JP2002233394A - Method for producing optically active 3-hydroxypyrrolidine compound - Google Patents

Abstract

(57) [Problem] To provide a method for producing an optically active 3-hydroxypyrrolidine compound. An optically active N-substituted 3-oxopyrrolidine compound is reacted with a) or b) below.
-Substituted-3-hydroxypyrrolidine compounds can be prepared. Enzyme having the amino acid sequence shown in a) SEQ ID NO: 1. b) a pyrrolidine-comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO: 1 and represented by the general formula (1)
The 3-one compound is converted to an optically active 3- represented by the general formula (2).
An enzyme having an enzymatic activity of reducing to a hydroxypyrrolidine compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing an optically active 3-hydroxypyrrolidine compound, and more particularly, to an optically active 3-hydroxypyrrolidine compound characterized by reacting a pyrrolidin-3-one compound with a certain enzyme. A method for producing the same.

[0002]

2. Description of the Related Art Optically active 3-hydroxypyrrolidine compounds represented by the following general formula (2) are useful as intermediates for pharmaceuticals (for example, Helvetica Chemical Act).
a Vol80 (1997) p892-896), and the development of an industrially advantageous production method thereof is desired.

[0003]

Means for Solving the Problems The present inventors diligently studied a method for producing an optically active 3-hydroxypyrrolidine compound. As a result, the pyrrolidin-3-one compound represented by the general formula (1) was replaced with SEQ ID NO: 1. An enzyme having the amino acid sequence shown or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in SEQ ID NO: 1,
In addition, an enzyme having an enzymatic activity to reduce the pyrrolidin-3-one compound represented by the general formula (1) to the 3-hydroxypyrrolidine compound represented by the general formula (2) is acted on to thereby form the compound represented by the general formula (2). The present inventors have found that the optically active 3-hydroxypyrrolidine compound shown can be obtained, and have completed the present invention.

That is, the present invention provides the following inventions. General formula (1)

Embedded image (Wherein, R represents a C1-C4 alkyl group or a benzyl group). A general formula (2) characterized by reacting the following a) or b) on a pyrrolidin-3-one compound represented by the following formula:

Embedded image (Wherein, R represents the same meaning as described above) (hereinafter referred to as the production method of the present invention) of an optically active 3-hydroxypyrrolidine compound represented by the formula: Enzyme having the amino acid sequence shown in a) SEQ ID NO: 1. b) a pyrrolidin-3-one compound having an amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO: 1 and represented by the general formula (1); An enzyme having an enzymatic activity of reducing to a 3-hydroxypyrrolidine compound. (Hereinafter a)
And b) are collectively referred to as the present enzyme. )

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the compound represented by the general formula (1) used in the production method of the present invention, C1-
Examples of the C4 alkyl group include a methyl group, an ethyl group,
Propyl group, 1-methylethyl group, butyl group, 1,1-
A dimethylethyl group is exemplified.

The gene sequence encoding the enzyme having the amino acid sequence shown in SEQ ID NO: 1 used in the production method of the present invention is shown in SEQ ID NO: 2 (Appl. Microbiol. Biotech.
nol (1999) 52,386-392). The gene having the nucleotide sequence encoding the amino acid sequence of the enzyme may be a naturally occurring gene, but may be one obtained by subjecting a naturally occurring gene to a mutation treatment (partial mutation introduction method, mutation treatment, etc.). You may.

The present enzyme can be produced, for example, by culturing a microorganism containing the gene represented by SEQ ID NO: 2 and producing an enzyme having the amino acid sequence represented by SEQ ID NO: 1 when the gene is expressed. . A microorganism that contains the gene represented by SEQ ID NO: 2 and that produces an enzyme having the amino acid sequence represented by SEQ ID NO: 1 when the gene is expressed is a naturally occurring microorganism,
A transformed microorganism into which a gene having the nucleotide sequence represented by SEQ ID NO: 2 has been introduced may be used.

