JP2011177029A - Method for producing optically-active amine derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an optically-active amine derivative. <P>SOLUTION: There is disclosed a method for producing an optically-active S-form amine derivative by applying an enzyme having an ability to stereo-selectively reduce an imine compound, a microorganism producing the enzyme or a processed material thereof to the imine derivative and collecting a formed optically-active amine derivative. The obtained optically-active amine derivative is useful as a raw material for synthesizing medicines. For example, it is possible to produce an optically-active compound represented by formula (IV) (in the formula, R represents a 1-3C alkyl group and n is an integer from 1 to 4). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing an optically active amine derivative used as a raw material or synthetic intermediate for various pharmaceuticals by stereoselectively reducing an imine derivative.

There are few known methods for producing optically active amine derivatives by stereoselective reduction of imine derivatives except for the following reports.
-Non-patent document 1: Tetrahedron Asymmetry, 19, 93, 96 (2008)-
Reduction of N-phenyl-1-phenylethylamine derivatives by Candida parapsilos However, in these known techniques, since the reduction product of N-benzylidenebenzylamine is not a chiral structure, the stereoselectivity of the reaction is unknown. is there. In addition, since the reduction reaction of N-((E) -N- (1-phenylethylidene) aniline derivatives by Candida parapsirosis is extremely weak, it is not suitable for industrial use. The reality of is completely unknown.

Moreover, the following methods are known as a manufacturing method of 2-methylpyrrolidine.
-Non-patent document 2; Acta. Pharm. Suec., 1978, 15, 255-263-
Method for obtaining chiral-2-methylpyrrolidine by reducing 2-methyl-1-pyrroline with sodium borohydride to racemic 2-methylpyrrolidine, followed by optical resolution using tartaric acid-Non-Patent Document 3; Org. Chem., 1989, 54, 209-216−
By converting the hydroxyl group of L-prolinol derived from L-proline to a chloro group with thionyl chloride, protecting the nitrogen atom with a benzyloxycarbonyl group, and then reducing radically with tributyltin hydride, Method for producing (R) -1-benzyloxycarbonyl-2-methylpyrrolidine and further deprotecting

-Patent Document 1; WO 2005/073388-
A method in which optically active 1,4-pentanediol is converted to disulfonate and then reacted with an amine to cyclize to obtain 2-methylpyrrolidine. However, each method has the following problems.
-Long process-Use of dangerous hydrogenation reagents-Yield does not exceed 50% due to resolution method

Tetrahedron Asymmetry, 19, 93,96 (2008) Acta. Pharm. Suec., 1978, 15, 255-263 J. Org. Chem., 1989, 54, 209-216

WO2005 / 073388

  An object of the present invention is to provide a method for producing an optically active amine derivative from an imine derivative. Furthermore, an object of the present invention is to provide an enzyme capable of producing an optically active amine derivative using an imine derivative as a substrate, or a microorganism expressing the enzyme. Another object of the present invention is to isolate a DNA encoding the enzyme having the desired properties and incorporate it into a vector to obtain a recombinant. In addition, another object is to provide a method for producing an optically active amine derivative using a recombinant.

  The present inventors diligently studied a method for satisfying such a high demand. And it discovered that an optically active amine derivative could be produced | generated by stereoselectively reducing an imine derivative using the stereoselective reduction capability which microorganisms have.

  As already mentioned, enzymes involved in biosynthesis that stereoselectively reduce imine derivatives are known. However, all known enzymes are enzymes involved in metabolism. Therefore, when applied to the production of pharmaceutical intermediates, the substrate specificity and activity required for industrial production cannot be expected. In particular, there has been no known method for obtaining an optically active cyclic alkylamine derivative by reducing a cyclic alkylimine derivative such as 2-methyl-1-pyrroline.

Therefore, as disclosed in an earlier international application (PCT / JP2009 / 065244) by the present applicant, a microorganism having substrate specificity and catalytic activity suitable for the production of pharmaceutical intermediates has been found. It was a very surprising result. Surprisingly, the stereoselective imine-reducing ability of microorganisms was sufficiently high, and was at a level that was not problematic for industrial use. For example, Streptomyces sp. GF3546 (Streptomyces sp. GF3546) has been found to be a microorganism having useful properties for solving the above-mentioned problems. Furthermore, the international application (PCT / JP2009 / 065244) has succeeded in highly purifying imine reductase (SsSIR) through the following steps after mass-culturing GF3546 and preparing a cell-free extract. Is disclosed.
-Hydrophobic chromatography using Phenyl Sepharose, and ion exchange chromatography using-MonoQ In the international application (PCT / JP2009 / 065244), DNA encoding the imine reductase (SEQ ID NO: 6) is simply used. It has also been disclosed that a recombinant bacterium that highly expresses the enzyme was constructed.

  The inventors then further performed an identity search using the amino acid sequence (SEQ ID NO: 5) of Streptomyces sp. GF 3546-derived imine reductase (SsSIR), and the amino acid sequence of unknown function registered in EMBL. Was found to have 57% identity to the imine reductase. Specifically, it is a predicted protein named C4EPL6_STRRS obtained from the results of genome analysis of Streptosporangium roseum, and its function is DDBJ (DNA Data Bank of Japan), and C4EPL6_STRRS is a beta-hydroxyacid It is predicted to be dehydrogenase, 3-hydroxyisobutyrate dehydrogenase. As a result of constructing a recombinant bacterium that highly expresses C4EPL6_STRRS and investigating its properties, the present inventors have found that it has an imine reducing activity completely different from that predicted by DDBJ, and completed the present invention.

式（ＩＩ）

（式中、Ｒ１、Ｒ２基は炭素数１〜３個のアルキル基を、Ｒ３基は、炭素数１〜３個のアルキル基もしくは水素を表し、Ｒ１とＲ３は環を巻いていてもよい）
〔２〕
式（Ｉ）の化合物が下記式（ＩＩＩ）で表されるイミン誘導体であり、式（ＩＩ）の化合物が式（ＩＶ）で表されるアミン誘導体である〔１〕に記載の光学活性なＳ体のアミン誘導体の製造方法。
式（ＩＩＩ）

式（ＩＶ）

（式中Ｒ基は炭素数１〜３個のアルキル基を、ｎは、１〜４の整数を表す）
〔３〕
式（ＩＩＩ）の化合物が２−メチル−１−ピロリンであり、式（ＩＶ）で表される化合物が、２−メチルピロリジンである、〔２〕に記載の光学活性なＳ体のアミン誘導体の製造方法。
〔４〕
下記（ａ）から（ｅ）のいずれかに記載のポリヌクレオチドによりコードされるイミン還元酵素。
（ａ）配列番号：２に記載された塩基配列を含むポリヌクレオチド、
（ｂ）配列番号：１に記載のアミノ酸配列からなるタンパク質をコードするポリヌクレオチド、
（ｃ）配列番号：１に記載のアミノ酸配列において、１若しくは複数のアミノ酸が置換、欠失、挿入、および／または付加したアミノ酸配列からなるタンパク質をコードするポリヌクレオチド、
（ｄ）配列番号：２に記載された塩基配列からなるＤＮＡとストリンジェントな条件下でハイブリダイズするポリヌクレオチド、
（ｅ）配列番号：１に記載のアミノ酸配列と８０％以上の同一性を有するアミノ酸配列をコードするポリヌクレオチド。
〔５〕
前記イミン還元酵素が、配列番号：１に記載のアミノ酸配列において、１個以上かつ５０個以下のアミノ酸が置換、欠失、挿入、および／または付加したアミノ酸配列からなるタンパク質である、〔４〕に記載のイミン還元酵素。
〔６〕
下記（ａ）から（ｅ）のいずれかに記載のポリヌクレオチドによりコードされるイミン還元活性を有するタンパク質を有効成分として含む、立体選択的イミン還元剤。
（ａ）配列番号：２に記載された塩基配列を含むポリヌクレオチド、
（ｂ）配列番号：１に記載のアミノ酸配列からなるタンパク質をコードするポリヌクレオチド、
（ｃ）配列番号：１に記載のアミノ酸配列において、１若しくは複数のアミノ酸が置換、欠失、挿入、および／または付加したアミノ酸配列からなるタンパク質をコードするポリヌクレオチド、
（ｄ）配列番号：２に記載された塩基配列からなるＤＮＡとストリンジェントな条件下でハイブリダイズするポリヌクレオチド、
（ｅ）配列番号：１に記載のアミノ酸配列と８０％以上の同一性を有するアミノ酸配列をコードするポリヌクレオチド。
〔７〕
下記（ａ）から（ｅ）のいずれかに記載のイミン還元酵素をコードするポリヌクレオチドと、ＮＡＤＰ＋を補酵素とする酸化還元反応を触媒することができる脱水素酵素をコードするポリヌクレオチドとが一緒に挿入された組換えベクター。
（ａ）配列番号：２に記載された塩基配列を含むポリヌクレオチド、
（ｂ）配列番号：１に記載のアミノ酸配列からなるタンパク質をコードするポリヌクレオチド、
（ｃ）配列番号：１に記載のアミノ酸配列において、１若しくは複数のアミノ酸が置換、欠失、挿入、および／または付加したアミノ酸配列からなるタンパク質をコードするポリヌクレオチド、
（ｄ）配列番号：２に記載された塩基配列からなるＤＮＡとストリンジェントな条件下でハイブリダイズするポリヌクレオチド、
（ｅ）配列番号：１に記載のアミノ酸配列と８０％以上の同一性を有するアミノ酸配列をコードするポリヌクレオチド。
〔８〕
脱水素酵素がグルコース脱水素酵素である、〔７〕に記載のベクター。
〔９〕
グルコース脱水素酵素がバシラス・サブチルス（Bacillus subtilis）に由来する、〔８〕に記載の組換えベクター。
〔１０〕
〔７〕〜〔９〕のいずれかに記載のベクターを発現可能に保持した形質転換体。
〔１１〕
〔１０〕に記載の形質転換体を培養する工程を含む、〔１〕〜〔３〕のいずれかに記載の光学活性なＳ体のアミン誘導体の製造方法。
〔１２〕
〔１〕記載のイミン還元酵素を生産する微生物がストレプトスポランジウム（Streptosporangium）属に属する微生物である〔１〕〜〔３〕のいずれかに記載の光学活性なＳ体のアミン誘導体の製造方法。
〔１３〕
式（I）で示されるイミン誘導体に、ストレプトスポランジウム（Streptosporangium）属に属する微生物またはその処理物を接触させる工程と、式（ＩＩ）で示される光学活性なＳ体のアミン誘導体を回収する工程を含む、光学活性なＳ体のアミン誘導体の製造方法。 That is, this invention provides the manufacturing method of the following optically active amines. Alternatively, the present invention relates to a microorganism or imine reductase useful for the method, a vector containing DNA encoding the enzyme, and a stereoselective imine reducing agent containing the enzyme.
[1]
The imine derivative represented by the formula (I) comprises an imine reductase encoded by the polynucleotide according to any one of (a) to (e) below, a microorganism producing the imine reductase, and a processed product thereof Production of an optically active S-form amine derivative comprising a step of contacting at least one enzyme active substance selected from the group and a step of recovering an optically active S-form amine derivative represented by the formula (II) Method.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(C) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1;
(D) a polynucleotide that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 2;
(E) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1.
Formula (I)

