JP4601049B2 - Microorganism expressing epoxide hydrolase derived from Bacillus subtilis and method for producing epoxide hydrolase using the same - Google Patents

Description

  The present invention relates to an epoxide hydrolase derived from Bacillus subtilis. More specifically, the present invention relates to a transformed microorganism that expresses an epoxide hydrolase derived from Bacillus subtilis and a method for producing an epoxide hydrolase derived from Bacillus subtilis using the microorganism.

  Epoxide hydrolase (EH: EC 3.3.2.3), which is a kind of hydrolase, hydrolyzes epoxide (a three-membered ring containing oxygen) to produce 1,2-diol. Epoxides are highly reactive due to strain in their structure. For example, in vivo, since epoxide can act as an alkylating agent, biopolymers such as DNA and proteins are alkylated to exhibit mutagenicity, carcinogenicity, cytotoxicity, and the like. EH converts the harmful epoxide compound generated by excessive oxidation in the living body into a less reactive and water-soluble diol, thereby metabolizing or excretion of the epoxide compound outside the body. Facilitate. Thus, EH has a function related to biological defense.

  EH is known to exist in microorganisms, and its properties have been studied (Patent Document 1, Non-Patent Documents 1 to 8). In these reports, EH has many monomeric enzymes, and many have an optimum pH in a neutral to weakly alkaline region. In general, EH has a low optimum temperature for enzyme reaction, and the stability of the enzyme itself does not seem to be very high. Furthermore, the gene of EH has been cloned (Patent Literature 1, Non-Patent Literature 3, 9 to 11).

By the way, if EH is allowed to act on a racemic epoxide and one of the enantiomers can be stereoselectively hydrolyzed, an optically active 1,2-diol and epoxide can be obtained. Both of these compounds can be useful compounds as chiral building blocks. Therefore, for example, by hydrolysis of styrene oxide by Aspergillus niger, a type of filamentous fungus, and hydrolysis of aliphatic and aromatic epoxides by Rhodococcus ruber, a type of actinomycetes, Attempts have been made to obtain optically active diols or epoxides (Patent Document 2). As for EH derived from Aspergillus niger, the higher-order structure of the protein has been elucidated by X-ray crystal structure analysis, and the improvement of stereoselectivity by identifying the catalytic site and site-specific mutation is also being investigated. (Patent Document 2 and Non-Patent Document 12). Furthermore, application studies aimed at synthesizing antifungal drugs have been attempted (Non-patent Documents 13 and 14). EH used in these reactions is mass-produced by gene recombination using Aspergillus niger and Escherichia coli as hosts, and is commercially available as a reagent. However, the price is very expensive and not suitable for industrial use.
International Publication No. 99/21972 Pamphlet International Publication No. 00/68394 Pamphlet Morrissey, Sea. (Morisseau, C.) et al., “European Journal of Biochemistry”, 1999, 263, 386. Cronenberg, N. A. E. (Kronenburg, N.A.E.) et al., “Biotechnology Letters”, 1999, 21, 519. Link, Earl. (Rink, R.) et al., “Journal of Molecular Catalyst: Enzyme”, 2000, 10, 539. Crowtil, W. (Kroutil, W.) et al., “Journal of Biotechnology”, 1998, 61, 143. Missitz, M. (Mischitz, M.) et al., “Biotechnol. Lett.”, 1995, 19, 893. Van der welf, m. Jay. (Van der Werf, M.J.) et al., “Journal of Bacteriology”, 1998, 108, 5052. Nakamura, tea. (Nakamura, T.) et al., "Applied Environmental Microbiology", 1994, 60, 4630. (Appl. Environ. Microbiol.) Misawa, e. (Misawa, E.) et al., “European Journal of Biochemistry”, 1998, 253, 173. Alland, M. (Arand, M.) et al., “Biochem. J.”, 1999, 244, 273. Visell, H. (Visser, H.), et al., “Applied Microvial Biotechnology”, 2000, 53, 415. Visell, H. (Visser, H.) et al., "Applied Environmental Microbiology (Appl. Environ. Microbiol.)", 1999, 65, 5159. Genuine, Zet. (Jinyu, Z.) et al., “Structure”, 2000, Vol. 8, p. 111. Montfort, N. (Monfort, N.) et al., “Tetrahedron”, 2004, 60, 601. Montfort, N. (Monfort, N.) et al., “Tetrahedron: Asymmetry”, 2002, 13, 2399.

  An object of this invention is to provide the method of expressing the level of EH which can be industrialized in microorganisms, and the method of stabilizing EH.

  The present invention provides a method for producing a microorganism having enhanced activity of epoxide hydrolase, comprising the step of culturing the microorganism in a medium containing starch at a concentration of 4% to 20%.

  In one embodiment, the epoxide hydrolase is derived from Bacillus subtilis.

  In another embodiment, the microorganism is a Bacillus bacterium.

  In a further embodiment, the microorganism is a recombinant microorganism, in which an amylase promoter or an epoxide hydrolase promoter is inserted upstream of a structural gene encoding the epoxide hydrolase.

  The present invention also provides a method for enhancing the epoxide hydrolase activity of a microorganism comprising the step of culturing the microorganism in a medium containing starch at a concentration of 4% to 20%.

  The present invention further provides a method for producing epoxide hydrolase, comprising the steps of culturing a microorganism in a medium containing starch at a concentration of 4% to 20%, and recovering epoxide hydrolase from the obtained culture. To do.

  The present invention also includes a step of culturing a microorganism having epoxide hydrolysis activity in a medium containing starch at a concentration of 4% to 20%, and immobilizing and / or drying the resulting culture. A method for producing a epoxide hydrolase is provided.

  The present invention further provides an epoxide hydrolase agent produced by the method for producing an immobilized epoxide hydrolase described above.

  According to the method of the present invention, a high expression strain of EH can be bred. In addition, according to the present invention, a microorganism having high activity and high stereoselective EH activity is provided. Furthermore, by immobilizing such a microorganism having high EH activity, the microorganism has excellent storage stability and operability. As a high biocatalyst, it can be used repeatedly.

  According to the method for producing a microorganism with enhanced activity of EH of the present invention, the microorganism exhibits a higher epoxide hydrolysis activity when cultured in a starch-containing medium having a concentration of 4 to 20 w / v%. . Such microorganisms can have an epoxide hydrolysis activity of at least 1.5 times or more, for example, 2 times to several hundred times higher than that when cultured in a medium usually used by those skilled in the art. This seems to be because the expression level of EH in the microorganism increases when cultured in a starch-containing medium having a concentration of 4 to 20 w / v%. For example, a recombinant microorganism (transformant) in which an appropriate promoter (for example, an amylase promoter) as described below is incorporated is several tens to several times that of a microorganism not subjected to such an operation. Can present hundred times as much activity.