Here, a method for preparing a transformed microorganism into which a gene having the nucleotide sequence of SEQ ID NO: 2 has been introduced will be described. As a host cell into which a gene having the nucleotide sequence represented by SEQ ID NO: 2 is introduced, for example, Escherichi
a, Bacillus, Corynebacterium, Staphylococcus, Stre
ptomyces, Saccharomyces, Kluyveromyces and Aspergil
Microorganisms belonging to the genus lus can be mentioned. Method for introducing the gene into a host cell is not particularly limited as long as the normal methods used depending on the cell as a host, for example, "Molecular Cloning: ALaboratory Manual 2 ^nd ed
ition "(1989), Cold Spring Harbor Laboratory Pres
s, `` Current Protocols in Molecular Biology '' (198
7), John Wiley & Sons, Inc. ISBNO-471-50338-X, etc.
oration: Gene Pulser / E.coli Pulser System '' Bio-Ra
d Laboratories, (1993) and the like.

The transformed microorganism into which the gene has been introduced is
Selection can be performed using the phenotype of the selectable marker gene contained in the vector used when introducing the gene into the host cell as an index. The transformed microorganism is carrying the gene, the vector DNA from the transformant microorganism
Is prepared, and the prepared DNA is subjected to a conventional method (confirmation of restriction enzyme site, nucleotide sequence, etc.) described in, for example, "Molecular cloning" (J. Sambrook et al., Cold Spring Harbor, 1989). Analysis, Southern hybridization, etc.).

Next, in order to produce the present enzyme used in the production method of the present invention, it contains the gene represented by SEQ ID NO: 2,
A method for culturing a microorganism that produces an enzyme having the amino acid sequence represented by SEQ ID NO: 1 when the gene is expressed will be described.

As a medium for culturing the microorganism, various media containing a carbon source, a nitrogen source, an organic salt, an inorganic salt and the like which are usually used for culturing a microorganism can be used.

Examples of the carbon source include sugars such as glucose, dextrin and sucrose, sugar alcohols such as glycerol, organic acids such as fumaric acid, citric acid and pyruvic acid, animal and vegetable oils, and molasses. The amount of these carbon sources added to the medium is usually 0.1 to 20% based on the total amount of the medium.
(W / v).

[0013] Nitrogen sources include meat extract, peptone, yeast extract, malt extract, soy flour, corn steep,
Natural organic nitrogen sources and amino acids such as liquor (Corn Steep Liquor), cottonseed flour, dried yeast, casamino acid, and ammonium salts and nitrates of inorganic acids such as sodium nitrate, ammonium chloride, ammonium sulfate and ammonium phosphate, and ammonium fumarate And ammonium salts of organic acids such as ammonium citrate, and organic or inorganic nitrogen sources such as urea. Of these, ammonium salts of organic acids, natural organic nitrogen sources, amino acids and the like can often be used as carbon sources. The amount of the nitrogen source to be added is usually about 0.1 to 30% (w / v) based on the total amount of the medium.

Examples of the organic and inorganic salts include chlorides, sulfates, acetates, carbonates and phosphates of potassium, sodium, magnesium, iron, manganese, cobalt, zinc and the like. Is sodium chloride,
Examples thereof include potassium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acetate, calcium carbonate, sodium carbonate, monopotassium hydrogen phosphate, and dipotassium hydrogen phosphate. The amount of organic or inorganic salts added is usually
It is good to be about 0.0001-5% (w / v).

Furthermore, a host cell into which a gene obtained by operably connecting a promoter of the type induced by allolactose, such as a tac promoter, a trc promoter, a lac promoter, and a gene encoding the present enzyme, is introduced. In some cases, as an inducer for inducing the production of the enzyme, for example, isopropyl thio-β-D-galactoside (IPT
G) may be added in a small amount to the medium.

The cultivation can be carried out in accordance with a method usually used for culturing microorganisms.
Reciprocating shaking culture, Jar Fermente
r) Methods such as liquid culture and solid culture, such as culture and tank culture, are possible. When using a jar fermenter,
It is necessary to introduce sterile air into the jar fermenter, usually using aeration conditions of about 0.1 to about 2 times / min of the culture volume. The culture temperature is preferably in the range of 35 to 42 ° C, and the pH of the medium is preferably in the range of about 6 to about 8. The culturing time varies depending on the culturing conditions, but is usually about 1 hour.
Days to about 5 days is desirable.

In the production method of the present invention, for example, the cells containing the present enzyme thus obtained, treated cells thereof, or purified products of the present enzyme can be used.

Here, the treated cells may be, for example, freeze-dried cells, organic solvent-treated cells, dried cells, crushed cells,
Autologous digest of cells, sonicated cells, cell extracts,
Alkali-treated cells can be mentioned, and those immobilized by these commonly used methods can also be mentioned.