Formula (II)

(Wherein R1 and R2 groups represent an alkyl group having 1 to 3 carbon atoms, R3 group represents an alkyl group having 1 to 3 carbon atoms or hydrogen, and R1 and R3 may be wound in a ring)
[2]
The optically active S according to [1], wherein the compound of formula (I) is an imine derivative represented by the following formula (III), and the compound of formula (II) is an amine derivative represented by formula (IV): A method for producing an amine derivative.
Formula (III)

Formula (IV)

(Wherein R represents an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 1 to 4)
[3]
The optically active S-form amine derivative according to [2], wherein the compound of formula (III) is 2-methyl-1-pyrroline and the compound represented by formula (IV) is 2-methylpyrrolidine. Production method.
[4]
An imine reductase encoded by the polynucleotide according to any one of (a) to (e) below.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(C) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1;
(D) a polynucleotide that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 2;
(E) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1.
[5]
The imine reductase is a protein comprising an amino acid sequence in which one or more and 50 or less amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1. [4] The imine reductase described in 1.
[6]
The stereoselective imine reducing agent which contains the protein which has the imine reduction activity encoded by the polynucleotide in any one of the following (a) to (e) as an active ingredient.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(C) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1;
(D) a polynucleotide that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 2;
(E) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1.
[7]
A polynucleotide encoding the imine reductase according to any one of (a) to (e) below and a polynucleotide encoding a dehydrogenase capable of catalyzing an oxidation-reduction reaction using NADP + as a coenzyme Recombinant vector inserted into.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(C) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1;
(D) a polynucleotide that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 2;
(E) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1.
[8]
The vector according to [7], wherein the dehydrogenase is glucose dehydrogenase.
[9]
The recombinant vector according to [8], wherein the glucose dehydrogenase is derived from Bacillus subtilis.
[10]
[7] A transformant that retains the vector according to any one of [9] in an expressible manner.
[11]
The method for producing an optically active S-form amine derivative according to any one of [1] to [3], comprising a step of culturing the transformant according to [10].
[12]
[1] The method for producing an optically active S-form amine derivative according to any one of [1] to [3], wherein the microorganism that produces the imine reductase according to [1] is a microorganism belonging to the genus Streptosporangium.
[13]
Contacting the imine derivative represented by formula (I) with a microorganism belonging to the genus Streptosporangium or a processed product thereof; and recovering the optically active S-form amine derivative represented by formula (II) A process for producing an optically active S-form amine derivative.

  INDUSTRIAL APPLICABILITY According to the present invention, there has been provided a method for efficiently producing an (S) isomer optically active amine by stereoselective reduction of imine using an imine reductase or a microorganism that expresses the enzyme. Also provided is an imine reductase responsible for the reaction. Furthermore, a DNA encoding an imine reductase was isolated, and a recombinant bacterium that highly expresses the enzyme was provided. The method of the present invention is industrially advantageous because an optically active amine can be synthesized using an imine derivative that can be synthesized at low cost as a substrate and utilizing the high stereoselectivity of microorganisms. The present invention is particularly useful for the production of (S) -2-methylpyrrolidine. (S) -2-methylpyrrolidine is a compound useful as an intermediate for synthesizing pharmaceuticals. According to the present invention, it is possible to easily and efficiently produce the compound by the action of microorganisms and enzymes, and to achieve a very high optical purity. The present invention has a remarkable effect of producing a compound with a high optical purity of 97.7% ee.

It is the figure which showed the process of construction | assembly of the plasmid (pSU-SRI1) in which the C4EPL6_STRRS imine reductase gene was inserted. It is the figure which showed the process of construction of the plasmid (pSG-SRI1) in which the C4EPL6_STRRS imine reductase gene and the glucose dehydrogenase gene derived from Bacillus subtilis were inserted. It is the figure which showed the process of construction of the plasmid (pSUS2MP1) in which the SsSIR imine reductase gene was inserted. It is the figure which showed the process of construction of the plasmid (pSG-S2MP) in which the SsSIR imine reductase gene and the glucose dehydrogenase gene derived from Bacillus subtilis were inserted.

式（ＩＩ）

（式中、Ｒ１、Ｒ２基は炭素数１〜３個のアルキル基を、Ｒ３基は、炭素数１〜３個のアルキル基もしくは水素を表し、Ｒ１とＲ３は環を巻いていてもよい）。 The present invention relates to an imine derivative represented by the formula (I), an enzyme protein (protein of SEQ ID NO: 1 or a variant thereof) having the ability to reduce the compound in a stereoselective manner, and a microorganism that expresses the protein. A step of contacting at least one enzyme active substance selected from the group consisting of a culture, a microbial cell and a processed product thereof, and a step of recovering an optically active S-form amine derivative represented by the formula (II) And an optically active S-form amine derivative.
Formula (I)

Formula (II)

(Wherein R1 and R2 groups represent an alkyl group having 1 to 3 carbon atoms, R3 group represents an alkyl group having 1 to 3 carbon atoms or hydrogen, and R1 and R3 may be wound in a ring) .

（式中、Ｒ基は炭素数１〜３個のアルキル基を、ｎは、１〜４の整数を表す）。 In the present invention, cyclic imine compounds are preferred as the compounds of formula (I). In the present invention, the cyclic imine compound includes, for example, an imine derivative represented by the following formula (III).
Formula (III)

(In the formula, R represents an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 1 to 4).

（式中、Ｒ基は炭素数１〜３個のアルキル基を、ｎは、１〜４の整数を表す）。 When the compound of the above formula (III) is used as a substrate, a compound represented by the formula (IV) can be obtained as an optically active amine derivative. The compound is a preferred optically active amine derivative that can be prepared according to the present invention.
Formula (IV)

(In the formula, R represents an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 1 to 4).

A preferable compound in the present invention is a compound in which, in the formula (III), R is, for example, a methyl group, an ethyl group, or a propyl group, and n is 2 to 3. More specifically, the following compounds can be shown as preferred imine derivatives in the present invention.
2-methyl-1-pyrroline 2-methyl-1-piperidein 2-ethyl-1-pyrroline 2-ethyl-1-piperidein

The following optically active amine derivatives can be produced using these compounds as substrates.
2-methyl-1-pyrroline → (S) -2-methylpyrrolidine 2-methyl-1-piperidein → (S) -2-methylpiperidine 2-ethyl-1-pyrroline → (S) -2-ethylpyrrolidine 2- Ethyl-1-piperidein → (S) -2-ethylpiperidine

  In the present invention, the ability to stereoselectively reduce an imine derivative refers to the ability to produce an S-form amine derivative using the imine derivative represented by the formula (I), preferably the formula (III) as a substrate. As the microorganism of the present invention, any microorganism can be used as long as it can express the imine reductase of SEQ ID NO: 1 or a variant thereof and can produce an S-form amine derivative. The microorganism used in the present invention can be obtained, for example, by examining the presence or absence of the imine reductase of SEQ ID NO: 1 or a variant thereof and the ability to produce an S-form amine derivative with respect to the following microorganisms.

