CN1950508A

CN1950508A - Process for producing optically active alcohol and carboxylic acid

Info

Publication number: CN1950508A
Application number: CN 200580011980
Authority: CN
Inventors: 出来岛康方; 川端润; 平冈宏敏; 上田诚; 上原久俊
Original assignee: API Corp
Current assignee: API Corp
Priority date: 2004-02-04
Filing date: 2005-02-04
Publication date: 2007-04-18

Abstract

It is an object of the present invention to provide a low-cost and efficient industrial process for producing (S)-2-pentanol, (S)-2-hexanol, 1-methylalkylmalonic acid and 3-methylcarboxylic acid with high optical purity. There is provided a process for producing (S)-2-pentanol and (S)-2-hexanol, comprising causing a certain type of microbe or transformed cells, a treatment mixture from the microbe or cells, a culture solution of the microbe or cells and/or a product of crude purification or purification of a carbonyl reductase fraction obtained from the microbe or cells to act on 2-pentanone and 2-hexanone, respectively.

Description

Preparation method with optically active alcohol and carboxylic acid

Technical field

The present invention relates to make the microorganism that belongs to Yi Shi yeast belong (Issatchenkia) etc., culture of this microbiological treatment thing and/or this microorganism and 2 pentanone or methyl-n-butyl ketone effect, preparation is as medicine, the method of industrial useful compound (the S)-2-amylalcohol of intermediate feed such as agricultural chemicals or (S)-2-hexanol, also relate to and make the transformant of expressing following DNA, this cell handled thing and/or this cell culture fluid and 2 pentanone or methyl-n-butyl ketone effect, the method of preparation (S)-2-amylalcohol or (S)-2-hexanol, wherein said dna encoding are by the mentioned microorganism gained, protein (carbonyl reductase) with ability of reducing carbonyl and synthesis of optically active alcohol.The invention still further relates to the preparation method of optical activity 1-methyl alkyl propanedioic acid and preparation method thereof and optical activity 3-methyl carboxylic acids.

Background technology

The method of known chemical preparation (S)-2-amylalcohol or (S)-2-hexanol for example has: in the presence of the branch-shape polymer that contains polyamidoamines and glucono-lactone, 2 pentanone is carried out reductive method (J.Am.Chem.Soc., 123 volumes, 5956-5961 page or leaf, calendar year 2001); Perhaps use optical activity boron that methyl-n-butyl ketone is carried out reductive method (spy opens flat 11-240894) etc.But they all can't satisfy the optical purity requirement of product.

In addition-aspect, the method that generates optically active alkylol cpd as thalline and/or this bacterial disposing thing with microorganism, knownly make microorganism and racemic ester cpds effect, by the optical selective ester hydrolysis, generate the method (spy opens flat 10-4998) of optically active alkylol cpd, but this has the low problem of yield, must give up problems such as not being needed stereochemistry alcohol or its ester in addition in addition.In addition, the compound that has ketone group as Stereoselective reduction, generate the method for optically active alkylol cpd, known have a method that microorganism and 2 pentanone or methyl-n-butyl ketone effect are prepared (Tetrahedron:Asymmetry, 14 volumes, 2659-2681 page or leaf, 2003), but the optical purity of product or production concentration can't meet the demands, the complex pretreatment of the thalline of reactions such as acetone treatment is fixed or be used for to thalline, and the adding concentration of substrate is low etc., can't practical application.

The active 1-methyl alkyl of known optical propanedioic acid is the useful compound as medical pesticide intermediate.Also the active 3-methyl carboxylic acids of known optical is the useful compound as medical pesticide intermediate too.

Optical activity 1-methyl butyl propanedioic acid is the compound (for example with reference to WO 00/24358) that can be used as the intermediate that shows neural inhibiting barbituric acid derivatives.In addition, can be used as pharmaceutical intermediates such as prostanoid (for example opening clear 62-265279) by optical activity 1-methyl alkyl propanedioic acid synthetic optical activity 3-methylhexanoic acid and optical activity 3-methyl enanthic acid with reference to the spy.

Existing people reports and uses optically active 2-amylalcohol to carry out synthetic (J.Am.Chem.Soc., 1950,72,4695) of (S)-1-methyl butyl propanedioic acid.But, in this method, in order to obtain optical activity 2-amylalcohol, adopted the 2-amylalcohol of racemic modification has been made phthalic monoester, cut apart the so very complicated method of posthydrolysis with brucine, in addition owing to be to cut apart, there is the amount of half to utilize, and for the alcohol that will cut apart, auxiliary group, the molecular weight of cutting apart group are big, and efficient is very low.

The also known example that the synthetic 3-methyl carboxylic acids of several decarboxylic reactions by 1-methyl alkyl propanedioic acid arranged (for example with reference to J.Am.Chem.Soc., 1950,72,4695 and NouveauJournal de Chime, 1985,9,557).But they all need to be difficult to control solvent-free, the disposable additive process of reaction and high temperature (180 ℃) etc., therefore are difficult to carry out industrial.

Also known to using additive, the method for the substrate different with the present invention being carried out at low temperatures decarboxylation.But in the presence of cupric oxide, the method that refluxes in acetonitrile is (with reference to J.Am.Chem.Soc., 1993,115,801) or the method that in the presence of sulfuric acid, heats (with reference to Org.Lett., 2002,4,1571), checking they during whether applicable to substrate of the present invention, all do not observe the noticeable response acceleration effect.Hence one can see that, uses method that additive makes decarboxylic reaction low temperatureization according to the difference of compound the applicable situation that can not be suitable for that also has to be arranged.

On the other hand, known have the example that reacts (for example with reference to J.Org.Chem. in solvent, under the temperature about 100-150 ℃, 1983,48,2994), still less than report about the solvent compliance test result, in addition, according to the substrate difference, necessary temperature of reaction is variant during decarboxylation, and solvent makes the effect of decarboxylic reaction low temperatureization indeterminate.

In addition, the secure contexts such as control for the carbonic acid gas that produces do not have consideration, the still residual a lot of unresolved problems of the industrializing implementation of decarboxylic reaction.

Synthetic method as the optical activity 1-methyl butyl malonic ester of the synthetic precursor that is considered to optical activity 1-methyl butyl propanedioic acid, known have by geraniol deutero-method (for example with reference to WO 00/24358), but have problems such as the rapid and low yield of multistep.In addition, synthesis method as optical activity 1-methyl amyl malonic ester, known have by asymmetric 1, the 4-addition reaction is carried out the synthetic method (for example with reference to Tetrahedron Asym., 2001,12,1151), but can't obtain enough optical purities (maximum 50%e.e.), can not practical application.