The purified product of the present enzyme can be produced, for example, by purifying the present enzyme from a culture of a microorganism having the present enzyme. As a method for purifying the present enzyme from a culture of a microorganism having the present enzyme, a method used in ordinary protein purification can be applied, and examples thereof include the following methods. First, cells are collected from the culture of the microorganism by centrifugation or the like, and then subjected to ultrasonic treatment, dynomill treatment, a physical disruption method such as French press treatment, or a cell lysing enzyme such as a surfactant or lysozyme. Crushing is performed according to the chemical crushing method used. Insoluble matter is removed from the obtained crushed liquid by centrifugation, filtration through a membrane filter, etc. to prepare a cell-free extract, which is then subjected to cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, gel chromatography, etc. The present reductase can be purified by fractionation using the separation and purification method described above as appropriate. Examples of the carrier used for chromatography include resin carriers such as cellulose, dextran, and agarose, into which a carboxymethyl (CM) group, a DEAE group, a phenyl group, a butyl group, or the like has been introduced. It is also possible to use a commercially available carrier-packed column, for example,
Q-Sepharose FF, Phenyl-Sepharose HP (trade names, all manufactured by Amersham Pharmacia Biotech), T
SK-gel G3000SW (trade name, manufactured by Tosoh Corporation)
And the like.

Next, the production method of the present invention will be described.
In the production method of the present invention, the reaction for converting the pyrrolidin-3-one compound represented by the general formula (1) to the optically active 3-hydroxypyrrolidine compound represented by the general formula (2) is carried out. This is achieved by allowing the present enzyme to act on a 3-one compound. The reaction is usually carried out in the presence of water, and water may be in the form of a buffer. In this case, examples of the buffer used include alkali metal phosphates such as sodium phosphate and potassium phosphate. And alkali metal salts of acetic acid such as sodium acetate and potassium acetate. In this case, the pH can be appropriately changed within a range in which the reaction proceeds.

When a buffer is used as a solvent, the amount of the solvent is the pyrrolidin-3-one compound 1 represented by the general formula (1).
It is usually 100 parts by weight or less based on parts by weight.

The reaction temperature is from 0 to 70 ° C, preferably from 10 to 40 ° C, in view of the stability and reaction rate of the enzyme.

The reaction can be carried out in the presence of an organic solvent in addition to water. As the organic solvent in this case, for example, tetrahydrofuran, t-butyl methyl ether,
Ethers such as isopropyl ether, hydrocarbons such as toluene, hexane, cyclohexane, heptane, isooctane and decane, t-butanol, methanol,
Examples thereof include alcohols such as ethanol, isopropanol and n-butanol, sulfoxides such as dimethyl sulfoxide, ketones such as acetone, nitriles such as acetonitrile, and mixtures thereof. The amount of the organic solvent used in the reaction is usually 1 to 1 part by weight of the pyrrolidin-3-one compound represented by the general formula (1).
It is at most 00 parts by weight, preferably at most 70 parts by weight.

The reaction may further comprise a coenzyme (eg, NAD
H, NADPH). The amount of the coenzyme used in the reaction is usually 0.5 times by weight or less, preferably 0.1 times by weight or less, based on the pyrrolidin-3-one compound represented by the general formula (1). When a coenzyme is added to the reaction, it is preferable to further add the following in order to increase the efficiency of the coenzyme. 1) Compounds such as formic acid, glucose, isopropanol, 2-butanol, 2-pentanol, 2-hexanol, 2-heptanol and 2-octanol The amount of these compounds used in this case is represented by the general formula (1) Is 100 weight times or less, preferably 10 weight times or less with respect to the pyrrolidin-3-one compound represented by 2) Dehydrogenases such as formate dehydrogenase and glucose dehydrogenase The amount of dehydrogenase used in this case is determined by the general formula (1)
0.1 with respect to the pyrrolidin-3-one compound represented by
It is not more than 50 times by weight, preferably not more than 0.05 times by weight.

The reaction is carried out, for example, by mixing water, a pyrrolidin-3-one compound represented by the general formula (1), the present enzyme, and if necessary, a coenzyme, an organic solvent and the like, and stirring and shaking. be able to.

The end point of the reaction can be determined, for example, by tracking the amount of the starting compound in the reaction solution by liquid chromatography, gas chromatography or the like. The range of the reaction time is usually from 5 minutes to 4 days.