  The concentration of starch in the medium may be 8-12 w / v%. Here, the starch-containing medium refers to a culture medium that contains starch more abundantly than those normally used by those skilled in the art for culturing microorganisms. The starch contained in the medium may be of any origin. For the purpose of preventing starch gelatinization in the medium, starch liquefied in advance may be used. Starch liquefaction can be carried out by methods known to those skilled in the art (for example, treatment with α-amylase, acid hydrolysis). In addition, culture | cultivation temperature can be about 30-37 degreeC normally.

  The species and genus of the microorganism are not particularly limited. The microorganism may be a bacterium or a yeast. In general, in one embodiment of the present invention, the microorganism can be a Bacillus bacterium, since many Bacillus bacteria have high epoxide hydrolysis activity.

  The microorganism may be a wild strain or a recombinant microorganism. In the case of a recombinant microorganism, the species and genus of the host are not particularly limited and can be appropriately selected by those skilled in the art. Examples thereof include Bacillus bacteria and E. coli. An appropriate EH or promoter can be incorporated into the recombinant microorganism as necessary.

  In the present invention, the EH expressed in the microorganism may be an EH originally possessed by the microorganism or an EH inserted by recombination. The origin of EH is not specifically limited, For example, it originates from Bacillus subtilis. EH may be a variant or derivative provided it can exhibit epoxide hydrolysis activity. As used herein, “variant” refers to a protein having at least 70, at least 80, or at least 90 percent amino acid sequence homology with natural EH and having EH activity. For example, natural EH includes proteins having one or more amino acid additions, deletions, or substitutions. “Derivative” refers to a fusion protein of natural EH and another peptide. Other peptides that are fused do not interfere with the basic folding and conformational structure of EH.

  In the present invention, for the purpose of expressing EH in a larger amount, a promoter suitable for EH expression may be incorporated upstream of a structural gene encoding natural or incorporated EH. Examples of such a promoter include an amylase promoter or an EH promoter that may be regulated to an arbitrary length. Furthermore, in addition to the promoter, other expression regulators such as enhancers and terminators may be incorporated. When these expression regulators are inserted, in order to further enhance the action of these factors, appropriate components can be added to the culture medium of microorganisms depending on the expression regulator.

  According to the method for producing EH of the present invention, EH can be recovered from the obtained culture after culturing the microorganism having EH described above in a medium containing starch at a concentration of 4% to 20%. According to this method, since the amount of EH expressed in the microorganism is large, EH can be produced more efficiently. The recovery of EH can be performed as follows, for example. The bacterial cells in the culture solution can be crushed by bead beater treatment or ultrasonic treatment, and then insoluble matter can be removed by centrifugation or the like to obtain a crude enzyme solution of EH. From this crude enzyme solution, EH can be further purified or isolated by means usually used by those skilled in the art, such as column chromatography.

  The EH highly expressed in the microorganism by the above-described method of the present invention may be fixed in the microbial cell (EH-fixed microbial cell). Examples of means for immobilizing EH in cells include means for immobilization using a fixing agent, means for drying, and means for immobilization after immobilization. Hereinafter, the EH-fixed cells obtained by each means are referred to as an immobilized cell, a dry cell, and an immobilized dry cell, respectively.

  The immobilized cells can be prepared by a comprehensive method using a fixing agent such as acrylamide, carrageenan, calcium alginate, and the like. The immobilized cells can be further stabilized by further crosslinking treatment using a combination of polyethyleneimine and glutaraldehyde or a combination of hexamethylenediamine and glutaraldehyde.

  The dried cells can be prepared by drying a culture solution containing the cells by freeze drying, air drying, acetone drying or the like. When preparing dry cells, glycerol may be added to the culture solution so as to be 20 w / v% and dried for the purpose of improving stability.

  The immobilized dried microbial cell can be prepared by drying the immobilized microbial cell using a means commonly used by those skilled in the art.

  The EH-fixed microbial cell prepared in this way can be used as a catalyst in the epoxide hydrolysis reaction, that is, as an epoxide hydrolase agent. Although the conventional EH agent was an enzyme agent produced by roughly purifying or purifying an enzyme, the EH agent of the present invention can be prepared very simply without purifying EH. In addition, the activity of EH can be maintained for a long time as compared with the purified enzyme, and handling is also easy. In particular, the immobilized dry cells can be stored at room temperature and can be used repeatedly. For example, even if it is used at least 10 times in the epoxide hydrolysis reaction, no decrease in the activity of the immobilized dry cells is observed.

  By allowing the microorganism having enhanced epoxide hydrolysis activity, EH derived from the microorganism, or EH agent to act on the racemic epoxide, for example, the following reaction proceeds.

  Only one enantiomer of the racemic epoxide is hydrolyzed by the microorganism, EH, or EH agent. Therefore, hydrolysis yields an optically active diol, and an epoxide, the other enantiomer that was not hydrolyzed (not affected by the enzyme), remains.

  Specifically, this reaction step is performed by adding microorganisms or EH to an appropriate buffer or medium, adding a racemate of epoxide, and stirring or shaking. The substrate (epoxide) concentration and the amount of bacterial cells or EH in the reaction solution in this step can be appropriately determined. The microorganism, EH, or EH agent used may be used alone, or several microorganisms or EHs of different origins may be mixed and used. Usually, the optimum pH of the reaction solution is about 6.5 to 8.0, and the reaction temperature is about 30 to 37 ° C. The reaction time is not particularly limited, and is usually at least 5 minutes, 30 minutes to 96 hours, 3 to 72 hours, or 6 to 48 hours.

  Next, the target optically active compound can be recovered from the enzyme reaction solution. Here, the target optically active compound is an optically active epoxide or an optically active diol. In the step of allowing the enzyme to act, only one enantiomer of the racemic epoxide is stereoselectively (with steric retention) hydrolyzed by the microorganism or EH. Therefore, since an optically active diol produced by hydrolysis and an optically active epoxide that has not been hydrolyzed may exist in the enzyme reaction solution, an epoxide or diol is appropriately used by those skilled in the art depending on the purpose. Recover by means. Specifically, an appropriate organic solvent is added to the enzyme reaction solution to extract epoxide and diol into an organic layer, and the extract is subjected to silica gel column chromatography or the like, so that they can be separated and recovered.

  Alternatively, only the optically active diol can be obtained as the target optically active compound. In this case, the above-mentioned microorganism, EH, or EH agent is allowed to act on the racemate, and then the resulting enzyme reaction solution is acid-treated. In this acid treatment step, the residual epoxide that has not been affected by the enzyme is hydrolyzed with steric inversion under acidic conditions. The diol produced by this acid treatment has the same absolute configuration as the diol produced by the previous enzymatic action due to steric inversion. Therefore, as shown in the following reaction formula, an optically active diol can be obtained from a racemic epoxide by acid-treating an enzyme reaction solution.