On the other hand, as can be by the synthetic method of optical activity 1-methyl alkyl propanedioic acid synthetic optical activity 3-methylhexanoic acid or optical activity 3-methyl enanthic acid, known have the Ba Dousuan derivative with asymmetric auxiliary group is carried out the addition reaction of organic copper reagent (for example with reference to Helv.Chim.Acta., 1985,68,212).But this method need import the high asymmetric auxiliary group of cost to intramolecularly, in addition, needs to use a large amount of organic copper reagents, and liquid waste disposal becomes problem, can not say so to be fit to the method for industry.The optical segmentation of known racemic compound (for example opening clear 62-265279) also with reference to the spy, but by cutting apart, the compound with target steric configuration obtains 50% at most only, and efficient is poor, discard the unwanted steric configuration compound of half amount, environmental stress is big.In addition, also known to deutero-methods such as citronellic acid (for example with reference to No. the 5136020th, United States Patent (USP) and Tetrahedron, 1977,33,289), but also there are problems such as the many and yield of step is low in it.

Synthesis example (the J.Am.Chem.Soc. that also has optical activity 1-methyl butyl propanedioic acid, 1950,72,4695), but wherein record: with the malonic ester coupling time, optical purity significantly reduces, afterwards after the bromination, even recrystallization repeatedly after deriving to optical activity 1-methyl butyl propanedioic acid also can only be increased to 70%e.e. with optical purity.That is, method in the past can't be synthesized the optical activity 1-methyl butyl propanedioic acid with the necessary high-optical-purity of medical pesticide intermediate, must study the synthetic method that optical purity is not reduced.

Also the active 1-methylheptyl of known optical propanedioic acid is (with reference to Nouveau Journal deChime, 1985,9,557) and with the optical activity 1-methyl-propyl propanedioic acid of radioelement mark (with reference to J.Am.Chem.Soc., 1980,102,7344) synthesis example.But, need long-time (12 hours or more than) when reacting with malonic ester, and needs a large amount of solvents (50 times of volumes) during with the dicarboxylic acid recrystallization, need high temperature (180 ℃) in the decarboxylic reaction, the cost-effective rate is low, and therefore many unfavorable factors are arranged aspect industrial implementation.And the optical purity for these compounds has only been reported specific rotation, and correct optical purity still is indeterminate.

Summary of the invention

But the object of the present invention is to provide industrialization, easy, prepare the novel preparation method that higher (the S)-2-amylalcohol of optical purity or (S)-2-hexanol, preferred optical purity are 99.0%e.e. or above (S)-2-amylalcohol or (S)-2-hexanol at low cost.Another object of the present invention is to provide and to obtain the low-cost of optical activity 1-methyl alkyl propanedioic acid and optical activity 3-methyl carboxylic acids and industrialized process for preparing efficiently with high-optical-purity.

The inventor is for solving above-mentioned problem, to (S)-2-amylalcohol or (S)-preparation method of 2-hexanol furthers investigate, found that: belong to the certain micro-organisms that Brettanomyces belongs to (Brettanomyces) etc. by use, with 2 pentanone or methyl-n-butyl ketone is substrate, can generate (S)-2-amylalcohol or (S)-2-hexanol simply, efficiently.And, from the Yi Shi yeast belong microorganism of one of mentioned microorganism, separate reductase 12-pentanone or methyl-n-butyl ketone, generate (S)-2-amylalcohol or (the S)-carbonyl reductase of 2-hexanol and DNA of this enzyme of encoding, its base sequence is analyzed.Also prepared the transformant of expressing this DNA, make this transformant, this cell handled thing and/or this cell culture fluid with as the 2 pentanone or the methyl-n-butyl ketone effect of raw material, find thus and can obtain target compound (S)-2-amylalcohol or (S)-2-hexanol with high-optical-purity and high density.

The inventor finds again: optical activity alcohol is transformed into leavings group, handle the compound of gained then with carbon nucleophile, can keep high-optical-purity to carry out replacement(metathesis)reaction, with the hydrolysis of gained optically active compound, then by partial crystallization, can be with high-optical-purity, prepare optical activity 1-methyl alkyl propanedioic acid efficiently.Also set up the easy and excellent preparation method of industrialization, this preparation method is transformed into optical activity 3-methyl carboxylic acids by decarboxylation with optical activity 1-methyl alkyl propanedioic acid, this moment is by using the additive of high polar solvent and/or promotion decarboxylation, can under the condition significantly gentleer, react, and can control the generation of carbonic acid gas than in the past method.

The present invention is achieved according to above understanding.

That is, the invention provides following invention.

1. the method for preparing (S)-2-amylalcohol, this method is to make by microorganism or transformant, this microorganism or cell handled thing, this microorganism or cell culture fluid, and/or thick purifying thing or the purifying thing and the 2 pentanone effect of the carbonyl reductase component that obtains from this microorganism or cell, the method of preparation (S)-2-amylalcohol, it is characterized in that: this microorganism or transformant are done the time spent at its viable bacteria body and 2 pentanone without the solvent pre-treatment, can generate (S)-2-amylalcohol of 95%e.e. or above optical purity, and its productive rate be 1mg (S)-2-amylalcohol/g dry mycelium weight/hour or more than.

2. the method for preparing (S)-2-hexanol, this method is to make by microorganism or transformant, this microorganism or cell handled thing, this microorganism or cell culture fluid, and/or thick purifying thing or the purifying thing and the methyl-n-butyl ketone effect of the carbonyl reductase component that obtains from this microorganism or cell, the method of preparation (S)-2-hexanol, it is characterized in that: this microorganism or transformant are done the time spent at its viable bacteria body and methyl-n-butyl ketone without the solvent pre-treatment, can generate (S)-2-hexanol of 95%e.e. or above optical purity, and its productive rate be 1mg (S)-2-hexanol/g dry mycelium weight/hour or more than.

3. the preparation method of (S)-2-amylalcohol of high-optical-purity or (S)-2-hexanol, it is characterized in that: make by being selected from Brettanomyces to belong to (Brettanomyces), mycocandida (Candida), Hortaea, Yi Shi yeast belong (Issatchenkia), rood yeast belong (Lodderomyces), Pichia (Pichia), Rhodotorula (Rhodotorula), genus arthrobacter (Arthrobacter), brevibacterium sp (Brevibacterium), Curtobacterium (Crutobacterium), soil bacillus (Geobacillus), Microbacterium (Microbacterium), pale Bacillaceae (Ochrobactrum), paracoccus (Paracoccus), rhizobium (Rhizobium), the microorganism of Rhod (Rhodococcus), this microbiological treatment thing, this microbial culture medium, and/or thick purifying thing or purifying thing and the 2 pentanone or the methyl-n-butyl ketone effect of the carbonyl reductase component that obtains from this microorganism, generate (S)-2-amylalcohol or (S)-2-hexanol.