  For example, the test microorganism is cultured in a medium containing an imine derivative represented by compound (I), preferably compound (III), and the optical purity of the optically active amine derivative produced in the culture is measured. As a result, if the production of an optically active (S) -amine derivative can be confirmed, the microorganism can be used in the present invention.

  Alternatively, test microorganisms previously grown in a medium are collected and suspended in an appropriate buffer. Further, the optical purity of the optically active amine derivative produced in this buffer solution is measured by contacting and reacting with the compound (I), preferably the imine derivative represented by the compound (III). If the production of an optically active amine derivative can be confirmed, the microorganism can be used in the present invention. At this time, if compound (I), preferably compound (III) is added to the medium during the cultivation of the test microorganism, induction of an enzyme for reducing this compound can be expected. If a reducing energy source is added during the reaction, more effective product accumulation can be expected. As the reducing energy source, glucose or the like can usually be used.

For example, if necessary, the microorganism is cultured in an appropriate medium containing 2-methyl-1-pyrroline, and the resulting microbial cell is treated with 10 mM 2-methyl-1-pyrroline, 25 mM potassium phosphate buffer (pH 7. 0), and if necessary, suspended in a reaction solution containing glucose, shaken at 25 ° C. for 24 hours, and analyzed the product by TLC or HPLC to confirm the ability of microorganisms to reduce 2-methyl-1-pyrroline. can do.
Examples of TLC conditions include a method of developing with a developing solution of 1-butanol / acetic acid / water = 2/1/1 using a Silica gel 60 F254 plate and coloring with ninhydrin.

The optical purity of the produced amine derivative can be measured, for example, by the following method.
The following components are added to 50 μL of a test sample, and the amine produced by reacting at 40 ° C. for 1 hour is converted to GITC, separated and quantified by HPLC.
2 mg / mL triethylamine 50 μL, and 8 mg / mL 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC) 100 μL

As conditions for HPLC, the following conditions can be used, for example.
-HPLC column: Wakosil II 5C18 (4.6 mm x 150 mm) manufactured by Wako Pure Chemical Industries, Ltd.
-Eluent: 10 mM phosphate buffer (pH 2.5): methanol = 55:45
-Flow rate: 1 mL / min
Detection: UV absorption at 254 nm Under the above conditions, (R) -2-methylpyrrolidine is eluted at 26.3 minutes and (S) -2-methylpyrrolidine derivative is eluted at 24.9 minutes.
A similar method can be used to examine the ability of the isolated imine reductase of SEQ ID NO: 1 or a variant protein thereof to produce an S derivative amine derivative.

  Needless to say, it is advantageous to use a microorganism with higher selectivity in order to obtain a product with high optical purity. Specifically, a microorganism capable of producing an optically active S-form amine derivative having a stereoselectivity of, for example, 60%, usually 70% or more, preferably 80% or more, more preferably 90% or more is used. Can do. As disclosed in the Examples of the present application, an optical purity (enantiomeric excess; ee) of 97.7% ee can be achieved by using a microorganism that expresses the enzyme of SEQ ID NO: 1.

  The “optically active amine derivative” in the present invention refers to an amine derivative in which one optical isomer is contained more than another optical isomer. In the present invention, a preferred optically active amine derivative has an optical purity (ee) of, for example, 60%, usually 70% or more, preferably 80% or more, and more preferably 90% or more. The optical purity of the optically active amine derivative can be confirmed using, for example, an optical resolution column. The “optical isomer” of the present invention may be generally referred to as “optically active form” and “enantiomeric”.

As disclosed in a previous international application (PCT / JP2009 / 065244) by the present applicant, for example, a microorganism belonging to the genus Streptomyces reduces the imine derivative represented by the above formula (I) and forms S form. To produce an amine derivative of Such microorganisms can be used for comparative analysis of the performance of the imine reductase of the present invention. It is also conceivable to introduce a gene encoding the enzyme of SEQ ID NO: 1 into such a microorganism.
More specifically, the following microorganisms can be shown as microorganisms belonging to the genus Streptomyces. This microorganism is a microorganism that produces a high optical purity (S) amine derivative.
Streptomyces sp. GF3546 (Streptomyces sp. GF3546)

The above microorganisms are deposited as follows (date of deposit; June 24, 2008).
(1) Name of depositary institution: National Institute of Technology and Evaluation (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
(3) Identification display and accession number:
Streptomyces sp. GF3546 (Accession number NITE P-593)

Furthermore, these microorganisms have been transferred to international deposits based on the Budapest Treaty as follows.
(1) International Depositary Authority: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2) Address: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
(3) Identification display and accession number Streptomyces sp. GF3546 (Streptomyces sp. GF3546)
; Accession number NITE BP-593 (Transfer date: August 4, 2009)

Microorganisms having the ability to stereoselectively reduce the imine derivative represented by the formula (I) or (III) can be found not only from these deposited strains but also from various microbiological resources. Microbial resources include microbial populations isolated from the natural environment, microbial strains stored in microbial depositories, and the like. Among them, a microorganism belonging to the genus Streptomyces is preferable as a microorganism having the ability to reduce the imine derivative represented by the formula (I) or (III) in a stereoselective manner. Therefore, for example, a microorganism having the target action can be selected by the selection method as described above for Streptomyces microorganisms stored in a microorganism depository. Deposited microorganisms can be sold, for example, from the following depository institutions.
Biological Resource Center (NBRC)
RIKEN (JCM)
Tokyo University of Agriculture strain storage room (IAM)
American Type Culture Collection (ATCC)
Deutsche Sammlung von Mikroorganismen (DSM)

A useful microorganism can also be selected based on the base sequence information of ribosomal DNA (rDNA). In general, it is widely known that rDNA base sequence information is useful as an index representing genetic proximity between organisms. In particular, DNA encoding 16S rRNA (hereinafter referred to as “16S rDNA”) is utilized as a tool for determining the genetic identity or similarity of microorganisms.
16S rRNA exists in all living organisms, and structural differences are observed between organisms, but the sequence is conserved to such an extent that alignment is possible. Since the frequency of horizontal propagation of 16S rRNA between organisms is low, a method used for classification is widely used.
Specifically, small subunit ribosomal RNA (SSU rRNA) is one of the nucleic acid molecules constituting the ribosome, which is the field of protein synthesis. Although the length of the base sequence is slightly different, the origin is considered to be the same from bacteria to human. Therefore, the base sequence of rRNA has high evolutionary conservation and is most frequently used in phylogenetic analysis of organisms (Microbiol. Rev., 58, 1-9 (1994)). SSU rRNA in prokaryotes is about 1500 base 16S rRNA. Phylogenetic classification using the base sequence of 16S rRNA is generally used for classification and identification of prokaryotes (ASM News, 65, 752-757 (1999), Biotechnology Experiments p113, Japanese Society for Biotechnology, Bafukan).
The current actinomycete classification system is constructed based on the similarity of 16S rDNA base sequences (Stackebrandt, et al. (1997) Int. J. Syst. Bacteriol., 47, 479-491). In the identification of strains, identification based on the homology of the 16S rDNA nucleotide sequence is not only at the genus level but also at the species level. Society) p19).

  By searching the base sequence information database for these base sequence information, a strain containing a highly identical base sequence in 16S rDNA can be found out of microorganisms for which 16S rDNA has already been determined. For example, public database service organizations such as DNA Data Bank of Japan (DDBJ) provide a search service for 16S rRNA (Prokaryotes) base sequence information. By selecting microorganisms belonging to the genus Streptomyces from the microorganisms thus found, microorganisms that can be used for comparative analysis, transformation, and the like with the imine reductase of the present invention can be obtained. If necessary, the action of the thus selected microorganism on the imine derivative can be confirmed in advance.

The microorganism to be used can be cultured according to a method known in the field of fermentation. As the medium, any of a synthetic medium or a natural medium can be used as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance and other nutrients. As the medium, a liquid medium or a solid medium can be used.
Specifically, as a carbon source, one or two or more types are appropriately selected from the following general carbon sources in consideration of the assimilation of microorganisms to be used.
Sugars: Natural carbohydrates:
Glucose, starch,
Fructose, starch hydrolysate,
Maltose, molasses,
Galactose, molasses,
wheat,
Corn, etc. Alcohols: Organic acids:
Glycerol, acetic acid,
Methanol, gluconic acid,
Such as ethanol, pyruvic acid,
Ketoglutaric acid,
Hydrocarbons such as citric acid:
Fats such as normal paraffin:
Palm kernel oil,
Soybean oil,
Olive oil, etc.

As the nitrogen source, one or more kinds of nitrogen sources are appropriately selected from the following general nitrogen sources in consideration of the assimilation of microorganisms to be used.
Organic nitrogen compounds:
Meat extract, peptone, yeast extract,
Soy hydrolyzate, milk casein, casamino acid,
Various amino acids, corn steep liquor,
Inorganic nitrogen compounds such as other animal, plant and microbial hydrolysates:
Ammonia, ammonium nitrate, ammonium sulfate,
Ammonium salts such as sodium chloride,
Nitrate such as sodium nitrate, urea, etc.