After completion of the reaction, for example, the reaction solution is extracted with an organic solvent such as hexane, heptane, tert-butyl methyl ether, ethyl acetate, toluene and the like, and the organic layer is dried and concentrated to obtain the desired product. Can be released. The target substance can be purified by column chromatography or the like, if necessary.

In the production method of the present invention, a) an amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO. And an enzyme having an enzymatic activity for reducing a pyrrolidin-3-one compound represented by the general formula (1) to an optically active 3-hydroxypyrrolidine compound represented by the general formula (2) can also be used. In this case, for example, DN shown in SEQ ID NO: 2
A, for example, a site-specific mutagenesis method, which is a well-known technique for causing a point mutation or the like in DNA; a method of selectively cleaving DNA, and then removing or adding a selected nucleotide to ligate the DNA; Or an amino acid sequence which can be prepared by performing the oligonucleotide mutant derivative method, wherein the amino acid sequence has one or several amino acids deleted, substituted or added in SEQ ID NO: 1, and has the general formula (1)
A pyrrolidin-3-one compound represented by the general formula (2)
A gene encoding an enzyme having an enzymatic activity to reduce to an optically active 3-hydroxypyrrolidine compound represented by is prepared, and the production method of the present invention is carried out by preparing, culturing, and reacting a transformant in the same manner as described above. it can.

[0029]

EXAMPLES The present invention will be described in more detail with reference to production examples and the like, but the present invention is not limited to these examples.

Production Example A liquid medium (tryptone 10 in 1000 ml of water) was placed in a flask.
g, 5 g of yeast extract and 5 g of sodium chloride were dissolved therein, and the pH was adjusted to 7.0 by dropwise addition of a 1N aqueous sodium hydroxide solution. ) Add 900ml and sterilize,
100 μg / ml ampicillin, isopropyl thio-β-
D-galactoside (IPTG) was added to a concentration of 0.4 mM, and the plasmid pUAR containing the DNA shown in SEQ ID NO: 2 (Accession number: FERM P-1812) was added thereto.
The transformant E. coli JM109 / pUAR obtained by transforming the E. coli JM109 strain in the usual manner in 7) was inoculated with 1 ml of a culture solution cultured in a liquid medium having the above composition, and cultured at 37 ° C. for 14 hours with shaking. The culture is centrifuged (15000 × g, 15
For 30 minutes at 4 ° C.), and the obtained cells are treated with a 50 mM monopotassium phosphate-dipotassium phosphate buffer (pH 7.0) 30 m
l, and the suspension is centrifuged (15000 xg,
(15 minutes, 4 ° C.) to obtain washed cells. NAD ⁺ was added to 50 ml of a 50 mM monopotassium phosphate-dipotassium phosphate buffer (pH 7.0) containing 5% of 2-propanol.
0.1 mmol and 6 g of the washed cells were added. Here, decane 5 containing 70 mg (0.31 mmol) of Nt-butyloxycarbonyl-3-oxopyrrolidine was used.
0 ml was added, and the mixture was stirred at room temperature for 24 hours. Thereafter, celite was added to the reaction solution, and the mixture was stirred for a while and filtered to separate a solution obtained. The aqueous layer was further extracted with ethyl acetate three times, and the obtained organic layers were all combined and concentrated to obtain (S) -Nt-butyloxycarbonyl-3-hydroxypyrrolidine (40 mg). The chemical purity of the product was analyzed by GC, and the optical purity was analyzed by HPLC using Daicel's Chiral Cell OF column (mobile layer: hexane / isopropyl alcohol = 80/20). (Chemical purity 1
(00%, optical purity: 100% ee) The absolute configuration of the product was determined by comparing an HPLC chart with a standard sample described in Reference Example described later. ¹ H-NMR (δ, ppm, CDCl ₃ ): 1.46
(S, 9H), 1.75-2.15 (m, 2H), 3.
25 to 3.85 (m, 4H), 4.45 (br, 1H)

Next, Nt-butyloxycarbonyl-
Reference examples 1 and 2 for determination of absolute configuration of 3-hydroxypyrrolidine and production of samples used for optical purity measurement
It writes in.