  Here, the acid used for the acid treatment may be an inorganic acid, and examples thereof include hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and perchloric acid. The concentration of the acid in the reaction solution is not particularly limited, but can usually be about 0.1M. Next, the produced optically active diol can be recovered by, for example, adding an alkaline solution to the acid treatment reaction solution to neutralize it, and then extracting with an appropriate organic solvent as described above.

  (Preparation Example 1) Preparation of 2-benzyloxymethyl-2-methyloxirane (epoxide 1)

  Sodium hydride (12.21 g, 305 mmol, washed with hexane) and anhydrous DMF (50 ml) were placed in a 1 L four-necked flask cooled in an ice bath, and dissolved in anhydrous DMF (90 ml) while stirring with a magnetic stirrer. Benzyl alcohol (30.0 g, 277 mmol) was slowly added dropwise. After completion of the dropwise addition, the reaction mixture was stirred for 1.5 hours (ice-cooling to room temperature), then ice-cooled again, and 3-chloro-2-methylpropene (30.14 g, 333 mmol) dissolved in DMF (50 ml) was dropped. did. After stirring at room temperature for 16 hours, water was slowly added while cooling with ice. After adding hexane and stirring, the organic layer was recovered, and the organic layer was washed with water and a saturated aqueous sodium chloride solution. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to obtain 45.3 g of (2-methylallyloxymethyl) -benzene (colorless transparent liquid, yield 100.8%).

  Next, the obtained (2-methylallyloxymethyl) -benzene (40.0 g, 247 mmol) was placed in a 300 L four-necked flask and dissolved in acetonitrile (20 ml) and methanol (100 ml). Furthermore, potassium hydrogen carbonate (7.41 g, 74 mmol) was dissolved in hydrogen peroxide solution (30% aqueous solution, 56 g, 494 mmol) and added dropwise. Since it separated into two layers in the middle, acetonitrile was added. The progress of the reaction was followed by TLC while stirring at room temperature. The TLC analysis conditions were developing solvent: hexane / ethyl acetate = 10: 1, and Rf value: (2-methylallyloxymethyl) -benzene = 0.6; epoxide = 0.3. Since the reaction stopped halfway, hydrogen peroxide solution (70 g, 621 mmol) was further added in three portions. Stirring was continued for 5 days, and it was confirmed by TLC that the raw materials almost disappeared, and the organic solvent was distilled off by concentration under reduced pressure. Hexane was added to recover the organic layer, which was washed with an aqueous sodium thiosulfate solution, water, and then saturated brine. After drying over magnesium sulfate and concentrating under reduced pressure, 39.76 g of 2-benzyloxymethyl-2-methyloxirane (epoxide 1) was obtained (colorless transparent liquid, yield 90.3%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ: 1.40 (3H, s, 2-Me), 2.63 (1H, d, J4.8, 1-H), 2.75 (1H, d, J4.8, 1-H), 3.44 (1H, d, J10.8, 3-H), 3.57 (1H, d, J10.8, 3-H), 4.54 (1H, d , J12.0, OC H HPh), 4.59 (1H, d, J12.0, OCH H Ph), 7.34 (5H, s, Ph).

The HPLC analysis conditions of the obtained epoxide 1 are as follows:
Column: CHIRALPAK AD. (250mm × 4.6mmI.D., Manufactured by Daicel)
Column temperature: 18 ° C
Eluent: Hexane / diglyme = 98/2
Flow rate: 0.5 ml / min Detection: UV 260 nm
Retention time: R-form = 21.7 minutes, S-form = 25.5 minutes.

(Preparation Example 2) Preparation of 2-benzyloxy-2-methylpropane-1,2-diol (diol 1b) THF (5 ml) and epoxide 1 (1.0 g, 5.6 mmol) were added to a 50 ml three-necked flask. And water (1 ml) was added little by little while stirring with a magnetic stirrer. Subsequently, concentrated sulfuric acid (7.5 μl, 5 mol% as H ⁺ ) was added and stirred at room temperature for 6 days. The progress of the reaction was followed by TLC. The TLC analysis conditions were developing solvent: hexane / ethyl acetate = 2: 1, and Rf value: epoxide = 0.7; diol = 0.1. After neutralization with an aqueous sodium hydrogen carbonate solution, ethyl acetate was added to recover the organic layer. Thereafter, the organic layer was washed with saturated brine, dried over magnesium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (hexane: ethyl acetate = 4: 1 to 1: 1) to obtain 559 mg of 3 -Benzyloxy-2-methylpropane-1,2-diol (diol 1b) was obtained (colorless transparent liquid, yield 50.9%).

The HPLC analysis conditions for epoxide 1 and the obtained diol 1b are as follows:
Column: CHIRALPAK AD. (250mm × 4.6mmI.D., Manufactured by Daicel)
Column temperature: 18 ° C
Eluent: Hexane / 2-propanol = 90/10
Flow rate: 0.75 ml / min Detection: UV 260 nm
Retention time: Epoxide 1 = 6.0 minutes, R-diol 1b = 13.6 minutes, S-diol 1b = 14.8 minutes.

  Preparation Example 3 Preparation of 2- (4-methoxyphenoxymethyl) -2-methyloxirane (epoxide 2)

  P-Methoxyphenol (36.6 g, 295 mmol) was placed in a 1 L four-necked flask and dissolved in DMF. Furthermore, when 44.8 g of potassium carbonate and 324 mmol were added and stirring was continued, the color changed from colorless and transparent to reddish purple. Subsequently, 3-chloro-2-methylpropene (32.04 g, 354 mmol) dissolved in DMF (30 ml) was added dropwise, and the mixture was stirred at room temperature for 1 day. Hexane was added to recover the organic layer, which was washed with an aqueous sodium carbonate solution, an aqueous sodium bicarbonate solution, water, and a saturated aqueous sodium chloride solution. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to obtain 34.3 g of 1-methoxy-4- (2-methylallyloxy) benzene (colorless transparent needle crystals, melting point about 37 ° C., yield). Rate 65.3%).