4. the preparation method of (S)-2-amylalcohol of high-optical-purity or (S)-2-hexanol, it is characterized in that: make the transformant of expressing following DNA, this cell handled thing, this cell culture fluid, and/or thick purifying thing or purifying thing and the 2 pentanone or the methyl-n-butyl ketone effect of the carbonyl reductase component that obtains from this cell, generate (S)-2-amylalcohol or (S)-2-hexanol, wherein, the carbonyl reductase that described dna encoding is obtained by following microorganism, described microorganism are selected from Brettanomyces and belong to (Brettanomyces), mycocandida (Candida), Hortaea, Yi Shi yeast belong (Issatchenkia), rood yeast belong (Lodderomyces), Pichia (Pichia), Rhodotorula (Rhodotorula), genus arthrobacter (Arthrobacter), brevibacterium sp (Brevibacterium), Curtobacterium (Crutobacterium), soil bacillus (Geobacillus), Microbacterium (Microbacterium), pale Bacillaceae (Ochrobactrum), paracoccus (Paracoccus), rhizobium (Rhizobium), Rhod (Rhodococcus).

5. claim 3 or 4 preparation method, it is characterized in that: described microorganism is to be selected from Brussels Brettanomyces (Brettanomyces bruxellensis), different Brettanomyces (Breettanomyces anomalus), candida famata (Candida famata), monilia krusei (Candida krusei), maltose candiyeast (Candida maltosa), candida tropicalis (Candida tropicalis), candida zeylanoides (Candida zeylanoides), Hortaea werneckii, Issatchenkia scutulata, Lodderomyces elongisporus, Angus pichia spp (Pichia angusta), Pichia besseyi, Pichia cactophila, Pichia segobiensis, Spartacus pichia spp (Pichia spartinae), happiness trehalose pichia spp (Pichia trehalophila), small rhodotorula (Rhodotorula minuta), oxidation Arthrobacter (Arthrobacter oxydans), polychrome Arthrobacter (Arthrobacterpolychromogenes), genus arthrobacter certain (Arthrobacter sp.), sulphur look Arthrobacter (Arthrobacter sulfurous), Brevibacterium butanicum, wilting bacillus pumilis (Curtobacterium flaccumfaciens), stearothermophilus ground bacillus (Geobacillusstearothermophilus), separate cutin microbacterium (Microbacterium keratanolyticum), longicorn microbacterium (Microbacterium saperdae), Microbacterium certain (Microbacteriumsp.), brick-red microbacterium (Microbacterium testaceum), human pallid bacillus (Ochrobactrum anthropi), pale Bacillaceae certain (Ochrobactrum sp.), the false pseudomonas bacillus (Pseudomonas ovalis) of ovum shape, Paracoccus denitrificans (Paracoccusdenitrificans), radiation root nodule bacterium (Rhizobium radiobacter), Rhod certain (Rhodococcus sp.), separate the microorganism of hydrocarbon rod bacillus (Corynebacterium hydrocarboclastum).

6. the preparation method of (S)-2-amylalcohol of high-optical-purity or (S)-2-hexanol, it is characterized in that: make transformant, this cell handled thing and/or this cell culture fluid and 2 pentanone or the methyl-n-butyl ketone effect of expressing each DNA in following (A)-(F), generation (S)-2-amylalcohol or (S)-2-hexanol:

(A) coding has the protein DNA of aminoacid sequence shown in the SEQ ID NO.1;

(B) DNA of coding following proteins, described protein have in aminoacid sequence shown in the SEQ ID NO.1 1 to a plurality of aminoacid deletion, interpolation or replace the aminoacid sequence of back gained and have the ability of reducing carbonyl, synthesis of optically active alcohol;

(C) DNA of coding following proteins, described protein have with aminoacid sequence shown in the SEQ ID NO.1 and have 50% or the aminoacid sequence of above homology and have the ability of reducing carbonyl, synthesis of optically active alcohol;

(D) has the DNA of base sequence shown in the SEQ ID NO.2;

(E) have shown in the SEQ ID NO.2 in the base sequence 1 to a plurality of base deletions, interpolation or replace the base sequence of back gained and have the DNA of the base sequence of coding following proteins, described protein has the ability of reducing carbonyl, synthesis of optically active alcohol;

(F) have base sequence with base sequence shown in the SEQ ID NO.2 or its complementary sequence hybridize under stringent condition, and have the DNA of the base sequence of coding following proteins, described protein has the ability of reducing carbonyl, synthesis of optically active alcohol.

7. the preparation method of (R) shown in the following general formula (5) or (S)-3-methyl carboxylic acids is characterized in that:

At high polar solvent and/or promote in the presence of the additive of decarboxylation, make having shown in the following general formula (1) optically active (R) or (S)-1-methyl alkyl propanedioic acid decarboxylation,

Wherein, in the formula (1), R ¹The expression carbonatoms is the alkyl of 3-5, and * represents unsymmetrical carbon; In the formula (5), R ¹Identical with aforementioned implication, * represents unsymmetrical carbon.

8. the preparation method of (R) shown in the following general formula (1) or (S)-1-methyl alkyl propanedioic acid is characterized in that:

Make the reaction of optical activity alcohol shown in the following general formula (2) and sulfonyl agent, obtain the optically active compound shown in the following general formula (3),

In the presence of alkali,, make the optically active compound shown in the following general formula (4) then, be hydrolyzed then with the carbon nucleophile reaction shown in the general formula (9),

Wherein, in the formula (1), R ¹The expression carbonatoms is the alkyl of 3-5, and * represents unsymmetrical carbon;

In the formula (2), R ¹Identical with aforementioned implication, * represents unsymmetrical carbon;

In the formula (3), R ¹Identical with aforementioned implication, X represents alkylsulfonyl oxygen base, and * represents unsymmetrical carbon;

In the formula (9), R ²And R ³Represent ester group, carboxyl or cyano group independently of one another, wherein R ²And R ³Can form ring texture together;

In the formula (4), R ¹, R ²And R ³Identical with aforementioned implication, * represents unsymmetrical carbon.

9. following general formula (1) expression, optical purity is 90%ee or above (R)-1-methyl alkyl propanedioic acid or (S)-1-methyl alkyl propanedioic acid

In the formula (1), R ¹The expression carbonatoms is the alkyl of 3-5, and * represents unsymmetrical carbon.

10. (R)-1-methyl alkyl propanedioic acid of claim 9 or (S)-1-methyl alkyl propanedioic acid, wherein R ¹Be n-propyl or normal-butyl.

11. the preparation method of optically active body shown in the general formula (6); this method comprises: make following microorganism or transformant, this microorganism or cell handled thing, this microorganism or cell culture fluid and/or thick purifying thing or the purifying thing and the 2 pentanone effect of the carbonyl reductase component that obtains from this microorganism or cell; be transformed into (S)-2-amylalcohol; (S)-2-amylalcohol and sulfonyl agent with gained reacts again; be transformed into the optically active body shown in the following general formula (6)

Wherein, in the formula (6), R ⁴The expression n-propyl, X represents alkylsulfonyl oxygen base; Described microorganism or transformant are to comprise to have microorganism or the transformant that reacts, generates the active carbonyl reductase of (S)-2-amylalcohol with 2 pentanone, it does the time spent at viable bacteria body and 2 pentanone without the solvent pre-treatment, can generate (S)-2-amylalcohol of 95%e.e. or above optical purity, and its productive rate be 10mg (S)-2-amylalcohol/g dry mycelium weight/hour or more than.