Reference Example 1 0.5 g of Nt-butyloxycarbonyl-3-oxopyrrolidine (manufactured by ACD) was dissolved in 4 g of ethanol.
Cooled to -20 ° C. To this was added 34 mg of sodium borohydride suspended in 1 ml of ethanol. afterwards,
The temperature was gradually raised to room temperature, and the mixture was further stirred for 1 hour. 1 ml of 10% citric acid aqueous solution, 10 ml of water and 20 ml of t-butyl methyl ether were added to the reaction solution, and the mixture was separated. The aqueous layer was further extracted with 20 ml of t-butyl methyl ether. The obtained organic layers were combined, washed with saturated saline, dried over anhydrous magnesium sulfate, and concentrated to obtain 0.5 g of Nt-butyloxycarbonyl-3-hydroxypyrrolidine. MS (m / z) 187 (M ⁺ ), 114, 87, 57 This Nt-butyloxycarbonyl-3-hydroxypyrrolidine was converted to a Chiral Cell OF column manufactured by Daicel (mobile layer: hexane / 2-propanol = 80/20). HPLC analysis using a UV detector (220 nm) gave two peaks having almost the same area ratio at retention times of 14 minutes and 16 minutes.

Reference Example 2 (R) -3-hydroxypyrrolidine (manufactured by Aldrich)
0.25 g was dissolved in 2.5 g of ethyl acetate, and 70.7 mg of N, N-dimethylaminopyridine was added. Under ice cooling, 0.625 g of di-t-butyl dicarbonate was added thereto, and the temperature was gradually raised to room temperature. Stir for an additional hour and add 1
2 ml of a 0% citric acid aqueous solution was added and the mixture was separated. The organic layer was washed successively with 2 ml of a saturated saline solution and 2 ml of a 7% aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated to obtain (R) -N-t.
0.39 g of -butyloxycarbonyl-3-hydroxypyrrolidine was obtained. HPLC analysis of this (R) -Nt-butyloxycarbonyl-3-hydroxypyrrolidine using a Chiralcel OF column (mobile layer: hexane / 2-propanol = 80/20, UV detector 220 nm) manufactured by Daicel Corporation. And 2 observed in racemic analysis
Of the peaks, only one of the peaks with a retention time of 16 minutes was given.

[0034]

According to the present invention, an optically active 3-hydroxypyrrolidine compound useful as a pharmaceutical intermediate can be produced.

[0035]