  Next, the obtained 1-methoxy-4- (2-methylallyloxy) benzene (31.0 g, 174 mmol) was placed in a 0.5 L four-necked flask, and acetonitrile (100 ml) and methanol (60 ml) were added. And dissolved. Next, potassium hydrogen carbonate (5.22 g, 52 mmol) was dissolved in water (10 ml), mixed with hydrogen peroxide (30% aqueous solution, 59.2 g, 522 mmol), and this was added dropwise to the above solution. On the way, the progress of the reaction was monitored by TLC. The analysis conditions of TLC are as follows: developing solvent: hexane / ethyl acetate = 10: 1, and Rf value: 1-methoxy-4- (2-methylallyloxy) benzene = 0.25; epoxide = 0.5 there were. To promote the reaction, stirring was continued while hydrogen peroxide (100 g, 881 mmol) and potassium bicarbonate (1.73 g, 17.3 mmol) were added in portions. After 8 days, the organic solvent was removed by concentration under reduced pressure, hexane was added to recover the organic layer, washed and dried, and then concentrated under reduced pressure to give 24.8 g of 2- (4-methoxyphenoxymethyl) -2. -Methyloxirane (epoxide 2) was obtained (pale yellow solid, yield 73.3%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ: 1.47 (3H, s, 2-Me), 2.71 (1H, d, J5.6, 1-H), 2.85 (1H, d, J5.2, 1-H), 3.76 (3H, s, OMe), 3.90 (1H, d, J10.8, 3-H), 3.97 (1H, d, J10.8, 3 -H), 7.34 (5H, s, Ph).

(Preparation Example 4) Preparation of 3- (4-methoxyphenoxy) -2-methylpropane-1,2-diol (diol 2b) Except that epoxide 2 obtained in Preparation Example 3 was used instead of epoxide 1 The same operation as in Preparation Example 2 was performed to obtain 765 mg of 3- (4-methoxyphenoxy) -2-methylpropane-1,2-diol (diol 2b) (white crystals, yield 70.0). %).

The HPLC analysis conditions for epoxide 2 and the obtained diol 2b are as follows:
Column: Chiralcel OD
Column temperature: 40 ° C
Eluent: hexane / isopropanol = 99/1 to 91/9
Flow rate: 1 ml / min Detection: UV 260 nm
Retention times: Epoxide 2 = 4.6 and 5.3 minutes, Diol 2b = 22.0 and 23.7 minutes.

  (Preparation Example 5) Preparation of 2-methyl-2-phenethyloxirane (Epoxide 3)

  Sodium hydride (2.4 g, 60 mmol), anhydrous THF (25 ml), and anhydrous DMSO (10 ml) were placed in a 300 L three-necked flask cooled in an ice bath and stirred with a magnetic stirrer. Next, trimethissulfoxonium iodide (13.2 g, 60 mmol) dissolved in DMSO (90 ml) was slowly added dropwise (hydrogen gas was generated). After completion of the dropwise addition, the mixture was stirred for about 50 minutes, and then 4-phenyl-2-butanone (7.41 g, 50 mmol) dissolved in DMSO (20 ml) was added dropwise. After stirring for about 5 hours (ice cooling → room temperature), water was slowly added. Petroleum ether was added, and the organic layer was collected, washed with water and saturated brine, and dried over sodium carbonate. Concentration under reduced pressure gave 6.9 g of 2-methyl-2-phenethyloxirane (epoxide 3) (colorless transparent liquid, yield 85.1%).

Preparation Example 6 Preparation of 2-methyl-4-phenylbutane-1,2-diol (diol 3b) In a 100 ml eggplant flask, THF (6 ml) and epoxide 3 (1.0 g, 6.2 mmol) were placed. Water was added little by little while stirring with a tic stirrer. Since cloudiness was observed when 2 ml of water was added, concentrated sulfuric acid (8.3 μl, 5 mol% with H ⁺ ) was added, and the mixture was stirred at room temperature for 4 hours. The mixture was neutralized with an aqueous sodium carbonate solution, concentrated under reduced pressure, and ethyl acetate was added to recover the organic layer. The organic layer was washed with saturated brine, dried over sodium carbonate, and concentrated under reduced pressure to obtain 1.01 g of 2-methyl-4-phenylbutane-1,2-diol (diol 3b) (colorless transparent liquid). Yield 90.9%).

Analysis conditions of TLC Developing solvent: hexane / ethyl acetate = 5: 1
Rf value: Epoxide 3 = 0.8, Diol 3b = 0.09
HPLC analysis conditions Column: Chiralcel OD
Column temperature: 40 ° C
Eluent: Hexane / isopropanol = 95/5 to 83/17 (30 minutes)
Flow rate: 1.0 ml / min Detection: UV 260 nm
Retention times: Epoxide 3 = 4.8 and 5.5 minutes, Diol 3b = 22.4 and 26.0 minutes.

(Experiment 1) Examination of epoxide hydrolysis using B. subtilis 168 strain
Bacillus subtilis has many strains having high epoxide hydrolysis activity. Therefore, the hydrolytic resolution of the three types of epoxides 1 to 3 was evaluated using the B. subtilis 168 strain, which is a genome decoding strain and a model strain of B. subtilis, as follows.

  B. subtilis 168 strain (B. subtilis JCM10629; RIKEN BioResource Center) cultured overnight in LB medium (peptone: 10 g / L, yeast extract: 5 g / L, NaCl: 5 g / L, pH 7.0) Concentrated about 15 times and resuspended in various buffers (pH 6: acetate buffer, pH 7: potassium phosphate buffer, pH 8 and 9: Tris-HCl buffer) did. 100 μl of the bacterial solution was placed in a 2 ml Eppendorf tube. Subsequently, 2 μl of epoxides 1 to 3 were added and vigorously stirred at 30 ° C. (substrate concentration 1.7%). After 48 hours, 250 μl of ethyl acetate was added to stop the reaction, and the mixture was further stirred for 30 minutes. The ethyl acetate layer was collected and subjected to HPLC analysis. The results are shown in Table 1.

  The E values (enantio ratio) in the table are determined according to the method of Shi et al. (Chen, CS et al., J. Am. Chem. Soc., 1982, 104, 7294) From the Ee (enantiomeric excess) of the product (diol), or from the conversion rate and the Ee of the product, it was calculated according to the following calculation formula.

  Epoxides 1 to 3 were all stereoselectively hydrolyzed by the B. subtilis 168 strain. When epoxides 1 and 2 were used as substrates, the optical purity of the remaining epoxide reached almost 100% after 6 hours from the start of the reaction. In particular, since epoxide 1 gave the highest E value (211) so far, it was clarified that the B. subtilis 168 strain has excellent EH activity in both reactivity and stereoselectivity. . As a result of reacting with epoxides 1 and 2 in different pH buffers, hydrolysis was high at pH 8. In a control experiment conducted in parallel, non-enzymatic hydrolysis was observed at pH 6.