12. the preparation method of optically active body shown in the general formula (6); this method comprises: make following microorganism or transformant, this microorganism or cell handled thing, this microorganism or cell culture fluid and/or thick purifying thing or the purifying thing and the methyl-n-butyl ketone effect of the carbonyl reductase component that obtains from this microorganism or cell; be transformed into (S)-2-hexanol; (S)-2-hexanol and sulfonyl agent with gained reacts again; be transformed into the optically active body shown in the following general formula (6)

Wherein, in the formula (6), R ⁴The expression normal-butyl, X represents alkylsulfonyl oxygen base; Described microorganism or transformant are to comprise to have microorganism or the transformant that reacts, generates the active carbonyl reductase of (S)-2-hexanol with methyl-n-butyl ketone, it does the time spent at viable bacteria body and methyl-n-butyl ketone without the solvent pre-treatment, can generate (S)-2-hexanol of 95%e.e. or above optical purity, and its productive rate be 10mg (S)-2-hexanol/g dry mycelium weight/hour or more than.

13. the method for claim 11 or 12, this method also comprises: in the presence of alkali, make the reaction of optically active body shown in the general formula (6) of gained and the carbon nucleophile shown in the general formula (9), be transformed into the step of the optically active compound shown in the following general formula (7)

In the formula (7), R ²And R ³Identical with aforementioned implication, R ⁴Be n-propyl or normal-butyl.

14. (R)-preparation method of 1-methyl butyl propanedioic acid or (R)-1-methyl amyl propanedioic acid, it is characterized in that: in the presence of alkali, make optically active body and the reaction of the carbon nucleophile shown in the general formula (9) shown in the general formula (6) that the method by claim 11 or 12 obtains, be transformed into the optically active compound shown in the following general formula (7)

Again with the hydrolysis of gained optically active compound, be transformed into (the R)-1-methyl butyl propanedioic acid shown in the following general formula (8) or (R)-1-methyl amyl propanedioic acid,

Wherein, in the formula (9), R ²And R ³Represent ester group, carboxyl or cyano group independently of one another, R ²And R ³Can form ring texture together;

In the formula (7), R ²And R ³Identical with aforementioned implication, R ⁴Be n-propyl or normal-butyl;

In the formula (8), R ⁴Identical with aforementioned implication.

15. (R)-preparation method of 3-methylhexanoic acid or (R)-3-methyl enanthic acid, it is characterized in that: in the presence of alkali, make optically active body and the reaction of the carbon nucleophile shown in the general formula (9) shown in the general formula (6) that the method by claim 11 or 12 obtains, be transformed into the optically active compound shown in the following general formula (7)

Again with the hydrolysis of gained optically active compound, be transformed into (the R)-1-methyl butyl propanedioic acid shown in the following general formula (8) or (R)-1-methyl amyl propanedioic acid, and then with gained (R)-1-methyl butyl propanedioic acid or (R)-decarboxylation of 1-methyl amyl propanedioic acid,

In the formula (8), R ⁴Identical with aforementioned implication.

The best mode that carries out an invention

Below embodiment of the present invention are described, but scope of the present invention is not limited by these contents.

1. use the preparation method of the optical activity alcohol that microorganism etc. carries out

The preparation method of (S)-2-amylalcohol of the present invention or (S)-2-hexanol is with microorganism or transformant, this microorganism or cell handled thing, this microorganism or cell culture fluid, and/or the thick purifying thing of the carbonyl reductase component that obtains by this microorganism or cell or purifying thing and 2 pentanone (or methyl-n-butyl ketone) effect, the method of preparation (S)-2-amylalcohol (or (S)-2-hexanol), it is characterized in that: the viable bacteria body and the 2 pentanone (or methyl-n-butyl ketone) without the solvent pre-treatment of this microorganism or transformant are done the time spent, can generate (S)-2-amylalcohol (or (S)-2-hexanol) of 95%e.e. or above optical purity, and its productive rate be 1mg (S)-2-amylalcohol (or (S)-2-hexanol)/g dry mycelium weight/hour or more than.

2 pentanone described in this specification sheets and methyl-n-butyl ketone refer to that all carbochain is the 2 pentanone and the methyl-n-butyl ketone of straight chain.

As mentioned above, feature for microorganism of using in the method for the present invention or transformant is: its viable bacteria body and 2 pentanone (or methyl-n-butyl ketone) without the solvent pre-treatment is being done the time spent, can generate (S)-2-amylalcohol (or (S)-2-hexanol) of 95%e.e. or above optical purity, and its productive rate be 1mg (S)-2-amylalcohol (or (S)-2-hexanol)/g dry mycelium weight/hour or more than.Solvent described here has: acetone, toluene, methyl-sulphoxide, 2-propyl alcohol, the method for pre-treatment has: thalline is soaked or thalline soaked and method such as drying under reduced pressure.In using the preparation method must carry out the microorganism of these processing or (S)-2-amylalcohol that transformant carries out or (S)-2-hexanol, this processing needs labour and expense, and is difficult to obtain the result etc. of reproducibility, and is therefore not preferred.

The optical purity of (the S)-2-amylalcohol that generates or (S)-2-hexanol is 95%e.e. or above get final product, preferred 98%e.e. or more than, further preferably 99%e.e. or more than.

Productive rate about (S)-2-amylalcohol, so long as 1mg (S)-2-amylalcohol/g dry mycelium weight/hour or above getting final product, be preferably 2mg (S)-2-amylalcohol/g dry mycelium weight/hour or more than, further preferred 5mg (S)-2-amylalcohol/g dry mycelium weight/hour or more than, further preferred 10mg (S)-2-amylalcohol/g dry mycelium weight/hour or more than, preferred especially 20mg (S)-2-amylalcohol/g dry mycelium weight/hour or more than.

Productive rate about (S)-2-hexanol, so long as 1mg (S)-2-hexanol/g dry mycelium weight/hour or above getting final product, preferred 2mg (S)-2-hexanol/g dry mycelium weight/hour or more than, further preferred 5mg (S)-2-hexanol/g dry mycelium weight/hour or more than, further preferred 10mg (S)-2-hexanol/g dry mycelium weight/hour or more than, further preferred again 20mg (S)-2-hexanol/g dry mycelium weight/hour or more than, further preferred again 50mg (S)-2-hexanol/g dry mycelium weight/hour or more than, preferred especially 100mg (S)-2-hexanol/g dry mycelium weight/hour or more than.

The preparation method's of (S)-2-amylalcohol of the present invention or (S)-2-hexanol a example can use the conversion bacterial strain of expressing following DNA to implement, and described dna encoding has the homologue of the protein of aminoacid sequence of SEQ ID NO.1 or this aminoacid sequence and has reducing carbonyl and the protein of the ability of synthesis of optically active alcohol (following it can be abbreviated as " carbonyl reductase ").

