BPY-1-2 is also genus Rhodococcus, respectively (Rhodococcus sp.), genus Rhodococcus genus (Rhodococcus sp.) and Rosedale O bakteo ( Roseobacter ( Celeribacter ) sp.) (FIGS. 4 and 5).

The phylogenetic diagram shown in Figure 4 is based on 1358 and 1434 base pairs of 16S rRNAs of strains 1-MIXS-4 and 1-NA-3-2, respectively, and 1-MIXS-4 and 1-NA-3-2 Rhodococcus sp. M1 5-2 (1414 bp, AY762055) and high homology of 99.93 and 99.77%, respectively. The phylogenetic diagram shown in FIG. 5 was made based on 1399 base pairs of 11-BPY-1-2 16S rRNA and 11- BPY -1-2 is Roseobacter sp. 204Z-13 (1435bp, GU584169) Celeribacter sp. Both strains of L-6 (1423bp, HM997022) were shown to have a high homology of 99.92% on NCBI.

Based on these results, each strain was deposited on August 14, 2012 to KCTC (Korean Colletion for Type Cultures, 125, Gwahak-ro, Yuseong-gu, Daejeon Metropolitan City). 1-MIXS-4 is Rhodococcus sp. YSMI04 was assigned accession number KCTC 12263BP, 1-NA-3-2 was Rhodococcus sp. YSNA32 was assigned accession number KCTC 12260BP, 11- BPY -1-2 was Roseobacter sp. It was named TSBP12 and was assigned accession number KCTC 12261BP.

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

Three strains showing epoxide hydrolysis activity were analyzed using GC to confirm whether they have 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. 6) and degradation rate (Table 2). Looking at the chromatogram of GC of Figure 6, As for 2-chlorostyrene oxide (2CSO), it was shown that all three strains degraded racemic 2CSO in both (R)-and (S)-. For the 3-chlorostyrene oxide (3CSO) substrate, all three strains showed optically selective activity, while the 1-MIXS-4 and 1-NA-3-2 strains were optically selective for (S) -3CSO while 11- BPY-1-2, in contrast, showed optical selectivity for (R) -3CSO. All three strains also showed optically selective activity against 4-chlorostyrene oxide (4CSO) substrate, while 1-MIXS-4 and 1-NA-3-2 strains were optically selective for (R) -4CSO while 11-BPY -1-2 conversely showed optical selectivity for (S) -4CSO. As a result, all three strains showed excellent epoxide hydrolysis activity 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 ) 1-MIXS-4
( Rhodococcus sp. YSMI04;
KCTC 12263BP) 1.22 0.77 0.42 0.43 1.71 0.28 1.33 1.74 1-NA-3-2
( Rhodococcus sp. YSNA32;
KCTC 12260BP) 1.03 1.77 0.03 0.07 1.04 0.44 1.13 1.66 11-BPY-1-2
Roseobacter sp. TSBP12;
KCTC 12261BP) 51.0 51.0 0.26 0.20 0.95 1.79 1.64 0.30

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

Next, under the same conditions as the above GC analysis conditions, the reaction time of three strains of whole cells and derivatives of styrene oxide (2CSO, 3CSO, 4CSO) substrate was investigated. As a result, the response of each substrate to each strain shows the GC results in FIGS. 7 to 9.

As a result of reacting the 2-chlorostyrene oxide (2CSO) substrate for up to 8 hours, it was shown that all three strains degraded racemic 2CSO little by little over time (FIG. 7).

The results for 3-chlorostyrene oxide (3CSO) substrates showed optically selective activity in all three strains as in the preliminary investigation, and 1-MIXS-4 and 1-NA-3-2 showed optical selectivity for (S) -3CSO. Whereas 11-BPY-1-2, in contrast, showed optical selectivity for (R) -3CSO. The time taken for the optical purity to be 100% was 1 hour for 1-MIXS-4, and 1-NA-3-2 showed 60% optical purity for 20 hours. 11-BPY-1-2 took 16 hours for the (R) -3CSO to achieve 100% optical purity (FIG. 8). Of particular note was that the 11-BPY-1-2 strain showed no optical selectivity for styrene oxide and 2CSO, but 3CSO showed optical selectivity and the selectivity was in the (R) -form as opposed to other strains.

The results for the 4-chlorostyrene oxide (4CSO) substrate were all three optically selective activity, such as 3CSO as in the previous investigation. Also, unlike 3CSO, 1-MIXS-4 and 1-NA-3-2 have optical selectivity for (R) -4CSO, while 11-BPY-1-2 has optical selectivity for (S) -4CSO. Appeared to be. The time taken for the optical purity to 100% was 12 hours for 1-MIXS-4, 11 hours for 1-NA-3-2, and 16 hours for 11-BPY-1-2 (FIG. 9). For 4CSO, 11-BPY-1-2 strain showed very good optical selectivity. Of particular note, as mentioned above, the 11-BPY-1-2 strain showed no optical selectivity for styrene oxide and 2CSO, but 3CSO and 4CSO showed optical selectivity. Is in the (R) -form, 4CSO is in the (S) -form. In contrast, the 1-MIXS-4 and 1-NA-3-2 strains showed optical selectivity of 3CSO as (S) -form and 4CSO as (R) -form, unlike 11-BPY-1-2. .

All of these results suggest that Rhodococcus genes 1-MIXS-4 and 1-NA-3-2 and Roseobacter genus 11- BPY -1-2 are not only strains but also interesting and novel in their properties and mechanisms. It is expected. In particular, the present invention was also the first to identify that the genus Roseobacter strain has optically selective epoxide hydrolysis activity.

Korea Biotechnology Research Institute KCTC12260BP 20120814 Korea Biotechnology Research Institute KCTC12261BP 20120814 Korea Biotechnology Research Institute KCTC12263BP 20120814

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Claims (12)

A method of preparing a chiral epoxide or diol, comprising reacting a Roseobacter sp. TSBP12 strain (Accession No. KCTC 12261BP) with an epoxide hydrolytic 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 chiral epoxide is (S) type chiral epoxide or (R) type chiral epoxide. The method of claim 1, wherein the diol is a chiral 1,2-diol compound. The method of claim 1, further comprising separating and purifying chiral epoxides or diols using gas chromatography. A biocatalyst composition for preparing chiral epoxides or diols from racemic epoxide substrates, comprising a genus TSBP12 strain (Accession No. KCTC 12261BP) having epoxide hydrolytic activity. 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. delete delete TSBP12 strain of genus Roseobacter with epoxide hydrolytic activity (Accession No. KCTC 12261BP).

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