  Subsequently, the B. subtilis 168 strain cultured overnight in LB medium was resuspended in 50 mM Tris-HCl buffer (pH 8). The bacterial solution (10 ml) was placed in a polypropylene tube (15 ml), 0.2 g of epoxides 1 and 2 were added (final substrate concentration 2%), and shaken at 30 ° C. During the sampling, 0.2 ml was sampled and the progress of the reaction was monitored by HPLC (FIG. 1). When the optical purity of the epoxide exceeded 99% or when the conversion rate reached 50% (epoxide 1: 9 hours, epoxide 2: 6 hours), ethyl acetate was added to extract the remaining epoxide and produced diol. (3 ml x 5 times). The diol and the epoxide were separated by silica gel chromatography (hexane: ethyl acetate = 5: 1 → 3: 1 → 1: 1), the respective masses were measured, and the yield was calculated (Table 2).

  From Table 2, it was found that optically active diols and epoxides can be produced quantitatively using the B. subtilis 168 strain.

(Experiment 2) Cloning of EH gene from B. subtilis 168 strain
The B. subtilis 168 strain has been genome-decoded and the information is publicly available (Kunst, F. et al., “Nature”, 1997, 390, 249). . The genome database of B. subtilis 168 strain was accessed and a search was performed using epoxide hydrolase as a keyword (http://bacillus.genome.jp/bsorf-bin/BSORF_data_view.pl?ACCESSION=BG12888). Since a gene similar to EH (EH-like protein, yfhM, 858 bp) was found, the nucleotide sequence information of this EH-like gene and the region before and after that was obtained. The obtained sequences include EH DNA sequence (SEQ ID NO: 1), EH amino acid sequence (SEQ ID NO: 2), long gene sequence including the EH-like gene (EH-Long: SEQ ID NO: 3), and EH-like gene It was a short gene sequence (EH-Short: SEQ ID NO: 4) including before and after. Primers were designed based on these DNA sequences. In designing the primer, the following two points were improved: The original EH gene start codon was GTG, but was changed to the more general start codon ATG; and 3 residues from the N-terminus Since the tRNA corresponding to GGA of the codon encoding the basic amino acid (glycine) is small in E. coli, it was changed to GGT, which is a codon that exists in large numbers.

B. subtilis 168 strain from a genomic DNA preparation kit (Gen GenTLE ^TM yeast; Takara Bio Inc.) genomic DNA was prepared using as a template, EHN1 (SEQ ID NO: 5) or EHN2 (SEQ ID NO: 6) EHC2 ( Two types of EH structural genes were amplified by PCR using SEQ ID NO: 7) as a primer. These EH genes were treated with EcoRI and SalI, and at the same time, pBluescript SK (Stratagene) was treated with the same restriction enzymes. After ligating the EH gene and the vector, competent cells of E. coli strain JM109 were transformed. Transformants were selected by blue-white selection. The agar plate for blue-white selection was prepared by adding 0.1 mM IPTG (isopropyl-thio-β-D-galactopyranoside) and 40 μg / ml X-gal (5-bromo-4-chloro-3) in LB medium. -Indolyl-β-D-galactopyranoside) was used. A white colony obtained by blue-white selection is picked up, and after liquid culture in LB medium containing ampicillin (50 μg / ml), a nucleic acid extraction device (Automatic Nucreic Acid Extracter MFX-2000; Toyobo Co., Ltd.) from 1.5 ml of the culture solution ) Was used to extract the plasmid. The size of the plasmid was confirmed by agarose gel electrophoresis, and colonies that could be confirmed to increase in size were selected one by one and sequence analysis was performed. A fragment having a size as expected (around 860 bp) was confirmed by agarose gel electrophoresis. Next, these genes were ligated to pBluescript II SK (-) (copy number: about 200) at EcoRI and SalI sites, and competent cells of E. coli JM109 strain were transformed. In order to confirm the presence of the target plasmid, the plasmid was extracted and the size was confirmed by electrophoresis. Expansion of the plasmid size was confirmed in 8 strains out of 9 strains using primer EHN1 and in 1 strain out of 10 strains with EHN2. An increase in plasmid size suggests that the gene of interest has been inserted. One of each strain (hereinafter referred to as EHN1 expression strain and EHN2 expression strain) was selected and analyzed for DNA base sequence to confirm that it was cloned correctly. In addition, EH activity was not observed in the host E. coli JM109 strain, but EHN1 or EHN2 expression strain was observed. Both activities were comparable. From this result, it was confirmed that the EH-like gene cloned from the genome information encodes a protein having EH activity.

(Experimental example 3) EH activity in wild strain and recombinant strain (3-A) Preparation of transformant having tac promoter The protein expression system using the above pBluescript II SK (-) is a weak class in E. coli. Although the classified lac promoter was used, a transformant having a stronger tac promoter was prepared. Using pKK223-3 (copy number: 50), which is a general expression vector having a tac promoter, the EH gene cloned above was ligated to pKK223-3 at the EcoRI site, and competent cells of E. coli JM109 strain were transformed. Converted. The direction of the EH gene was confirmed by cleaving using a restriction enzyme site inside the EH gene and examining the size of the resulting DNA fragment, and a strain inserted in the forward direction was selected.

(3-B) Preparation of transformant (E. coli) having EH promoter
PCR was performed using the genomic DNA of B. subtilis 168 strain as a template, and the EH gene including the upstream and downstream regions was cloned. The primer used for PCR was EHNlongBam (SEQ ID NO: 8) or a combination of EHNshortBam (SEQ ID NO: 9) and CBam (SEQ ID NO: 10). By cloning, two types of EH genes including a promoter region (P _eh : 0.2 to 1.2 kb) and a terminator region (T _eh : 0.2 kb) were obtained. The structure of these genes is shown in FIG. The cloned gene was ligated to pBluescript SK (-) at the BamHI site, and competent cells of E. coli JM109 strain were transformed. When a plasmid was extracted from the clone obtained by transformation and the size was confirmed by electrophoresis, one 2.5 kb gene (EH-Long) and one 1.3 kb gene (EH-Short) each (EH -Long and EH-Short) and two (EH-Long × 2 and EH-Short × 2) inserted strains were obtained (FIG. 3). The gene insertion direction has not been confirmed.

(3-C) Preparation of a transformant (Bacillus subtilis) having an EH promoter Since EH is an enzyme produced by Bacillus subtilis, using a protein expression system of Bacillus subtilis itself, It can be assumed that the mismatch is eliminated and a high expression level can be achieved. Therefore, a Bacillus subtilis protein expression system was constructed.