In this specification sheets, the carbonyl reduction enzymic activity is meant the carbonyl that reduces in the carbonyl containing compound, the activity of making the optical activity alcohols asymmetricly.Described activity can followingly be calculated: contain carbonyl containing compound as substrate and contain NADPH or the reaction solution of NADH as coenzyme in, make target protein, have express this proteinic ability transformant, transformant handled thing or nutrient solution as enzyme and reaction solution effect, the absorbancy by assaying reaction liquid changes the minimizing initial velocity that calculates NADPH in the reaction solution or NADH.

Carbonyl reductase used in the present invention is so long as can make 2 pentanone or enzyme that methyl-n-butyl ketone generates (S)-2-amylalcohol or (S)-2-hexanol gets final product.When measuring the carbonyl reduction enzymic activity of carbonyl reductase used in the present invention, use the compound that contains carbonyl as substrate, carbonyl containing compound is not limited to 2 pentanone or methyl-n-butyl ketone, also can preferably use structurally similar compounds such as its substituent derivative.Described example has: 1-acetoxy-3-chloro-2-acetone etc.

As putting down in writing in this specification sheets, the base sequence of the aminoacid sequence of carbonyl reductase and this aminoacid sequence of coding is clear and definite, therefore as described later, the base sequence of part or all of aminoacid sequence according to the coding carbonyl reductase, the preparation probe uses this probe, separates the DNA of coding carbonyl reductase from have the active any microorganism of carbonyl reductase, then based on this, adopt conventional gene engineering method to obtain.

Can also be as for finishing the present invention does, from have the active microorganism of carbonyl reductase, promptly from having the microorganism of DNA of coding carbonyl reductase, for example be selected from purifying in the microorganism of Brettanomyces genus, mycocandida, Hortaea, Yi Shi yeast belong, rood yeast belong, Pichia, Rhodotorula, genus arthrobacter, brevibacterium sp, Curtobacterium, native gemma Pseudomonas, Microbacterium, pale Bacillaceae, paracoccus, rhizobium, Rhod, the preferred Yi Shi yeast belong zymic culture.

Yi Shi yeast belong yeast can preferably use Issatchankia scutulata var.scutulata, for example the carbonyl reductase from Issatchankia scutulata var.scutulata JCM 1828 bacterial strains uses as carbonyl reductase of the present invention, and is then excellent especially in the preparation of optical activity alcohol.This bacterial strain can be preserved facility (JapanCollection of Microorganism (JCM)) available from Japanese RIKEN microflora.

When being used for preparation (S)-2-amylalcohol with 2 pentanone, the DNA of carbonyl reductase or this enzyme of encoding preferably uses from the carbonyl reductase of the microorganism that belongs to Brettanomyces genus, mycocandida, Hortaea, rood yeast belong or Pichia or the DNA of this enzyme of encoding.

Further preferably use from Brussels Brettanomyces, candida tropicalis, candida zeylanoides, Hortaea werneckii, Lodderomyces elongisporus, Pichiasegobiensis, the Spartacus pichia spp, Arthrobacter globiformis, the oxidation Arthrobacter, the polychrome Arthrobacter, the wilting bacillus pumilis, the stearothermophilus ground bacillus, brick-red microbacterium, human pallid bacillus, pale Bacillaceae certain (ovum shape pseudomonas), the carbonyl reductase of radiation root nodule bacterium or the DNA of this enzyme of encoding.

Specifically, especially preferably use from Brussels Brettanomyces NBRC 0629, Brussels Brettanomyces NBRC 0797, candida tropicalis NBRC 0006, candida zeylanoides CBS 6408, candida zeylanoides JCM 1627, Hortaea werneckii NBRC4875, Lodderomyces elongisporus NBRC 1676, Pichia segobiensis JCM10740, Spartacus pichia spp JCM 10741, Arthrobacter globiformis NBRC 12137, oxidation Arthrobacter DSM 20120, polychrome Arthrobacter DSM 342, wilting bacillus pumilis ATCC12813, stearothermophilus ground bacillus NBRC 12550, stearothermophilus ground bacillus IAM11002, stearothermophilus ground bacillus IAM 11004, stearothermophilus ground bacillus IAM12043, brick-red microbacterium JCM 1353, human pallid bacillus ATCC 49237, certain (ovum shape pseudomonas) NBRC 12950 of pale Bacillaceae, certain (ovum shape pseudomonas) NBRC 12952 of pale Bacillaceae, certain (ovum shape pseudomonas) NBRC 12953 of pale Bacillaceae, the carbonyl reductase of radiation root nodule bacterium IAM 12048 or the DNA of this enzyme of encoding.

When being used for preparation (S)-2-hexanol with methyl-n-butyl ketone, the DNA of carbonyl reductase or this enzyme of encoding preferably uses from the carbonyl reductase of the microorganism that belongs to Brettanomyces genus, mycocandida, Yi Shi yeast belong, rood yeast belong, Pichia or Rhodotorula or the DNA of this enzyme of encoding.

Wherein especially preferably use from the carbonyl reductase of different Brettanomyces, candida famata, Crewe Si Shi candiyeast, maltose candiyeast, candida zeylanoides, Issatchankia scutulata, Lodderomyces elongisporus, Angus pichia spp, Pichia cactophila, Pichiasegobiensis, happiness trehalose pichia spp and little rhodotorula or the DNA of this enzyme of encoding.

Specifically, preferably use from different Brettanomyces NBRC 0627, candida famata ATCC 10539, Crewe Si Shi candiyeast NBRC 1664, Crewe Si Shi candiyeast JCM2284, Crewe Si Shi candiyeast JCM 2341, maltose candiyeast NBRC 1977, candida zeylanoides CBS 6408, Issatchankia scutulata var.scutulata JCM 1828, Lodderomyces elongisporus NBRC 1676, Angus pichia spp NBRC 1024, Angus pichia spp NBRC 1071, Pichia cactophila JCM 1830, Pichiasegobiensis JCM 10740, happiness trehalose pichia spp JCM 3651, and little rhodotorula NBRC 0879, certain DSM 20407 of genus arthrobacter, sulphur look Arthrobacter (Sulfur tyrothricin (Brevibacterium sulfureum)) JCM 1338, Brevibacterium butanicumATCC 21196, Sulfur tyrothricin JCM 1485, wilting bacillus pumilis ATCC 12813, separate cutin microbacterium NBRC 13309, longicorn microbacterium JCM 1352, certain NBRC15615 of Microbacterium, human pallid bacillus ATCC 49237, certain (ovum shape pseudomonas) NBRC 12952 of pale Bacillaceae, certain (ovum shape pseudomonas) NBRC 12953 of pale Bacillaceae, Paracoccus denitrificans NBRC 12442, radiation root nodule bacterium IAM 12048, radiation root nodule bacterium IAM 13129, the carbonyl reductase of certain ATCC 15960 of Rhod or the DNA of this enzyme of encoding.