In addition, an inducer can be used to enhance the stereoselective reduction ability of the microbial imine derivative. As the inducer, an imine derivative can be used depending on the microorganism to be used.
Furthermore, as an inorganic salt, one or two or more kinds can be appropriately selected and used from a trace amount of magnesium, manganese, potassium, calcium, sodium, copper, zinc and other phosphates, hydrochlorides, nitrates, acetates and the like. . Moreover, you may add antifoaming agents, such as vegetable oil, surfactant, and silicone, in a culture solution as needed.

Culturing can be performed in a liquid medium containing the above-mentioned medium components using a normal culture method. For example, the following culture method can be used.
Shaking culture
Aeration-agitation culture
Continuous culture
Feeding culture, fed batch culture

The culture conditions can be appropriately selected depending on the type of microorganism, the type of culture, and the culture method. There is no particular limitation as long as the strain to be used grows and can have the stereoselective reduction ability of the imine derivative. The following conditions can be shown as general culture conditions.
The pH at the start of the culture is adjusted to 4 to 10, preferably 6 to 8. Cultivation is carried out at a temperature of 15 to 70 ° C, preferably 20 to 40 ° C. It is not particularly limited if possible. Usually, the cells are cultured for 1 to 14 days, preferably 1 to 7 days.

  In the present invention, the treated product of microbial cells means a product obtained by treating the cells under conditions that maintain the target enzyme activity. The maintenance of the enzyme activity means that the enzyme activity obtained from viable cells is generally maintained at 20% or more, usually 30% or more, preferably 50% or more, more preferably 70% or more. The enzymatic activity of the living microbial cell and its treated product can be quantitatively compared by comparing the enzymatic activity of the living microbial cell with the enzymatic activity of the treated product obtained from the same amount of living microbial cell. The treated product in the present invention may have an enzyme activity higher than that of viable cells.

  As a method for obtaining such a treated product, freeze-drying, dehydration drying with an organic solvent, self-digestion of bacterial cells, extraction of enzyme active fraction, sonication and the like can be used. By such a treatment method, for example, freeze-dried microbial cells, acetone-dried microbial cells, microbial cell autolysates, microbial cell extracts, microbial cell pulverized materials, sonicated microbial cells, and the like can be obtained.

  These processes can be applied singly or by overlapping a plurality of processes. For example, after culturing a microorganism with an appropriate culture solution, the cells can be disrupted and a cell-free extract can be prepared by centrifugation or the like. The cells can be crushed by mechanical crushing, ultrasonic waves, high-pressure treatment, enzyme digestion, autolysis, and the like. The cell-free extract is included in the treated product of the microbial cells in the present invention.

In the present invention, a partially purified or completely purified enzyme from the microorganism expressing the imine reductase of SEQ ID NO: 1 or a variant thereof can be used. Similar to the imine reductase disclosed in the international application (PCT / JP2009 / 065244), the imine reductase of the present invention has the physicochemical properties shown in the following (1) to (2).
(1) Action: 2-methyl-1-pyrroline is reduced in a NADPH-dependent manner to produce (S) -2-methylpyrrolidine. Depending on NADP +, (S) -2-methylpyrrolidine is oxidized to produce 2-methyl-1-pyrroline; and (2) Coenzyme dependency: NADPH is used as a coenzyme in the reduction reaction. NADP + is used as a coenzyme for the oxidation reaction.

The imine reductase of the present invention can be purified from a microorganism expressing the imine reductase of SEQ ID NO: 1 or a variant thereof, or a cell-free extract thereof. Specifically, substantially pure imine reductase can be obtained from a cell-free extract by appropriately combining the following purification steps.
Splitting by solubility using ammonium sulfate, acetone, etc.,
Ion exchange chromatography,
Hydrophobic chromatography,
Affinity chromatography using 2 ′, 5′-ADP sepharose, blue or red sepharose,
Gel filtration

For example, after collecting microorganisms, the imine reductase of the present invention can be isolated as a substantially pure enzyme through the following purification steps.
Disruption of cells with ultrasound or glass beads and preparation of cell-free extract,
Isolation of imine reductase activity fractions by hydrophobic chromatography using Phenyl Sepharose and ion exchange chromatography using MonoQ

  In the present invention, “substantially pure” means that the enzyme is substantially free of biological polymer compounds and chemical substances other than the enzyme. Biological polymer compounds other than the enzyme include, for example, proteins, saccharides, lipids, and nucleic acids derived from bacterial cells and culture solutions. The substantially pure enzyme in the present invention has a purity of at least 75% or more, usually 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, or 99% or more. Methods for determining enzyme purity are known. For example, methods for determining protein purity by various types of chromatography and polyacrylamide gel electrophoresis are well known.

  Alternatively, the present invention provides an isolated imine reductase having the physicochemical properties (1) and (2). In the present invention, an isolated imine reductase means that it is separated from the natural environment in which it exists. Therefore, the imine reductase separated from the cells is included in the isolated imine reductase in the present invention.

  The substantially pure imine reductase or the isolated imine reductase in the present invention may be purified or isolated and then made into an enzyme composition. For example, an enzyme composition containing the imine reductase of the present invention can be prepared by combining purified or isolated imine reductase with a suitable carrier. As the carrier, an inert protein such as albumin, a saccharide such as sucrose, a sugar alcohol, or the like can be used. The enzyme composition can be liquid or in a dry state. An enzyme composition obtained by freeze-drying an aqueous solution containing an enzyme and a carrier is one of the preferred compositions in the present invention. A composition containing such a protein having imine reducing activity can be used as a stereoselective imine reducing agent. That is, the stereoselective imine reducing agent of the present invention contains a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and / or a variant thereof as an active ingredient.

The activity of the imine reductase of the present invention can be quantified by the following method. That is, the reaction is carried out at 30 ° C. in a reaction solution having the following composition (1 mL), and the decrease in absorbance at 340 nm resulting from the decrease in NADPH is measured.
10 mM 2-methyl-1-pyrroline,
100 mM potassium phosphate buffer (pH 7.5),
0.1 mM NADPH and 1 U of enzyme were used as the amount of enzyme that catalyzes the decrease of 1 μmol of NADPH per minute under the above conditions.

  The present invention relates to a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. The present invention also includes a protein homolog (also referred to herein as a variant) consisting of the amino acid sequence set forth in SEQ ID NO: 1. The imine reductase homologue (variant) of the present invention comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 1. A protein functionally equivalent to the protein consisting of the amino acid sequence described in 1. In the amino acid sequence shown in SEQ ID NO: 1, for example, mutation of amino acid residues of 100 or less, usually 50 or less, preferably 30 or less, more preferably 15 or less, still more preferably 10 or less, or 5 or less is allowed. That is, the imine reductase of the present invention contains, for example, mutations of 1 or more, 5 or more, 10 or more, 30 or more, 50 or more, or 100 or more amino acid residues with respect to the amino acid sequence shown in SEQ ID NO: 1. sell.

  The present invention relates to a polynucleotide encoding an imine reductase and a homolog (variant) thereof. In the present invention, the polynucleotide can be an artificial molecule including an artificial nucleotide derivative in addition to a natural polynucleotide such as DNA or RNA. The polynucleotide of the present invention can also be a DNA-RNA chimeric molecule. The polynucleotide encoding the imine reductase of the present invention includes, for example, the base sequence shown in SEQ ID NO: 2. The base sequence shown in SEQ ID NO: 2 encodes a protein containing the amino acid sequence shown in SEQ ID NO: 1, and the protein containing this amino acid sequence constitutes a preferred embodiment of the imine reductase according to the present invention.

  The polynucleotide homologue (variant) encoding the imine reductase of the present invention is an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence set forth in SEQ ID NO: 1. And a polynucleotide encoding a protein having the physicochemical properties (1) to (2). Those skilled in the art will be able to introduce site-directed mutagenesis into the polynucleotide of SEQ ID NO: 2 (Nucleic Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular. Cloning 2ndEdt., Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach IRL Press pp.200 (1991)), etc. to introduce polymorphs by introducing substitutions, deletions, insertions, and / or addition mutations as appropriate. Nucleotide homologues can be obtained.

  In addition, the polynucleotide homologue (variant) in the present invention is a polynucleotide that can hybridize under stringent conditions with the polynucleotide comprising the base sequence shown in SEQ ID NO: 2, and has the physicochemical properties (1) Also included are polynucleotides that encode proteins having (2) to (2). The polynucleotide capable of hybridizing under stringent conditions is one or more selected from any of at least 20, preferably at least 30, for example, 40, 60, or 100 consecutive sequences in SEQ ID NO: 2. The probe DNA is used as a probe DNA and hybridized under the conditions described in the manual (wash: 42 ° C, primary wash buffer containing 0.5 x SSC) using, for example, ECL direct nucleic acid labeling and detection system (manufactured by GE Healthcare). Refers to a polynucleotide.

  Furthermore, the homologue (variant) of the polynucleotide in the present invention is at least 70%, alternatively at least 75%, preferably at least 80%, alternatively at least 85%, more preferably 90% or more with the amino acid sequence shown in SEQ ID NO: 1. Or a polynucleotide encoding a protein having 95% or more identity. Protein identity searches include databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, and DAD, databases related to DNA sequences such as DDBJ, EMBL, and Gene-Bank, and databases related to predicted amino acid sequences based on DNA sequences. For example, it can be performed through the Internet using programs such as BLAST and FASTA.