  First, PCR was performed using the genomic DNA of B. subtilis 168 strain as a template, and the EH gene including the upstream and downstream regions was cloned. The primer used for PCR was EHNlongBam (SEQ ID NO: 8) or a combination of EHNshortBam (SEQ ID NO: 9) and Eco-EH-terR (SEQ ID NO: 11). The obtained gene was treated with BamHI and EcoRI, respectively, and ligated to pUB110 (obtained from ATCC) pretreated with the same restriction enzyme (16 ° C., 2 to 16 hours) to construct the plasmid shown in FIG. B. subtilis MT-2 (neutral protease-deficient host: sold by Ritsumeikan University, Faculty of Science and Engineering, Department of Chemistry and Biotechnology, Prof. Miki Kubo) was transformed by the tent cell method to obtain EH-Long strain and EH-Short strain.

(3-D) Measurement of EH activity Using the transformants of Escherichia coli and Bacillus subtilis obtained in (A) to (C) above, EH activity was measured as follows. B. subtilis 168 wild strain or transformant was cultured in LB medium (peptone: 10 g / L, yeast extract: 5 g / L, NaCl: 5 g / L, pH 7.0), and then collected by centrifugation. Resuspended in 50 mM Tris-HCl buffer (pH 8.0), 100 μl was placed in a 2 ml Eppendorf tube. 2 μl of Epoxide 1 was added to the tube and stirred vigorously at 30 ° C. for 30 minutes. The reaction was then stopped by adding 250 μl of ethyl acetate and stirred for an additional 30 minutes. The ethyl acetate layer was collected and subjected to HPLC analysis. The amount of the product and the remaining substrate was calculated from a calibration curve prepared in advance, and the base mass hydrolyzed in 1 hour per 1 ml of the culture solution was defined as EH activity. The results are shown in Table 3.

  When the EH promoter was functioned in a Bacillus subtilis host, it showed higher activity than in E. coli. Moreover, the activity value was significantly different between EH-Long and EH-Short. When E. coli was used as a host, the activity of EH-Long was higher than that of EH-Short, whereas in the case of Bacillus subtilis host, the expression level was reversed. There was no change in stereoselectivity due to differences in expression host and promoter (data not shown).

(Experimental Example 4) Preparation of transformants having various promoters (4-A) Preparation of transformants (B. subtilis) having amylase promoter It is known that Bacillus subtilis secretes and produces a large amount of protein outside the cells. Since its safety is well known by eating experience, it has been developed as a protein mass expression system. However, a successful example is only about the enzyme secreted out of a microbial cell, and Bacillus subtilis is hardly utilized as an expression system of the intracellular enzyme. Only by linking the structural gene of maltose phosphorylase downstream of the amylase promoter derived from Bacillus amyloliquefaciens NBRC 15535, which is known as a high amylase-producing bacterium, and expressing it in Bacillus subtilis using pUB110, the expression level is more than 250 times. (Inoue, Y. et al., “Bioscience, Biotechnol. Biochem.”, 2002, 66, 2594). . Therefore, the promoter sequence and terminator sequence of B. amyloliquefaciens amylase gene (Accession No. J01542) were obtained from DDBJ (http://srs.ddbj.nig.ac.jp/cgi-bin/wgetz?-id+ 2XtAQ1OFfGs + -e + [DDBJRELEASE: 'BACAAM']).

First, Xba-PamyF (SEQ ID NO: 12) and Bam-PamyR (SEQ ID NO: 13) using the genomic DNA of B. amyloliquefaciens NBRC15535 strain (purchased from the Biological Resource Center of the National Institute of Technology and Evaluation (NBRC)) as a template As a primer, the upstream region of the amylase gene (249 bp including the promoter) was amplified by PCR to obtain an amylase promoter site (P _amy ). At that time, an XbaI site was linked to the 5′-end and a BamHI site was linked to the 3′-end. In addition, EH-5Bam (SEQ ID NO: 14) and EHC3 (SEQ ID NO: 15: without terminator) or Eco-EH-terR (SEQ ID NO: 8: with terminator) were used as primers with the genomic DNA of B. subtilis 168 strain as a template. A PCR was performed to amplify the EH gene (without and with a terminator). At this time, a BamHI site was linked to the 5′-end and an EcoRI site was linked to the 3′-end. These _Pamy and EH genes were each treated with BamHI and ligated at the BamHI site. This composite gene was ligated to pUB110 at XbaI site and EcoRI site (16 ° C., overnight) to construct EH expression plasmids pUB-P _amy -EH and pUB-P _amy -EH-T _eh , respectively. These were used to transform B. subtilis MT-2 by the competent cell method, and B. subtilis MT-2 / pUB-P _amy -EH (hereinafter referred to as Pamy-12 strain: see FIG. 5) and B. subtilis MT-2 / pUB-P _amy -EH-T _eh (hereinafter referred to as Amy-5 strain) was prepared.

In addition, the terminator region (293 bp) of the amylase gene was amplified by PCR using the genomic DNA of B. amylolichefaciens NBRC15535 strain as a template and Eco-Tamy3F (SEQ ID NO: 16) and Acc-Tamy223R (SEQ ID NO: 17) as primers. The amylase terminator site (T _amy ) was obtained. Downstream of pUB-P _amy -EH, T _amy was inserted at the EcoRI and AccI sites to construct pUB-P _amy -EH-T _amy (FIG. 6). The constructed pUB-P _amy -EH-T _amy was incorporated into B. subtilis MT-2 to obtain B. subtilis MT-2 / pUB-P _amy -EH-T _amy (hereinafter referred to as Tamy-2 strain).

(Example 1) EH activity of microorganisms cultured in starch-containing medium
B. subtilis 168 wild strain or the transformant containing the amylase promoter obtained in Experimental Example 3 was added to a starch-containing medium (polypeptone S: 50 g / L, amylase-treated starch: 120 g / L, (NH ₄ ) _2. HPO ₄ : 10 g / L, CaCl ₂ · 2H ₂ O: 2 g / L, MgSO ₄ · 7H ₂ O: 2 g / L, corn steep liquor: 10 g / L, pH 7.0) for 4 days. After culturing, it was diluted 2 to 20 times with 50 mM Tris-HCl buffer (pH 8.0), and 100 μl was placed in a 2 ml Eppendorf tube. 2 μl of Epoxide 1 was added to the tube and stirred vigorously at 30 ° C. for 30 minutes. The reaction was then stopped by adding 250 μl of ethyl acetate and stirred for an additional 30 minutes. The ethyl acetate layer was collected and subjected to HPLC analysis. The amount of the product and the remaining substrate was calculated from a calibration curve prepared in advance, and the base mass hydrolyzed in 1 hour per 1 ml of the culture solution was defined as EH activity. The results are shown in Table 4.