The method that obtains carbonyl reductase from the culture of microorganism can be used conventional enzyme purification method, for example can carry out in accordance with the following methods.Mentioned microorganism is cultivated on conventional substratum in yeast culture such as YM substratum, treat that fully reclaim the propagation back, have in the damping fluid of DTT reductive agents such as (dithiothreitol (DTT)) or phenylmethylsulfonyl fluoride proteinase inhibitor such as (PMSF) brokenly in adding, make cell-free extract.Will be according to the appropriate combination such as affinity chromatography of the classification (saltouing etc.) of protein solubility or cationic exchange, anionresin, gel-filtration, hydrophobic, hydroxyapatite chromatography, chelating, dyestuff, use antibody etc. by organic solvent deposit or by sulfuric acid amine etc., can be from cell-free extract purifying.

Shown in the embodiment of this specification sheets, anion-exchange chromatography by using DEAE Sepharose Fast Flow (Amersham Biosciences manufacturings) to carry out, use the hydrophobic interaction chromatogram that ButylSepharose4 Fast Flow (Amersham Biosciences manufacturings) carries out, the anion-exchange chromatography that use MonoQ (Amersham Biosciences manufacturing) carries out, the gel filtration chromatography that use Superdex 200 (Amersham Biosciences manufacturing) carries out etc., can be purified to electrophoresis detection and be single band almost.

As mentioned above purifying, contain a kind of subunit from the carbonyl reductase of Issatchankia scutulata var.scutulata JCM1828 bacterial strain (following this enzyme can be called " IsADH1 "), the molecular weight that this subunit is measured according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter to be referred as SDS-PAGE) is about 40,000Da, the definite molecular weight of gel-filtration that carries out with Superdex 200 HR10/30 (Amersham Biosciences manufacturing) is about 40,000Da.More than can inference: IsADH1 be contain a kind of about 40, the monomer of the subunit of 000Da.

The DNA of coding carbonyl reductase for example can separate by following method.

At first, wait the purifying carbonyl reductase according to the method described above, analyze the N-terminal aminoacid sequence then, cut off by enzymes such as lysyl endopeptidase, V8 proteolytic enzyme again, by purified peptide fragments such as reversed-phase liquid chromatographies, by the protein sequencer analysis of amino acid sequence, can determine a plurality of aminoacid sequences thus.

According to the aminoacid sequence design PCR primer of determining, be template with the chromosomal DNA or the cDNA library of the microorganism strains of producing carbonyl reductase, use the PCR primer that designs by aminoacid sequence, carry out PCR, can obtain the part of DNA of the present invention thus.Be probe with the gained dna fragmentation again, the restriction enzyme digestion product of chromosomal DNA of producing the microorganism strains of carbonyl reductase is imported phage, plasmid etc., transformed into escherichia coli, utilize gained library or cDNA library, by colony hybridization, plaque hybridization etc., the DNA of the carbonyl reductase that can obtain encoding.

Base sequence to the dna fragmentation that obtained by PCR is analyzed, the PCR primer that is used for extension outside known DNA by the gained sequences Design, use the cDNA of the microorganism strains of producing carbonyl reductase, by RACE method (cDNA terminal rapid amplifying) (MolecularCloning 3rd Ed., Cold Spring Harbor Laboratory Press, hereinafter referred to as " molecular cloning "), also can obtain DNA of the present invention.

Like this, the base sequence of the DNA of, coding carbonyl reductase IsADH1 isolating by the chromosomal DNA of Issatchankia scutulata var.scutulata JCM 1828 bacterial strains is shown in SEQID NO.2.

The DNA of coding carbonyl reductase IsADH1 removes by aforesaid method cloned genes group DNA or the cDNA, and also as described in this manual, its base sequence has been clearly, so also can wait by the chemosynthesis based on SEQ ID NO.2 and obtain.

The homologue of DNA of coding IsADH1 is meant in not damaging the active scope of carbonyl reductase, has in the aminoacid sequence of SEQ ID NO.1 through disappearance, replaces or add the aminoacid sequence that one or more amino acid obtain.Here, " a plurality of " specifically refer to 20 or below, preferred 10 or below, more preferably 5 or below.

The homologue of IsADH1 be meant have with aminoacid sequence at least 50% shown in the SEQ ID NO.1 above, preferred 70% or above, more preferably 80% or the protein of above homology.

In passing, above-mentioned proteinic homology search can be an object with DNA Databank ofJAPAN (DDBJ) etc. for example, uses FASTA or BLAST supervisor to carry out.Use the aminoacid sequence of SEQ ID NO.1, with DDBJ is object, use blast program to carry out the homology retrieval, the result is in known protein matter, that show highest homology is protein Ydr541cp protein (SEQ ID NO.3: searching number AAB64983), show 42% homology from the Unknown Function of Saccharomyces cerevisiae (Saccharomyces cerevisiae).

The DNA of coding IsADH1 is DNA or its homologue of the above-mentioned IsADH1 of coding, is that coding has the active protein DNA of carbonyl reductase.

The above-mentioned protein DNA of encoding for example has the DNA that comprises base sequence shown in the SEQ ID NO.2.

The homologue of DNA of coding IsADH1 comprises following DNA: this dna encoding do not damage the active scope of carbonyl reductase contain in the aminoacid sequence of SEQ ID NO.1 through disappearance, replace or add one or more amino acid and the protein of aminoacid sequence.Here, a plurality of specifically be meant 60 or below, preferred 30 or below, more preferably 10 or below.

Those skilled in the art are by using site-directed mutagenesis method (Nucleic Acids Res., 10 volumes, 6487 pages (1982), Methods in Enzymol., 100 volumes, 448 pages (1983), MolecularCloning, PCR-A Practical Approach, IRL Press, 200 pages (1991)) etc., in the DNA of SEQ ID NO.2, import suitably and replace, disappearance, insert and/or add variation, the homologue of the DNA of the IsADH1 that then can obtain encoding.

According to aminoacid sequence or its part of IsADH1 or DNA or its part of the IsADH1 that encodes, for example in DNA Databank of JAPAN databases such as (DDBJ), carry out homology search, also can obtain the base sequence information of the dna homology thing of coded protein of the present invention.Those skilled in the art obtain this dna fragmentation according to this base sequence information by the PCR to preservation strain.

The all right following acquisition of the homologue of the DNA of coding IsADH1: the DNA of use coding IsADH1 or its part are as probe, by colony hybridization method, plaque hybridization method or southern blotting technique hybrid method etc., under stringent condition with by DNA, hybridize, obtain the DNA of hybridization with the active any microorganism preparation of carbonyl reductase." part " of the DNA of coded protein of the present invention is that its length is enough to the DNA that uses as probe, be specially 15bp or more than, preferred 50bp or more than, more preferably 100bp or more than.

Various hybridization can be carried out according to the method for record in " molecular cloning " etc.