  The present invention relates to a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. The present invention also includes a protein homolog (variant) consisting of the amino acid sequence set forth in SEQ ID NO: 1. The homologue of the imine reductase of the present invention consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence described in SEQ ID NO: 1, and is described in SEQ ID NO: 1. A protein functionally equivalent to a protein consisting of the amino acid sequence of In the present invention, being functionally equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 means that the protein has the physicochemical properties shown in (1) to (2) above. Those skilled in the art will be able to introduce site-directed mutagenesis into the DNA of SEQ ID NO: 2 (Nucleic Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning 2ndEdt. , Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach IRL Press pp.200 (1991)), etc., to introduce imine reductase by appropriately introducing substitution, deletion, insertion, and / or addition mutation A polynucleotide encoding the homologue of can be obtained. By introducing a polynucleotide encoding the homologue of the imine reductase into a host and expressing it, it is possible to obtain the imine reductase homologue described in SEQ ID NO: 1.

  Further, the homologue (variant) of the imine reductase of the present invention is at least 70%, alternatively at least 75%, preferably at least 80%, alternatively at least 85%, more preferably 90, with the amino acid sequence shown in SEQ ID NO: 1. % Or more, or a protein having 95% or more identity. Protein identity searches include databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, and DAD, databases related to DNA sequences such as DDBJ, EMBL, and Gene-Bank, and databases related to predicted amino acid sequences based on DNA sequences. For example, it can be performed through the Internet using programs such as BLAST and FASTA.

  The polynucleotide encoding the imine reductase of the present invention can be isolated by, for example, the following method.

  Primers for PCR are designed based on the base sequence described in SEQ ID NO: 2, and the chromosome of an enzyme-producing strain (a strain of the genus Streptosporandium such as Streptosporanium roseum, for example, Streptosporandium roseum DSM 43021) The DNA of the present invention can be obtained by performing PCR using DNA or a cDNA library as a template.

  Furthermore, using the obtained DNA fragment as a probe, a restriction enzyme digest of a chromosomal DNA of an enzyme producing strain is introduced into a phage, plasmid, etc., and a library or cDNA library obtained by transforming E. coli is used. The polynucleotide of the present invention can be obtained by colony hybridization, plaque hybridization and the like.

  In addition, the base sequence of the DNA fragment obtained by PCR is analyzed, and from the obtained sequence, a PCR primer for extending outside the known DNA is designed, and the chromosomal DNA of the enzyme production strain is transformed with an appropriate restriction enzyme. After digestion, by performing reverse PCR using DNA as a template by a self-cyclization reaction (Genetics 120, 621-623 (1988)), the RACE method (Rapid Amplification of cDNA End, “PCR Experiment Manual” p25-33, It is also possible to obtain the polynucleotide of the present invention by HBJ Publishing Bureau).

Polynucleotides that can be used in the present invention include genomic DNA cloned by the method as described above, or cDNA, as well as DNA obtained by synthesis.
An imine reductase expression vector is provided by inserting the thus isolated polynucleotide encoding the imine reductase according to the present invention into a known expression vector.

  In addition, the imine reductase of the present invention can be obtained from a recombinant by culturing a transformant transformed with this expression vector.

  In order to express imine reductase in the present invention, a microorganism to be transformed is transformed with a recombinant vector containing a polynucleotide encoding a polypeptide having imine reductase and expresses imine reductase activity. There is no particular limitation as long as it is an organism that can be used. Examples of the microorganisms that can be used include the following microorganisms.

Host vector system such as Escherichia genus Bacillus genus Pseudomonas genus Serratia genus Brevibacterium genus Corynebacterium genus Streptococcus genus Lactobacillus genus Bacteria Rhodococcus spp. Streptomyces spp. Developed host vector systems such as Streptomyces spp. Saccharomyces spp. Kluyveromyces spp. Schizosaccharomyces spp. Yeast neurospo that have been developed for host vector systems such as Zygosaccharomyces genus Yarrowia genus Trichosporon genus Rhodosporidium genus Pichia genus Candida genus (Neurospora) genus Aspergillus (Aspergillus) genus Cephalosporium (Cephalosporium) genus Trichoderma (Trichoderma) mold that has been developed host vector system such as the genus

  A procedure for producing a transformant and construction of a recombinant vector suitable for the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al., Molecular cloning, Cold Spring Harbor Laboratories). In order to express the imine reductase gene using NADPH of the present invention as an electron donor in microorganisms, the DNA is first introduced into a plasmid vector or a phage vector that is stably present in the microorganism, and the genetic information thereof is introduced. Must be transcribed and translated.

  For that purpose, a promoter corresponding to a unit that controls transcription / translation may be incorporated upstream of the 5′-side of the DNA strand of the present invention, and more preferably a terminator may be incorporated downstream of the 3′-side. As the promoter and terminator, it is necessary to use a promoter and terminator known to function in a microorganism used as a host. Regarding the vectors, promoters, and terminators that can be used in these various microorganisms, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan”, especially regarding yeast, Adv. Biochem. Eng. 43, 75-102 (1990), Yeast 8, 423-488 (1992), etc.

  For example, in the genus Escherichia, particularly Escherichia coli, pBR and pUC plasmids can be used as plasmid vectors, and lac (β-galactosidase), trp (tryptophan operon), tac, trc (lac, trp). Promoters derived from λ phage PL, PR, and the like. Moreover, as a terminator, a terminator derived from trpA, phage, or rrnB ribosomal RNA can be used. Among these, a vector pSE420D (described in JP-A 2000-189170) obtained by partially modifying a multicloning site of commercially available pSE420 (manufactured by Invitrogen) can be suitably used.

  In the genus Bacillus, pUB110-type plasmids, pC194-type plasmids and the like can be used as vectors, and can be integrated into chromosomes. Further, apr (alkaline protease), npr (neutral protease), amy (α-amylase) and the like can be used as a promoter and terminator.

  In the genus Pseudomonas, host vector systems have been developed for Pseudomonas putida, Pseudomonas cepacia, and the like. A broad host range vector (including genes necessary for autonomous replication derived from RSF1010) pKT240 based on the plasmid TOL plasmid involved in the degradation of toluene compounds can be used, and a lipase (special) can be used as a promoter and terminator. (Kaihei 5-284773) genes and the like can be used.

  In the genus Brevibacterium, particularly in Brevibacterium lactofermentum, plasmid vectors such as pAJ43 (Gene 39, 281 (1985)) can be used. As the promoter and terminator, promoters and terminators used in Escherichia coli can be used as they are.

  In the genus Corynebacterium, particularly Corynebacterium glutamicum, plasmid vectors such as pCS11 (Japanese Patent Laid-Open No. 57-183799) and pCB101 (Mol. Gen. Genet. 196, 175 (1984) are available. is there.

  In the genus Streptococcus, pHV1301 (FEMS Microbiol. Lett. 26, 239 (1985), pGK1 (Appl. Environ. Microbiol. 50, 94 (1985)), etc. can be used as a plasmid vector.

  In the genus Lactobacillus, pAMβ1 (J. Bacteriol. 137, 614 (1979)) developed for Streptococcus can be used, and those used in Escherichia coli as a promoter can be used. .

  In the genus Rhodococcus, a plasmid vector isolated from Rhodococcus rhodochrous can be used (J. Gen. Microbiol. 138,1003 (1992)).

  In the genus Streptomyces, plasmids can be constructed according to the method described in Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory Manual Cold Spring Harbor Laboratories (1985). In particular, in Streptomyces lividans, pIJ486 (Mol. Gen. Genet. 203, 468-478, 1986), pKC1064 (Gene 103,97-99 (1991)), pUWL-KS (Gene 165,149). -150 (1995)) can be used. The same plasmid can also be used in Streptomyces virginiae (Actinomycetol. 11, 46-53 (1997)).

  In the genus Saccharomyces (especially Saccharomyces cerevisiae), YRp, YEp, YCp, and YIp plasmids can be used, and homologous recombination with ribosomal DNA existing in multiple copies in the chromosome is used. The integration vector (EP537456 and the like) is very useful because it can introduce a gene in multiple copies and can stably hold the gene. ADH (alcohol dehydrogenase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PHO (acid phosphatase), GAL (β-galactosidase), PGK (phosphoglycerate kinase), ENO (enolase) Promoters and terminators such as can be used.

  In Kluyveromyces genus, especially Kluyveromyces lactis, 2 μm-type plasmid derived from Saccharomyces cerevisiae, pKD1 series plasmid (J. Bacteriol. 145, 382-390 (1981)), involved in killer activity PGK11-derived plasmids, autonomously proliferating gene KARS-type plasmids in the genus Kleberomyces, vector plasmids (eg EP 537456 etc.) that can be integrated into the chromosome by homologous recombination with ribosomal DNA and the like can be used. In addition, promoters and terminators derived from ADH, PGK and the like can be used.