[Sequence List] SEQUENCE LISTING <110> Sumitomo Chemical Co., Ltd. <120> Preparation of optically active 3-hyudroxypyrorizinone compound <130> P153362 <150> JP 2000-372706 <151> 2000-12-09 <160> 2 <210> 1 <211> 385 <212> PRT <213> Corynebacterium sp. <400> 1 Met Lys Ala Ile Gln Tyr Thr Arg Ile Gly Ala Glu Pro Glu Leu Thr 1 5 10 15 Glu Ile Pro Lys Pro Glu Pro Gly Pro Gly Glu Val Leu Leu Glu Val 20 25 30 Thr Ala Ala Gly Val Cys His Ser Asp Asp Phe Ile Met Ser Leu Pro 35 40 45 Glu Glu Gln Tyr Thr Tyr Gly Leu Pro Leu Thr Leu Gly His Glu Gly 50 55 60 Ala Gly Lys Val Ala Ala Val Gly Glu Gly Val Glu Gly Leu Asp Ile 65 70 75 80 Gly Thr Asn Val Val Val Tyr Gly Pro Trp Gly Cys Gly Asn Cys Trp 85 90 95 His Cys Ser Gln Gly Leu Glu Asn Tyr Cys Ser Arg Ala Gln Glu Leu 100 105 110 Gly Ile Asn Pro Pro Gly Leu Gly Ala Pro Gly Ala Leu Ala Glu Phe 115 120 125 Met Ile Val Asp Ser Pro Arg His Leu Val Pro Ile Gly Asp Leu Asp 130 135 140 Pro Val Lys Thr Val Pro Leu Thr Asp Ala Gly Leu Thr Pro Tyr His 145 15 0 155 160 Ala Ile Lys Arg Ser Leu Pro Lys Leu Arg Gly Gly Ser Tyr Ala Val 165 170 175 Val Ile Gly Thr Gly Gly Leu Gly His Val Ala Ile Gln Leu Leu Arg 180 185 190 His Leu Ser Ala Ala Thr Val Ile Ala Leu Asp Val Ser Ala Asp Lys 195 200 205 Leu Glu Leu Ala Thr Lys Val Gly Ala His Glu Val Val Leu Ser Asp 210 215 220 Lys Asp Ala Ala Glu Asn Val Arg Lys Ile Thr Gly Ser Gln Gly Ala 225 230 235 240 Ala Leu Val Leu Asp Phe Val Gly Tyr Gln Pro Thr Ile Asp Thr Ala 245 250 255 Met Ala Val Ala Gly Val Gly Ser Asp Val Thr Ile Val Gly Ile Gly 260 265 270 Asp Gly Gln Ala His Ala Lys Val Gly Phe Phe Gln Ser Pro Tyr Glu 275 280 285 Ala Ser Val Thr Val Pro Tyr Trp Gly Ala Arg Asn Glu Leu Ile Glu 290 295 300 Leu Ile Asp Leu Ala His Ala Gly Ile Phe Asp Ile Gly Gly Gly Asp 305 310 315 320 Leu Gln Ser Arg Gln Arg Cys Arg Ser Val Ser Thr Thr Gly Cys Arg 325 330 335 Asn Ala Gln Arg Pro Cys Gly Cys Gly Pro Trp Ser Val Val Pro Thr 340 345 350 Ala Val Glu Arg Gln Arg Lys Asn Thr Asp Ala Arg Pro Asn Ser Ile 355 36 0 365 Arg Pro Gly Ile Ser Val Arg Asn Ser Val Cys Ala Ser Cys Thr Pro 370 375 380 Arg 385 <210> 2 <211> 1158 <212> DNA <213> Corynebacterium sp. <220> <221> CDS <222 > (1) .. (1158) <400> 2 atg aag gcg atc cag tac acg cga atc ggc gcg gaa ccc gaa ctc acg 48 Met Lys Ala Ile Gln Tyr Thr Arg Ile Gly Ala Glu Pro Glu Leu Thr 1 5 10 15 gag att ccc aaa ccc gag ccc ggt cca ggt gaa gtg ctc ctg gaa gtc 96 Glu Ile Pro Lys Pro Glu Pro Gly Pro Gly Glu Val Leu Leu Glu Val 20 25 30 acc gct gct ggc gtc tgc cac tcg gac gac ttc atc atg ctg ccc 144 Thr Ala Ala Gly Val Cys His Ser Asp Asp Phe Ile Met Ser Leu Pro 35 40 45 gaa gag cag tac acc tac ggc ctt ccg ctc acg ctc ggc cac gaa ggc 192 Glu Glu Gln Tyr Thr Tyr Gly Leu Pro Leu Thr Leu Gly His Glu Gly 50 55 60 gca ggc aag gtc gcc gcc gtc ggc gag ggt gtc gaa ggt ctc gac atc 240 Ala Gly Lys Val Ala Ala Val Gly Glu Gly Val Glu Gly Leu Asp Ile 65 70 75 80 gga acc aat gtc gtc gtc tac ggg cct tgg ggt tgc ggc aac tgt tgg 288 Gly Thr Asn Val Val Val Tyr Gly Pro Tr p Gly Cys Gly Asn Cys Trp 85 90 95 cac tgc tca caa gga ctc gag aac tat tgc tct cgc gcc caa gaa ctc 336 His Cys Ser Gln Gly Leu Glu Asn Tyr Cys Ser Arg Ala Gln Glu Leu 100 105 110 gga atc aat cct ccc ggt ctc ggt gca ccc ggc gcg ttg gcc gag ttc 384 Gly Ile Asn Pro Pro Gly Leu Gly Ala Pro Gly Ala Leu Ala Glu Phe 115 120 125 atg atc gtc gat tct cct cgc cac ctt gtc ccg atc ggt gac ctc Ile Val Asp Ser Pro Arg His Leu Val Pro Ile Gly Asp Leu Asp 130 135 140 ccg gtc aag acg gtg ccg ctg acc gac gcc ggt ctg acg ccg tat cac 480 Pro Val Lys Thr Val Pro Leu Thr Asp Ala Gly Leu Thr Pro Tyr His 145 150 155 160 gcg atc aag cgt tct ctg ccg aaa ctt cgc gga ggc tcg tac gcg gtt 528 Ala Ile Lys Arg Ser Leu Pro Lys Leu Arg Gly Gly Ser Tyr Ala Val 165 170 175 gtc att ggt acc ggc ggt ctc gtc gct att cag ctc ctc cgc 576 Val Ile Gly Thr Gly Gly Leu Gly His Val Ala Ile Gln Leu Leu Arg 180 185 190 cac ctc tcg gcg gca acg gtc atc gct ttg gac gtg agc gcg gac Aag Ala Seru Val Ile Ala Leu Asp Val Ser Ala Asp Lys 195 200 205 ctc gaa ctg gca acc aag gta ggc gct cac gaa gtg gtt ctg tcc gac 672 Leu Glu Leu Ala Thr Lys Val Gly Ala His Glu Val Val Leu Ser Asp 210 215 220 aag gac gcg gcc gag aac gtc cgc aag atc act gga agt caa ggc gcc 720 Lys Asp Ala Ala Glu Asn Val Arg Lys Ile Thr Gly Ser Gln Gly Ala 225 230 235 240 gca ttg gtt ctc gac ttc gtc ggc tac cag acc acc atc gcg 768 Ala Leu Val Leu Asp Phe Val Gly Tyr Gln Pro Thr Ile Asp Thr Ala 245 250 255 atg gct gtc gcc ggc gtc gga tca gac gtc acg atc gtc ggg atc ggg 816 Met Ala Val Ala Gly Val Gly Ser Asp Val Thr Ile Val Gly Ile Gly 260 265 270 gac ggc cag gcc cac gcc aaa gtc ggg ttc ttc caa agt cct tac gag 864 Asp Gly Gln Ala His Ala Lys Val Gly Phe Phe Gln Ser Pro Tyr Glu 275 280 285 gct tcg gtg aca gtt cc gtt tgg ggt gcc cgc aac gag ttg atc gaa 912 Ala Ser Val Thr Val Pro Tyr Trp Gly Ala Arg Asn Glu Leu Ile Glu 290 295 300 ttg atc gac ctc gcc cac gcc ggc atc ttc gac atc ggc ggt gga gap Leu IuAla His Ala Gly Ile Phe Asp Ile Gly Gly Gly Asp 305 310 315 320 ctt cag tct cga caa cgg tgc cga agc gta tcg acg act ggc tgc cgg 1008 Leu Gln Ser Arg Gln Arg Cys Arg Ser Val Ser Thr Thr Gly Cys Arg 325 330 335 aac gct cag cgg ccg tgc ggt tgt ggt ccc tgg tct gta gta ccg aca 1056 Asn Ala Gln Arg Pro Cys Gly Cys Gly Pro Trp Ser Val Val Pro Thr 340 345 350 gcg gta gaa cga cag cgg aaa aac act gat gcc cgg ccg aat tcg att 1104 Ala Val Glu Arg Gln Arg Lys Asn Thr Asp Ala Arg Pro Asn Ser Ile 355 360 365 cgg ccg ggc atc agt gtc aga aat tcg gtg tgc gct agc tgc acg cct 1152 Arg Pro Gly Ile Ser Val Ar Val Cys Ala Ser Cys Thr Pro 370 375 380 cga tga 1158 Arg 385