In this specification sheets, " DNA of hybridize under stringent condition " is meant and uses DNA as probe, under stringent condition, by using the colony hybridization method, the plaque hybridization method, perhaps southern blotting technique hybrid method etc. and the base sequence of the DNA that obtains, about stringent condition, for example in colony hybridization method and plaque hybridization method, be to use and be fixed with from the DNA of colony or plaque or the filter membrane of this dna fragmentation, in the presence of 0.7-1.0M sodium-chlor, hybridize at 65 ℃, (1 * SSC consists of 150mM sodium-chlor to use 0.1-2 * SSC solution then, the 15mM Trisodium Citrate), the condition of washing filter membrane under 65 ℃ condition.

By DNA isolating as mentioned above, the coding carbonyl reductase is inserted known expression vector in effable mode, can provide the carbonyl reductase expression vector.By cultivating the transformant that transforms with this expression vector, can obtain carbonyl reductase by this transformant.Perhaps, transformant also can be incorporated in the known host chromosome DNA in effable mode by the DNA of the carbonyl reductase of will encoding and obtain.

The preparation method of transformant is specific as follows: DNA of the present invention is imported can be in microorganism in the plasmid vector or phage vector of stable existence, the expression vector that makes up is imported in this microorganism, the dna direct of carbonyl reductase of perhaps will encoding imports in the host genome, makes this genetic information transcribe, translate.

At this moment, if the DNA of coding carbonyl reductase do not contain can be in host microorganism expression promoter, then need suitable promotor is incorporated into 5 '-one side upstream of DNA chain of the present invention, more preferably also terminator is incorporated into 3 '-one side downstream.This promotor and terminator are so long as known can getting final product at promotor and the terminator as performance function in host's the microorganism, be not particularly limited, about these carrier, promotor and terminators etc. that can in various microorganisms, utilize, for example in " microbiology basis lecture 8 hereditary sub-engineerings are upright altogether publishes " description is being arranged, particularly about yeast, at Adv.Biochem.Eng.43,75-102 (1990), Yeast 8 are documented among the 423-488 (1992) etc.

Be used to make carbonyl reduction expression of enzymes of the present invention, as the host microorganism that transforms object so long as host itself does not have detrimentally affect to get final product to this reaction, be not particularly limited, microorganism shown below is specifically arranged.

The bacterium that the host carrier system of being established that belongs to Escherichia (Escherichia), bacillus (Bacillus), Rhodopseudomonas (Pseudomonas), serratia (Serratia), brevibacterium sp (Brevibacterium), corynebacterium (Corynebaterium), streptococcus (Streptococcus), lactobacillus (Lactobacillus) etc. obtains.

The actinomycetes that the host carrier system of being established that belongs to Rhod (Rhodococcus), streptomyces (Streptomyces) etc. obtains.

That is established belongs to saccharomyces (Saccharomyces), Crewe Vickers yeast belong (Kluyveromyces), the fragmentation saccharomyces, fragmentation saccharomyces (Schizosaccharomyces), detection of zygosaccharomyces (Zygosaccharomyces), Ye Shi yeast belong (Yarrowia), Trichosporon (Trichosporon), red teliosporeae (Rhodosporidium), Hansenula anomala belongs to (Hansenula), Pichia (Pichia), the yeast that the host carrier system of mycocandida (Candida) etc. obtains.

The mould that the host carrier system of being established that belongs to neurospora (Neurospora), Aspergillus (Aspergillus), Cephalosporium (Cephalosporium), Trichoderma (Trichoderma) etc. obtains.

In the mentioned microorganism, the preferred Escherichia of host, bacillus, brevibacterium sp, corynebacterium, preferred especially Escherichia, corynebacterium.

Transformant preparation order is suitable for host's structure of recombinant vectors and host's cultural method and can carries out (for example method of record in " molecular cloning ") according to technology commonly used in molecular biology, biotechnology, the field of genetic engineering.

Below, specifically provide the example of the preferred method for transformation, carrier, promotor, terminator etc. of preferred host microorganism, each microorganism, but the present invention is not limited to these examples.

In the Escherichia, particularly in the bacillus coli (Escherichia coli), it is plasmid that plasmid vector has pBR, pUC, and promotor from lac (beta-galactosidase enzymes), trp (tryptophan operon), tac, trc (fusion of lac, trp), lambda particles phage PL, PR etc. etc. is arranged.Terminator has from trpA, from phage, from terminator of rrnB RNA etc.

In the bacillus, carrier can be that pUB110 is that plasmid, pC194 are plasmid etc., can also be incorporated in the karyomit(e).Promotor and terminator can utilize the promotor of enzyme genes such as Sumizyme MP, neutral protease, α-Dian Fenmei or terminator etc.

In the Pseudomonas, the host carrier system of the routine of the pseudomonasputida that carrier is set up to some extent (PseudomonasPutida), onion pseudomonas (Pseudomonas cepacia) etc. or the plasmid relevant with the decomposition of toluene compound, based on broad host range carrier (comprising the required gene of the self-replicating) pKT240 (Gene of TOL plasmid from RSF1010 etc., 26,273-82 (1983)).

In brevibacterium sp, the particularly brevibacterium (Brevibacterium lactofermentum), carrier can exemplify pAJ43 plasmid vectors such as (Gene, 39,281 (1985)).Promotor and terminator can utilize various promotors and the terminator that uses in intestinal bacteria.

In corynebacterium, the particularly corynebacterium glutamicum (Corynebacterium glutamicum), carrier has: pCS11 (spy opens clear 57-183799), pCB101 plasmid vectors such as (Mol.Gen.Genet.196,175 (1984)).

In saccharomyces, the particularly Saccharomyces cerevisiae, carrier has: YRp system, YEp system, YCp system, YIp are plasmid.Can utilize promotor, the terminator of various enzyme genes such as alcoholdehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, acid phosphatase, beta-galactosidase enzymes, phosphoglyceric kinase, Hydratase, phosphoenolpyruvate.

In the fragmentation saccharomyces, carrier can exemplify Mol.Cell.Biol.6, the plasmid vector from grain wine fragmentation sugar yeast of 80 (1986) middle records.Particularly, pAU224 is sold by the precious wine company that makes, and can easily utilize.

In the Aspergillus; black aspergillus (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae) etc. obtain the research of fullest in mould; can utilize to plasmid or in karyomit(e) and integrate; also can be used to from thalline exoproteinase or diastatic promotor (Trends inBiotechnology 7,283-287 (1989)).

Except that above-mentioned, as long as set up the host carrier system that adapts to various microorganisms, then they can suitably use.

Except that microorganism, various host's carrier decorum in plant, animal, have also been made up, particularly (Nature 315 in the animals such as insect of using silkworm, 592-594 (1985)) or made up the system of great expression heterologous protein and the system that uses cell-free protein synthetic systems such as intestinal bacteria cell-free extract or wheatgerm in the plants such as vegetable seed, corn, potato, they all can preferably utilize.