  In the genus Schizosaccharomyces, plasmid vectors containing ARS (genes involved in autonomous replication) derived from Schizosaccharomyces pombe and selection markers that complement the auxotrophy derived from Saccharomyces cerevisiae are available ( Mol. Cell. Biol. 6, 80 (1986)). Moreover, the ADH promoter derived from Schizosaccharomyces pombe can be used (EMBO J. 6, 729 (1987)). In particular, pAUR224 is commercially available from Takara Bio and can be used easily.

  In the genus Zygosaccharomyces, plasmid vectors derived from pSB3 (Nucleic Acids Res. 13, 4267 (1985)) derived from Zygosaccharomyces rouxii can be used, and the PHO5 promoter derived from Saccharomyces cerevisiae In addition, a promoter of GAP-Zr (glyceraldehyde-3-phosphate dehydrogenase) derived from Tigosaccharomyces rouxi (Agri. Biol. Chem. 54, 2521 (1990)) can be used.

  In the genus Pichia, a host vector system has been developed in Pichia Angusta (formerly Hansenula polymorpha). As vectors, genes involved in autonomous replication derived from Pichia Angusta (HARS1, HARS2) can be used, but since they are relatively unstable, multicopy integration into chromosomes is effective (Yeast 7, 431- 443 (1991)). Further, AOX (alcohol oxidase), FDH (formic acid dehydrogenase) promoter, etc. induced by methanol or the like can be used. In addition, host vector systems utilizing genes (PARS1, PARS2) involved in Pichia-derived autonomous replication have been developed for Pichia pastoris (Mol. Cell. Biol. 5, 3376 (1985)). Strong promoters such as high concentration culture and methanol-inducible AOX can be used (Nucleic Acids Res. 15, 3859 (1987)).

  In the genus Candida, host vector systems in Candida maltosa, Candida albicans, Candida tropicalis, Candida utilis, etc. Has been developed. In Candida maltosa, ARS derived from Candida maltosa has been cloned (Agri. Biol. Chem. 51, 51, 1587 (1987)), and vectors using this have been developed. In Candida utilis, a strong promoter has been developed for the chromosome integration type vector (Japanese Patent Laid-Open No. 08-173170).

  In the genus Aspergillus, Aspergillus niger and Aspergillus oryzae are the most studied among molds, and integration into plasmids and chromosomes is available. Promoters derived from external proteases or amylases are available (Trends in Biotechnology 7, 283-287 (1989)).

  In the genus Trichoderma, a host vector system using Trichoderma reesei has been developed, and an extracellular cellulase gene-derived promoter or the like can be used (Biotechnology 7, 596-603 (1989)).

  In addition to microorganisms, various host and vector systems have been developed in plants and animals, especially in insects using moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes and other plants. A system for expressing a large amount of heterologous protein has been developed and can be suitably used.

The microorganisms having imine reductase producing ability used in the present invention are all strains belonging to the genus Streptosporanium having the ability to produce imine reductase, mutant strains, variants, gene manipulation techniques of the present invention. Includes transformed strains that have acquired the ability to produce enzymes. Examples of the strain belonging to the genus Streptosporandium having the ability to produce an imine reductase include the following strains.
Streptosporangium roseum DSM 43021
Streptosporandium Amethyst Genes Sub sp. Amethystogenes NBRC 13986 (Streptosporangium amethystogenes subsp. Amethystogenes)
Streptosporangium canum NBRC 106320
Streptosporangium longisporum NBRC 13141
Streptosporangium vulgare NBRC 13985
Particularly preferred is Streptosporangium roseum DSM 43021.
The strains with DSM numbers can be obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), and the strains with NBRC numbers can be obtained from the National Institute of Technology and Evaluation, Biological Genetic Resources Division. You can get it.

The method for producing an optically active amine derivative of the present invention comprises an enzyme active substance having imine reductase activity having the ability to stereoselectively reduce a substrate compound represented by formula (I) or formula (III). Contacting the substrate compound under conditions that allow the substrate compound to be reduced by the substance. In the present invention, an enzyme active material means an enzyme protein having the ability to reduce the substrate compound represented by formula (I) or formula (III) in a stereoselective manner (the protein of SEQ ID NO: 1 or its protein). Variant), or a substance derived from a microorganism that expresses the protein, the substance maintaining its ability. Specifically, for example, the following elements are included in the enzyme active substance in the present invention as long as the imine reductase activity is maintained.
i) the protein of SEQ ID NO: 1 having the ability to stereoselectively reduce the substrate compound represented by formula (I) or formula (III) or a variant thereof
ii) Microorganisms expressing the protein of i)
iii) Culture of microbial cells of ii)
iv) Processed microbial cells of ii)

  In the present invention, the microorganism can be a culture or a microbial cell. A culture refers to a state in which the microorganism is present together with a medium that supports the survival and growth of the microorganism. For example, a liquid medium containing microorganisms is a culture. On the other hand, in the present invention, “microbial cell” is used as a term indicating the microorganism itself. A microbial cell refers to the microbial cell collect | recovered from the culture. For example, microbial cells can be recovered from the liquid medium by centrifugation. By washing the cells recovered from the culture, the medium components on the surface of the cells can be removed.

In the present invention, the enzyme active substance can be brought into contact with the substrate compound as it is or immobilized. Various methods are known for immobilizing microbial cells or processed products thereof. For example, the following methods are known as immobilization methods for microorganisms and processed products thereof.
Polyacrylamide method Sulfur-containing polysaccharide gel method (κ-carrageenan gel method, etc.)
Alginate gel method Agar gel method Ion exchange resin method Alternatively, in a membrane bioreactor using an ultrafiltration membrane or the like, a microorganism and its treated product can be brought into contact with a substrate compound to be reacted.

  The stereoselective imine reduction reaction by the microorganism in the present invention is performed by culturing the microorganism under an appropriate culture condition in which the enzyme is induced, and the obtained culture solution, or the cells collected from the culture solution or the cells. It can be carried out by adding an imine derivative as a reaction substrate to the treated product and incubating. Further, an imine stereoselective reduction reaction can be carried out while culturing by adding an imine derivative as a substrate at the start of culture of the microorganism or during the culture.

In the stereoselective reduction method using a microorganism in the present invention, the microorganism is cultured under an appropriate culture condition in which an enzyme is induced, and the obtained culture solution, or the cells collected from the culture solution or the treated cells thereof are used. A stereoselective reduction reaction can be performed by adding a reaction substrate. Further, it can be carried out in parallel with the culture for 1 to 7 days at the same pH and temperature range as the culture conditions.
As reaction conditions, the following conditions can be shown, for example.
pH 4.0 to 9.0, preferably pH 6.0 to 8.0,
Temperature 15 to 50 ° C., preferably 20 to 40 ° C.,
The reaction time is contacted for 4 hours to 7 days.
In general, if the culture and reaction of microorganisms are performed separately, it is possible to minimize the introduction of impurities in the medium into the reaction system. As a result, advantageous results can be expected in the purification of the product after the reaction.

In the enzymatic reduction reaction, a more efficient reaction can be expected by performing the reaction in the presence of saccharides or the like as a source of reduction energy. The compound added as a reducing energy source can be added according to the amount of the imine derivative as a reaction substrate. More specifically, the following reducing energy sources can be used.
Sugars: Glucose, glucose-6-phosphate, sucrose, etc. Organic acids: Malic acid, citric acid, formic acid, etc. Amino acids: Alanine, glutamic acid, lysine, etc. Alcohol: Ethanol, isopropanol, etc. Other inorganic compounds such as phosphorous acid May be available as

  The reduction reaction in the present invention proceeds in the presence of a hydrogen donor. Therefore, the conditions under which the substrate compound can be reduced in the present invention can be provided by incubating the substrate compound and the enzyme active substance in the presence of a hydrogen donor. Any hydrogen donor can be used as long as it assists the reduction reaction in the present invention. A preferred hydrogen donor in the present invention is NADPH or NADH. When using the imine reductase derived from Streptosporandium roseum DSM 43021 and the homologues or enzyme active substances that have been successfully isolated by the present inventors, NADPH is preferably used.

  The regeneration of NADP + produced from NADPH in association with the above reduction reaction to NADPH can be performed using NADP + reducing ability (glycolytic system, methylotrophic C1 compound utilization pathway, etc.) possessed by microorganisms. These NADP + reducing ability can be enhanced by adding glucose, ethanol, or the like to the reaction system. Moreover, it can also carry out by adding the microorganisms which have the capability to produce | generate NADPH from NADP +, its processed material, and an enzyme to a reaction system. For example, microorganisms containing glucose dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase, etc.), processed products thereof, and partially purified or purified enzymes can be used for NADPH. Playback can be performed. The components constituting the reaction necessary for NADPH regeneration are added to the reaction system for the production of the optically active amine according to the present invention, the immobilized one is added, or the membrane can be exchanged for NADPH. Can be contacted.

  In addition, when a living cell of a microorganism transformed with a recombinant vector containing the DNA of the present invention is used in the method for producing an optically active amine, an additional reaction system for NADPH regeneration can be eliminated. There is. That is, by using a microorganism having high NADPH regeneration activity, an efficient reaction can be performed without adding an enzyme for NADPH regeneration in a reduction reaction using a transformant. Furthermore, genes such as glucose dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase, etc.) that can be used for NADPH regeneration encode the imine reductase of the present invention. By introducing it into the host simultaneously with DNA, it is possible to perform more efficient simultaneous expression of an NADPH regenerating enzyme and an imine reductase and an imine reduction reaction. In order to introduce these two or more genes into the host, a method of transforming the host with a recombinant vector in which genes are separately introduced into a plurality of vectors different from the origin of replication in order to avoid incompatibility, A method of introducing both genes into a single vector, a method of introducing both genes into a chromosome, or the like can be used.