  All the strains having an amylase promoter had very high EH activity, but a difference in EH activity was observed depending on the presence or absence of a terminator. By inserting the EH terminator, the Emy activity in the Amy-5 strain was about 1.5 times that of the Pamy-12 strain, and the Emy activity was further increased in the Tamy-2 strain into which the amylase terminator was inserted. In addition, in microorganisms cultured in a starch-containing medium, not only a strain having an amylase promoter but also a B. subtilis 168 strain which is a wild strain or a strain having an EH promoter is 10 times or more compared to the case of being cultured in an LB medium. Increased EH activity was observed. From this, it is speculated that the starch-containing medium acted not only on the amylase promoter but also on the EH promoter to increase EH activity.

  Therefore, other wild strains were also cultured in starch-containing medium and then measured for EH activity. The results are shown in Table 5.

  By culturing in a starch-containing medium, EH activity was increased for both bacterial and yeast species. In particular, Bacillus subtilis IAM 1186 was 7.2 times more active in bacteria, and Candida colliculosa JCM 2199 was 9.3 times more active in yeast.

(Example 2) Preparation of dry cells Amy-5 strain was cultured in a starch-containing medium for 4 days, and then collected by centrifugation. The collected cells were lyophilized at -20 ° C. and stored for 3 days, added with 20 v / v% glycerol, lyophilized at −20 ° C. and stored for 3 days, or air-dried at room temperature. Subsequently, 20 times the volume of 50 mM Tris-HCl buffer (pH 8) of the culture solution before drying was added to each dried cell and resuspended, and 100 μl was placed in a 2 ml Eppendorf tube. 2 μl of Epoxide 1 was added and stirred vigorously at 30 ° C. After 30 minutes, 250 μl of ethyl acetate was added to stop the reaction, and the mixture was further stirred for 30 minutes. The ethyl acetate layer was collected and subjected to HPLC analysis. The results are shown in Table 6.

  When the cells were frozen, the activity was halved, but when the cells were frozen in the presence of 20% glycerol, they were hardly inactivated. Addition of glycerol seems to be effective for maintaining EH activity even in storage in cells. On the other hand, in the air-dried cells, the activity was reduced to about ½.

(Example 3) Preparation of immobilized cells-1
Employing the inclusion method, which is a general technique for immobilizing bacterial cells, Amy-5 strain cultured in starch-containing medium is immobilized using carrageenan and calcium alginate, which are typical inclusion agents. It was.

  κ-carrageenan (4%) was dissolved at 100 ° C., and a carrageenan aqueous solution (about 3 ml) and a bacterial solution (corresponding to twice the concentration of the culture solution, 1 ml) were quickly mixed. The mixture was solidified by standing at room temperature, ground and subjected to the reaction.

  A sodium alginate aqueous solution (1 ml) having a concentration twice the final concentration and a bacterial solution (equivalent to twice the concentration of the culture solution, 1 ml) are mixed, and the solution is added to a calcium chloride aqueous solution (0.13 M) using a 0.45 mm syringe. It was dripped. The mixture was stirred for 1 hour or longer, and the gelled bacterial cell beads were collected and used for the reaction.

  For evaluating the activity of the immobilized cells, the carrageenan-immobilized cells were used as a catalyst having a concentration equivalent to the culture solution. Calcium alginate-immobilized cells were about 1.4 times concentrated because about 0.7 g of immobilized cells were obtained from 1% sodium alginate (1 ml) and bacterial liquid (1 ml). Used for reaction. A 20-fold volume of 50 mM Tris-HCl buffer (pH 8) of each immobilized cell was added and resuspended, and 100 μl was placed in a 2 ml Eppendorf tube. 2 μl of Epoxide 1 was added and stirred vigorously at 30 ° C. After 30 minutes, 250 μl of ethyl acetate was added to stop the reaction, and the mixture was further stirred for 30 minutes. The ethyl acetate layer was collected and subjected to HPLC analysis.

  In carrageenan-immobilized cells, EH activity was halved, but when immobilized with calcium alginate, there was no adverse effect of immobilization, and almost complete activity was maintained. In addition, no change in stereoselectivity was observed when each immobilized cell was used as a catalyst. Although the reaction was attempted in heptane to increase the substrate concentration, no improvement in activity was observed.

(Example 4) Preparation of immobilized cells-2
It is known that the concentration of the aqueous solution and the ease of gelation differ depending on the component ratio of the two types of sugars (mannuronic acid and guluronic acid) that make up alginic acid (the more gelled, the more easily gelled). Therefore, the Pamy-5 strain was immobilized using sodium alginate having a different composition, and selection of the optimum alginate was selected. In addition, the sodium alginate concentration was optimized.

  First, three types of sodium alginate having different component ratios of mannuronic acid (M) and guluronic acid (G), that is, Kimika Argin I-7, Kimika Argin IL-6G (all of which are manufactured by Kimika Co., Ltd.), and MANUCOL MV ( ISP (made by International Specialty Products). EH activity was measured by the same procedure as described in Example 3 above. As a result, MANUCOL MV with a high mannuronic acid content showed high activity (Table 7). The concentration of MANUCOL MV was optimally 0.9 to 1.0% (Table 8).

(Example 5) Crosslinking treatment of immobilized cells Crosslinking treatment of immobilized cells using a combination of polyethyleneimine and glutaraldehyde and a combination of 1,6-hexamethylenediamine and glutaraldehyde as a crosslinking agent. Went.

  The 1% calcium alginate-immobilized cells prepared in Example 3 (Amy-5 strain) were immersed in a 2% aqueous polyethyleneimine solution (molecular weight 60,000 to 80,000, neutralized with hydrochloric acid) for 5 minutes. The cells were collected, immersed in a 0.5% glutaraldehyde aqueous solution, and slowly stirred for 30 minutes with a magnetic stirrer. The immobilized cells were collected by filtration, washed 3 times with 50 mM Tris-HCl buffer (pH 8), and excess water was absorbed on the filter paper. Stored at 4 ° C. until use.

  Similarly, 1% calcium alginate-immobilized cells prepared in Example 3 (Amy-5 strain) were immersed in a 1% 1,6-hexamethylenediamine aqueous solution (neutralized with hydrochloric acid) for 10 minutes. The cells were collected, immersed in a 1% glutaraldehyde aqueous solution, and slowly stirred for 30 minutes with a magnetic stirrer. The immobilized cells recovered by filtration were washed 3 times with 50 mM Tris-HCl buffer (pH 8) to absorb excess water on the filter paper. Stored at 4 ° C. until use.

  Immediately after the cross-linking treatment, 10 days and 31 days, 20 times volume of 50 mM Tris-HCl buffer (pH 8) of each immobilized microbial cell was added and resuspended, and 100 μl was placed in a 2 ml Eppendorf tube. 2 μl of Epoxide 1 was added and stirred vigorously at 30 ° C. After 30 minutes, 250 μl of ethyl acetate was added to stop the reaction, and the mixture was further stirred for 30 minutes. The ethyl acetate layer was collected and subjected to HPLC analysis. The results are shown in Table 9.