Claims (4)

[Claims] 1. A compound of the general formula (1) (Wherein R represents a C1-C4 alkyl group or a benzyl group). The following general formula (2) is characterized by reacting the following a) or b) on a pyrrolidin-3-one compound represented by the following formula: ] (Wherein, R represents the same meaning as described above.) A method for producing an optically active 3-hydroxypyrrolidine compound represented by the formula: Enzyme having the amino acid sequence shown in a) SEQ ID NO: 1. b) a pyrrolidin-3-one compound having an amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO: 1 and represented by the general formula (1); An enzyme having an enzymatic activity of reducing an optically active 3-hydroxypyrrolidine compound. 2. An optically active compound represented by the general formula (2) according to claim 1, wherein the asymmetric carbon atom at the 3-position of pyrrolidine in the general formula (2) is rich in the absolute configuration of the (S) configuration.
-A method for producing a hydroxypyrrolidine compound. 3. The compound of the formula (2) wherein the asymmetric carbon atom at the 3-position of pyrrolidine has the absolute configuration of (S).
The method for producing an optically active 3-hydroxypyrrolidine compound represented by the general formula (2) according to claim 1, wherein the content is 0% or more. 4. The method for producing an optically active 3-hydroxypyrrolidine compound represented by the general formula (2) according to claim 1, wherein R is a 1,1-dimethylethyl group. .