When introducing multiple genes into a single vector, methods such as linking promoter- and terminator-related regions related to expression control to each gene, or expressing as an operon containing multiple cistrons such as the lactose operon. Is possible.
For example, glucose dehydrogenase derived from Bacillus subtilis or Thermoplasma acidophilum can be used as an enzyme for NADPH regeneration, specifically, imine reductase and Bacillus subtilis For example, pSG-SRI1, which is a recombinant vector into which the gene for glucose dehydrogenase is introduced, is preferably used.

  If there is a change in the pH of the reaction solution due to the decomposition of the amine derivative, adjust the pH to a certain range using an appropriate acid or alkali, and maintain a better reactivity. Can be continued.

When viable cells are used in the present invention, the reaction time may be shortened if a surfactant is added to the reaction solution. The surfactant used for this purpose is not particularly limited as long as it increases the permeability of the cell wall of viable cells, and is cetylpyrimidinium bromide, cetyltrimethylammonium bromide, Triton X-100, paraisooctyl. Phenyl ether, Tween 80, Span 60, etc. are mentioned, and it is preferable to use about 0.0001 to 1% with respect to the reaction solution.
In addition, the same effect may be expected by adding an organic solvent to the reaction solution. The surfactant used for this purpose is not particularly limited as long as it increases the permeability of the cell wall of viable cells, and examples thereof include organic solvents such as toluene and xylene. It is preferable to use about 1%.
Alternatively, cells that have been improved in cell wall permeability by collecting cells and pretreating them with water or a buffer containing a surfactant and an organic solvent may be used without adding them to the reaction solution.

  The imine derivative, which is a reaction substrate in the present invention, can be used at an appropriate concentration so that the target product can be efficiently produced. The imine derivative may be toxic to the bacterial cells and the enzyme that catalyzes the reaction, and high concentration reaction may not always lead to efficient production, and therefore requires attention. The concentration of the imine derivative in the reaction solution can be, for example, 0.05 to 50% w / v, preferably 0.1 to 10% w / v. The imine derivative can be added by any method such as batch (batch method), divided addition (fed batch method) or continuous addition (feed method). Furthermore, without adding imine as a substrate, ketone and amine as imine raw materials can be added to the reaction solution to produce imine in the reaction solution and supplied as a substrate.

  The imine derivative to be added is prevented from being colored by being handled in an inert gas atmosphere, and an improvement in product quality can be expected. In anticipation of such an effect, the raw material can be added to the reaction solution in an inert gas atmosphere. Further, coloring can be prevented by carrying out the enzyme reaction in an inert gas atmosphere. As the inert gas used here, for example, nitrogen, argon, helium, or the like can be used. In particular, nitrogen is an inert gas that can be easily obtained. Alternatively, argon has a higher specific gravity than air and therefore does not diffuse easily. Therefore, even in an open operation, the environment under an inert gas atmosphere can be maintained, which is advantageous in terms of operation.

The present invention includes a step of recovering an optically active amine derivative obtained by contacting a substrate compound with an enzyme active substance. Specifically, the optically active amine derivative can be isolated from the reaction solution by combining various chromatographies and crystallization by stereoselective reduction of the imine derivative.
The optically active amine derivative accumulated in the reaction solution can be extracted with, for example, the following solvent.
Ethyl acetate,
Butyl acetate,
toluene,
Xylene,
Hexane,
Heptane,
Methyl isobutyl ketone,
Methyl-t-butyl ether,
Organic solvents such as halogens

The extracted amine derivative can be further purified by the following chromatography. Prior to purification, treatment with acid or alkali, dehydration with a dehydrating agent, or the like can be combined as necessary.
Ion exchange chromatography,
Adsorption chromatography

For example, the optically active amine derivative can be collected by removing insoluble substances such as bacterial cells from the reaction solution by centrifugation, followed by solvent extraction under alkaline conditions and concentration under reduced pressure.
The liquid from which insoluble substances such as bacterial cells have been removed is adsorbed on an ion exchange resin capable of adsorbing amine derivatives and eluted with an alkali such as ammonia water, sodium hydroxide aqueous solution, sodium hydroxide methanol solution or sodium hydroxide ethanol solution. Then, a highly concentrated solution can be obtained simply.
In addition, the liquid from which insoluble substances such as bacterial cells have been removed is adsorbed on an ion exchange resin or the like that can adsorb amine derivatives. If it is eluted, a high concentration solution can be easily obtained.
In addition, the optically active amine derivative can be collected as crystals by subjecting it to hydrochloric acid chloride with hydrochloric acid after solvent extraction. In order to increase the recovery rate, the solvent may be concentrated under reduced pressure, or the solvent may be replaced with a solvent having good crystallinity.
In order to increase the recovery rate, the solvent can be concentrated under reduced pressure, or the solvent can be replaced with a solvent having good crystallinity.

  Alternatively, the optical purity of the optically active amine derivative may be increased by collecting it as a salt crystal with an acid. After solvent extraction, the optically active amine derivative can be collected as crystals by chlorinating with hydrochloric acid. As an acid for purifying the salt, a mineral acid such as hydrochloric acid or sulfuric acid, or an organic acid such as mandelic acid, citric acid or tartaric acid can be used. The salt of the amine derivative thus obtained is dissolved in a strong acid or strong alkali, then neutralized with a suitable alkali or acid, and neutralized, and easily separated from other impurities. Can do. For the strongly acidic aqueous solution that dissolves the salt of the amine derivative, for example, a mineral acid such as hydrochloric acid or sulfuric acid can be used. Moreover, NaOH aqueous solution and ammonia water can be utilized for strong alkaline aqueous solution.

Moreover, it can be easily separated from other impurities by heating to an aqueous solution, cooling and recrystallization.
Furthermore, it can refine | purify further highly by performing the silica gel column chromatography which melt | dissolves the obtained crystal | crystallization in a small amount of water, and elutes with a suitable developing phase. For the developing phase of silica gel gel chromatography, for example, butanol, acetic acid, or a mixed solvent thereof with water can be used.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this.
Example 1 Cloning of imine reductase homolog C4EPL6_STRRS
PCR primers (SEQ ID NO: 3 and SEQ ID NO: 4) were synthesized based on the DNA sequence (SEQ ID NO: 2) corresponding to the predicted protein C4EPL6_STRRS (SEQ ID NO: 1) registered in EMBL. 50 pmol of each primer, dNTP 10 nmol, Streptosporangium roseum DSM 43021-derived chromosomal DNA (purchased from DSMZ) 50 ng, Prime STAR DNA polymerase buffer (TAKARA), Prime STAR DNA polymerase 2.5 U (TAKARA) 50 Using a μL reaction solution, 30 cycles of denaturation (98 ° C, 10 seconds), annealing (58 ° C, 5 seconds) and extension (72 ° C, 1 minute), using GeneAmp PCR System 9700 (Applied Biosystems) I went. The obtained DNA fragment was purified and recovered by GFX PCR DNA and Gel Band Purification Kit (GE Healthcare).
The recovered DNA fragment was double-digested with BspH I and Xba I, double-digested with Nco I and Xba I (pse420 modified from the multicloning site of plasmid vector pSE420 from Invitrogen, JP 2000-189170) and Ligation was performed using Takara Ligation Kit, and E. coli JM109 strain was transformed.
The transformed strain was grown in an LB medium containing ampicillin, plasmid was extracted from the grown transformant, and a plasmid in which the imine reductase gene was confirmed to be inserted was designated as pSU-SRI1. The process of plasmid construction is shown in FIG.

[Example 2] Activity evaluation of C4EPL6_STRRS
E. coli JM109 strain transformed with plasmid pSU-SRI1 expressing C4EPL6_STRRS was cultured overnight at 30 ° C. in a liquid LB medium containing ampicillin, 0.1 mM IPTG was added, and further cultured for 4 hours.
Bacteria are collected by centrifugation, suspended in 100 mM potassium phosphate buffer (pH 7.5) containing 1 mM DTT, and then sealed using a sealed ultrasonic crusher UCD-200TM (Cosmo Bio). The body was crushed. The cell disruption solution was centrifuged, and the supernatant was recovered as a cell extract. Add 100 μL of cell extract to 100 mM phosphate buffer (pH 7.5) containing 10 mM 2-methyl-1-pyrroline and 10 mM NADPH, and measure the absorbance at 340 nm while maintaining at 30 ° C. Enzyme activity was measured by following the decrease. 1 U was defined as the amount of enzyme that catalyzes the consumption of 1 μmol of NADPH per minute under the above reaction conditions. As a result, the imine reduction activity was 11.4 mU / mg.