  When the activity was measured immediately after the crosslinking treatment, a decrease in activity was observed in the crosslinked cells. When polyethyleneimine was used as the base, it decreased to 71%, and the activity was reduced by half in the crosslinking treatment using hexamethylenediamine having a high crosslinking density. When the activity was measured again after storing at 4 ° C. for 1 month, it decreased to 46% before storage in the non-crosslinked cells, but the retention of activity was high in the crosslinked cells (89 and 96%). . From these results, it was clarified that the cells cross-linked with a high concentration of glutaraldehyde are inactivated immediately after the cross-linking, but stability is improved and long-term storage is possible.

(Example 6) Drying treatment of immobilized bacterial cells The calcium alginate-immobilized bacterial cells and cross-linked bacterial cells of the Amy-5 strain prepared in Examples 3 and 5 were dried for 1 hour using a concentrator. It was. About these dry microbial cells, EH activity was measured by operation similar to the said Example 2. FIG. The results are shown in Table 10.

  In the case of the immobilized cells, the loss of activity due to the drying treatment was slight, and the activity of 90% or more was maintained. Furthermore, when the dried and immobilized cells were allowed to stand at room temperature and the residual activity was measured every month, the activity tended to decrease little by little. The activity of calcium alginate-immobilized cells (without cross-linking treatment) decreased to less than half after one month in wet cells, but it was possible to retain 80% activity after two months by drying treatment. . From this, it is estimated that the preservation | save for about six months-one year at room temperature is possible. From the above experiment, it was found that combining calcium alginate immobilization, crosslinking treatment, and drying treatment is effective for long-term storage of Amy-5 strain.

(Example 7) Repeated use of immobilized cells The dried immobilized cells were weighed and swollen with 100 μl of 50 mM Tris-HCl buffer (pH 8) for 1 hour. After excess water was removed by suction with a pipette, the same buffer (100 μl) was added again, 2 to 5 μl of epoxide 1 was added, and the mixture was reacted at 30 ° C. After 1 hour, only the aqueous layer was removed by suction and subjected to HPLC analysis. The buffer solution and the substrate were added again to the remaining immobilized cells, and the second and subsequent reactions were performed up to 10 times. The results are shown in FIG.

  As shown in FIG. 8, there was no significant loss of activity due to repeated use at least 10 times. Further, no significant difference was observed depending on the presence or absence of crosslinking and the type of crosslinking agent. Even after 10 uses, it was confirmed that the conversion rate reached 50% by extending the reaction time (data not shown).

Example 8 Substrate Specificity of Immobilized Bacteria Using Amy-5 calcium alginate-immobilized bacteria prepared in Example 5 above, EH activity against epoxides 1 to 4 was examined. Epoxide 4 was prepared as follows.

  Preparation of 2-methyl-2-phenoxymethyloxirane (epoxide 4)

  Sodium hydride (7.53 g, 188 mmol, washed with hexane) and anhydrous THF (30 ml) were placed in a 1 L three-necked flask cooled in an ice bath, and dissolved in anhydrous DMSO (200 ml) while stirring with a magnetic stirrer. Trimethylsulfoxonium iodide (39.6 g, 180 mmol) was slowly added dropwise. After completion of the dropping, the mixture was stirred at room temperature for about 30 minutes, and 1-phenoxy-2-propanone (24.57 g, 164 mmol) dissolved in DMSO (100 ml) was added dropwise. After stirring at room temperature for about 5 hours, an aqueous ammonium chloride solution was slowly added while cooling with ice. The organic component was extracted with hexane, washed with water and saturated brine, and dried over magnesium sulfate. Concentration under reduced pressure gave 10.63 g of 2-methyl-2-phenoxymethyloxirane (epoxide 4) (colorless transparent liquid, yield 39.5%). The results are shown in Table 11.

  There was no change in stereoselectivity compared to wild strains and free cells.

  According to the present invention, a microorganism that highly expresses highly active and highly stereoselective EH is provided. Further, by immobilizing the microorganism, it can be used repeatedly as a biocatalyst having excellent storage stability and high operability. Therefore, an optically active compound can be industrially produced from a racemic epoxide using the microorganism of the present invention as an organic synthesis reagent. The optically active compound thus obtained can be a useful compound as a chiral building block. Furthermore, when a 2,2-disubstituted epoxide having two substituents on one carbon atom of the epoxide ring is stereoselectively hydrolyzed, an optically active tertiary alcohol which is very difficult to chemically synthesize is produced. Which can be derived into various compounds. Therefore, the microorganism of the present invention is very useful for the production of pharmaceuticals and agricultural chemicals.

2 is a graph showing the time course of Ee of epoxide and diol in the hydrolysis reaction of epoxide by B. subtilis 168 strain. It is a schematic diagram of the gene structure containing the EH promoter and EH terminator of the EH gene. It is a schematic diagram which shows the structure of the plasmid constructed by inserting the EH-Long gene and the EH-Short gene into pBluescript SK (-). It is a schematic diagram which shows the structure of the plasmid constructed by inserting the EH-Long gene and the EH-Short gene into pUB110. It is a schematic diagram showing the construction of EH expression vector having an amylase promoter _(pUB-P amy -EH). It is a schematic diagram which shows construction of an EH expression vector (pUB-P _amy -EH-T _amy ) having an amylase terminator. It is a graph which shows the relationship between the repetition use frequency of the fixed microbial cell of Amy-5 stock | strain, and a diol yield.

Claims (6)

  A method of enhancing the epoxide hydrolase activity of a microorganism comprising culturing the microorganism in a medium containing starch at a concentration of 4% to 20%.   A method for producing an epoxide hydrolase, comprising a step of culturing a microorganism in a medium containing starch at a concentration of 4% to 20%, and a step of recovering the epoxide hydrolase from the obtained culture.   A method for immobilizing an epoxide hydrolase comprising: culturing a microorganism having epoxide hydrolyzing activity in a medium containing starch at a concentration of 4% to 20%; and immobilizing and / or drying the resulting culture. Production method.   The microorganism according to any one of claims 1 to 3, wherein the microorganism is a recombinant microorganism, and an amylase promoter or an epoxide hydrolase promoter is inserted upstream of a structural gene encoding the epoxide hydrolase in the microorganism. The method described in 1. An epoxide hydrolase agent produced by the method according to claim 3 or 4. A recombinant microorganism having an amylase promoter or an epoxide hydrolase promoter inserted upstream of a structural gene encoding epoxide hydrolase, and obtained by culturing in a medium containing starch at a concentration of 4% to 20%. A recombinant microorganism with enhanced activity of epoxide hydrolase.

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