[Example 3] Construction of plasmid pSG-SRI1 co-expressing C4EPL6_STRRS and glucose dehydrogenase derived from Bacillus subtilis Plasmid pSE-BSG1 (Japanese Patent Laid-Open No. 2000-189170) expressing a glucose dehydrogenase gene derived from Bacillus subtilis The glucose dehydrogenase gene derived from Bacillus subtilis double digested with two restriction enzymes I and HindIII was ligated using pSU-SRI1 and Takara Ligation Kit double digested with the same enzyme, and C4EPL6_STRRS and glucose PSG-SRI1, which is a plasmid capable of simultaneously expressing dehydrogenase, was obtained. The process of plasmid construction is shown in FIG.

[Example 4] Co-expression of C4EPL6_STRRS and glucose dehydrogenase by E. coli
The Escherichia coli JM109 strain transformed with pSG-SRI1 was cultured overnight at 30 ° C. in a liquid LB medium containing ampicillin, 0.1 mM IPTG was added, and further cultured for 4 hours.
The cells are collected by centrifugation, then suspended in 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM DTT, and the cells using a sealed ultrasonic crusher UCD-200TM (Cosmo Bio). Was crushed. The cell disruption solution was centrifuged, the supernatant was collected as a cell extract, and the imine reduction activity and glucose dehydrogenation activity against 2-methyl-1-pyrroline were measured. The glucose dehydrogenation activity was measured at 30 ° C. in a reaction solution containing 100 mM potassium phosphate buffer (pH 6.5), 2.5 mM NADP +, 100 mM glucose, and a cell extract. 1 U was defined as the amount of enzyme that catalyzes the production of 1 μmol NADPH per minute under the above reaction conditions. As a result, the imine reduction activity was 5.13 mU / mg, and the glucose dehydrogenation activity was 9.36 mU / mg.

[Example 5] Production of (S) -2-methylpyrrolidine by recombinant Escherichia coli
E. coli strain JM109 transformed with pSG-SRI1 was cultured overnight at 30 ° C. in 10 mL of liquid LB medium containing ampicillin, 0.1 mM IPTG was added, and further cultured for 4 hours.
The cells are collected by centrifugation, then suspended in 10 mL of 100 mM potassium phosphate buffer (pH 7.5) containing 42.0 mM 2-methyl-1-pyrroline and 84.0 mM glucose, and shaken overnight at 25 ° C. Went. After the reaction, the optical purity of the produced 2-methylpyrrolidine and the concentrations of 2-methyl-1-pyrroline and 2-methylpyrrolidine in the reaction solution were analyzed. As a result, the optical purity of the produced (S) -2-methylpyrrolidine was 97.7% ee, and the reaction yield was 99.4%.
The optical purity measurement method is as follows.
The following components were added to 50 μL of the reaction solution, and the amine produced by reacting at 40 ° C. for 1 hour was converted to GITC, separated and quantified by HPLC.
2 mg / mL triethylamine 50 μL and
8 mg / mL 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC) 100 μL
The HPLC conditions are as follows.
-HPLC column: Wakosil II 5C18 (4.6 mm x 150 mm) manufactured by Wako Pure Chemical Industries, Ltd.
− Eluent: 10 mM phosphate buffer (pH 2.5): methanol = 55: 45
− Flow rate: 1 mL / min
-Detection: UV absorption at 254 nm Under the above conditions, (R) -2-methylpyrrolidine was eluted at 26.3 minutes and (S) -2-methylpyrrolidine at 24.9 minutes. Optical purity was determined based on UV absorption at 254 nm of each eluted fraction.

ここで、pSUS2MP1およびpSG-S2MPはそれぞれ、イミン還元酵素SsSIRを発現するプラスミド（図３）、イミン還元酵素SsSIRと枯草菌由来のグルコース脱水素酵素を共発現するプラスミド（図４）である。
なお、SsSIRの酵素活性、光学純度の測定は以下のようにして行った。
pSG-S2MPで形質転換された大腸菌ＪＭ１０９株をアンピシリンを含む５ｍＬの液体ＬＢ培地で終夜３０℃培養し、０．１ｍＭ ＩＰＴＧを加え、さらに４時間培養を行った。菌体を遠心分離により集菌した後、２０ｍＭ ２−メチル−１−ピロリン、４０ｍＭ グルコースを含む１００ｍＭリン酸カリウム緩衝液（ｐＨ７．０）１ｍＬに懸濁し、終夜２５℃で振とう反応を行った。反応後、生成した２−メチルピロリジンの光学純度、及び、反応液中における２−メチル−１−ピロリンと２−メチルピロリジンの濃度を実施例５と同様にして分析した。その結果、生成した（Ｓ）−２−メチルピロリジンの光学純度は９２％ｅｅであった。
上記の表から明らかなように、C4EPL6_STRRSの使用により、SsSIRを上回る光学純度を達成することができた。 [Example 6]
A table comparing the enzyme activities and reaction results of SsSIR and C4EPL6_STRRS is shown below.
[Table 1]

Here, pSUS2MP1 and pSG-S2MP are respectively a plasmid that expresses imine reductase SsSIR (FIG. 3) and a plasmid that co-expresses imine reductase SsSIR and glucose dehydrogenase derived from Bacillus subtilis (FIG. 4).
The enzyme activity and optical purity of SsSIR were measured as follows.
E. coli JM109 strain transformed with pSG-S2MP was cultured overnight at 30 ° C. in 5 mL of liquid LB medium containing ampicillin, 0.1 mM IPTG was added, and further cultured for 4 hours. The cells were collected by centrifugation, then suspended in 1 mL of 100 mM potassium phosphate buffer (pH 7.0) containing 20 mM 2-methyl-1-pyrroline and 40 mM glucose, and shaken at 25 ° C. overnight. . After the reaction, the optical purity of the produced 2-methylpyrrolidine and the concentrations of 2-methyl-1-pyrroline and 2-methylpyrrolidine in the reaction solution were analyzed in the same manner as in Example 5. As a result, the optical purity of the produced (S) -2-methylpyrrolidine was 92% ee.
As is clear from the above table, optical purity exceeding SsSIR could be achieved by using C4EPL6_STRRS.

  The present invention provides a method for efficiently producing an optically active amine derivative by stereoselective reduction using a microorganism-derived imine reductase. Since the method of the present invention can be expected to have high stereoselectivity and optical purity (97.7% ee), it is industrially advantageous. The optically active amine derivative produced by the present invention is useful as a raw material for synthesizing optically active pharmaceuticals. For example, (S) -2-methylpyrrolidine is a useful compound as a pharmaceutical raw material.

Claims (13)

The imine derivative represented by the formula (I) comprises an imine reductase encoded by the polynucleotide according to any one of (a) to (e) below, a microorganism producing the imine reductase, and a processed product thereof Production of an optically active S-form amine derivative comprising a step of contacting at least one enzyme active substance selected from the group and a step of recovering an optically active S-form amine derivative represented by the formula (II) Method.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(C) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1;
(D) a polynucleotide that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 2;
(E) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1.
Formula (I)

Formula (II)

(Wherein R1 and R2 groups represent an alkyl group having 1 to 3 carbon atoms, R3 group represents an alkyl group having 1 to 3 carbon atoms or hydrogen, and R1 and R3 may be wound in a ring) The optically active compound according to claim 1, wherein the compound of the formula (I) is an imine derivative represented by the following formula (III), and the compound of the formula (II) is an amine derivative represented by the formula (IV). A process for producing an S derivative amine derivative.
Formula (III)

Formula (IV)

(Wherein R represents an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 1 to 4)   3. The optically active S-form amine derivative according to claim 2, wherein the compound of formula (III) is 2-methyl-1-pyrroline, and the compound represented by formula (IV) is 2-methylpyrrolidine. Production method. An imine reductase encoded by the polynucleotide according to any one of (a) to (e) below.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(C) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1;
(D) a polynucleotide that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 2;
(E) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1.   The imine reductase is a protein comprising an amino acid sequence in which one or more and 50 or less amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1. The imine reductase described in 1. The stereoselective imine reducing agent which contains the protein which has the imine reduction activity encoded by the polynucleotide in any one of the following (a) to (e) as an active ingredient.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(C) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1;
(D) a polynucleotide that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 2;
(E) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1. A polynucleotide encoding the imine reductase according to any one of (a) to (e) below and a polynucleotide encoding a dehydrogenase capable of catalyzing an oxidation-reduction reaction using NADP + as a coenzyme Recombinant vector inserted into.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(C) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1;
(D) a polynucleotide that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 2;
(E) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1.   The vector according to claim 7, wherein the dehydrogenase is glucose dehydrogenase.   The recombinant vector according to claim 8, wherein the glucose dehydrogenase is derived from Bacillus subtilis.   A transformant retaining the vector according to any one of claims 7 to 9 in an expressible manner.   The manufacturing method of the amine derivative of the optically active S body in any one of Claims 1-3 including the process of culture | cultivating the transformant of Claim 10.   The method for producing an optically active S-form amine derivative according to any one of claims 1 to 3, wherein the microorganism that produces the imine reductase according to claim 1 is a microorganism belonging to the genus Streptosporangium.   Contacting the imine derivative represented by formula (I) with a microorganism belonging to the genus Streptosporangium or a processed product thereof; and recovering the optically active S-form amine derivative represented by formula (II) A process for producing an optically active S-form amine derivative.

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