The present invention is by making transformant, this transformant handled thing and/or nutrient solution and reaction substrate 2 pentanone or the methyl-n-butyl ketone effect that is obtained by aforesaid method etc., can make the carbonyl asymmetric reduction of this compound, preparation (S)-2-amylalcohol or (S)-2-hexanol, wherein above-mentioned transformant is that the transformant of possessing recombinant DNA maybe is incorporated into this DNA in the chromosomal DNA and the transformant that obtains, and wherein said recombinant DNA is incorporated into the DNA of proteinic base sequence with aminoacid sequence of coding tool SEQ ID NO.1 in the carrier and obtains.

The present invention can be with following transformant, or above-mentioned recombinant DNA is incorporated into the transformant that obtains in the chromosomal DNA, this transformant handled thing and/or nutrient solution and reaction substrate 2 pentanone or methyl-n-butyl ketone effect, make the carbonyl asymmetric reduction of this compound, preparation (S)-2-amylalcohol or (S)-2-hexanol, wherein said transformant possess will have the DNA of base sequence of coding following proteins be incorporated in the carrier and the recombinant DNA that obtains, described protein is to contain aminoacid sequence with SEQ IDNO.1 to have 50% or the aminoacid sequence of above homology, and has reducing carbonyl, the protein of the ability of synthesis of optically active alcohol.

The present invention belongs to by being selected from Brettanomyces, mycocandida, Hortaea, the Yi Shi yeast belong, the rood yeast belong, Pichia, Rhodotorula, genus arthrobacter, brevibacterium sp, Curtobacterium, ground bacillus belongs to, Microbacterium, pale Bacillaceae, paracoccus, rhizobium, the microorganism of Rhod, this microbiological treatment thing, thick purifying thing or purifying thing and the reaction substrate 2 pentanone or the methyl-n-butyl ketone effect of this microbial culture medium and/or the carbonyl reductase component that obtains by this microorganism, carbonyl that can this compound of asymmetric reduction, preparation (S)-2-amylalcohol or (S)-2-hexanol.

When being used for preparation (S)-2-amylalcohol with 2 pentanone, the preferred use belongs to the Brettanomyces genus, mycocandida, Hortaea, the microorganism of rood yeast belong or Pichia, especially preferably use Brussels Brettanomyces, candida tropicalis, candida zeylanoides, Hortaeawerneckii, Lodderomyces elongisporus, Pichia segobiensis, the Spartacus pichia spp, Arthrobacter globiformis, the oxidation Arthrobacter, the polychrome Arthrobacter, the wilting bacillus pumilis, the stearothermophilus ground bacillus, brick-red microbacterium, human pallid bacillus, pale Bacillaceae certain (ovum shape pseudomonas), the radiation root nodule bacterium, concrete preferred use the: Brussels Brettanomyces NBRC0629, Brussels Brettanomyces NBRC 0797, candida tropicalis NBRC 0006, candida zeylanoides CBS 6408, candida zeylanoides JCM 1627, Hortaea werneckiiNBRC 4875, Lodderomyces elongisporus NBRC 1676, Pichia segobiensisJCM 10740, Spartacus pichia spp JCM 10741, Arthrobacter globiformis NBRC 12137, oxidation Arthrobacter DSM 20120, polychrome Arthrobacter DSM 342, wilting bacillus pumilis ATCC12813, stearothermophilus ground bacillus NBRC 12550, stearothermophilus ground bacillus IAM11002, stearothermophilus ground bacillus IAM 11004, stearothermophilus ground bacillus IAM12043, brick-red microbacterium JCM 1353, human pallid bacillus ATCC 49237, certain (ovum shape pseudomonas) NBRC 12950 of pale Bacillaceae, certain (ovum shape pseudomonas) NBRC 12952 of pale Bacillaceae, certain (ovum shape pseudomonas) NBRC 12953 of pale Bacillaceae, radiation root nodule bacterium IAM 12048.

When being used for preparation (S)-2-hexanol, preferably belong to the microorganism of Brettanomyces genus, mycocandida, Yi Shi yeast belong, rood yeast belong, Pichia or Rhodotorula with methyl-n-butyl ketone.

Wherein, preferred especially different Brettanomyces, candida famata, Crewe Si Shi candiyeast, maltose candiyeast, candida zeylanoides, Issatchankia scutulata, Lodderomyces elongisporus, Angus pichia spp, Pichia cactophila, Pichiasegobiensis, happiness trehalose pichia spp and the little rhodotorula of using.

Concrete preferred use the: different Brettanomyces NBRC 0627, candida famata ATCC10539, Crewe Si Shi candiyeast NBRC 1664, Crewe Si Shi candiyeast JCM 2284, Crewe Si Shi candiyeast JCM 2341, maltose candiyeast NBRC 1977, candida zeylanoides CBS 6408, Issatchankia scutulata var.scutulata JCM 1828, Lodderomyces elongisporus NBRC 1676, Angus pichia spp NBRC 1024, Angus pichia spp NBRC 1071, Pichia cactophila JCM 1830, Pichiasegobiensis JCM 10740, happiness trehalose pichia spp JCM 3651, and little rhodotorula NBRC 0879, certain DSM 20407 of genus arthrobacter, sulphur look Arthrobacter (Sulfur tyrothricin) JCM 1338, Brevibacterium butanicum ATCC 21196, Sulfur tyrothricin JCM1485, wilting bacillus pumilis ATCC 12813, separate cutin microbacterium NBRC 13309, longicorn microbacterium JCM 1352, certain NBRC 15615 of Microbacterium, human pallid bacillus ATCC49237, certain (ovum shape pseudomonas) NBRC 12952 of pale Bacillaceae, certain (ovum shape pseudomonas) NBRC 12953 of pale Bacillaceae, Paracoccus denitrificans NBRC 12442, radiation root nodule bacterium IAM 12048, radiation root nodule bacterium IAM 13129, certain ATCC 15960 of Rhod.

Reaction substrate 2 pentanone or methyl-n-butyl ketone are 0.01-90%w/v at concentration of substrate usually, use in the scope of preferred 0.1-30%w/v.Reaction substrate can be in reaction disposable interpolation when initial, but when enzyme has inhibition to substrate, from the angle that reduces influence or improve product and accumulate the angle of concentration and consider continuously preferred or intermittent the interpolation.

Among the preparation method of the present invention, make above-mentioned transformant or have the active microorganism of carbonyl reduction and carbonyl containing compounds (reaction substrate) such as 2 pentanone or methyl-n-butyl ketone are done the time spent, can directly use this transformant or microorganism cells, also can use this cell handled thing for example to carry out that freeze-drying cell handled thing that handle or that obtain after physics or enzyme fragmentation, the carbonyl reductase component in this cell extract with the form of thick purifying thing or purifying thing or they are fixed on polyacrylamide gel, carrageenin gel etc. to the material on the carrier of representative etc.The amount of transformant that adds in reaction solution or microorganism cells and/or this cell handled thing is to count about 0.1-50%w/v according to wet thallus weight usually with the concentration of this cell, preferred 1-20%w/v, add in the reaction solution, during prepared products such as use enzyme, can obtain the specific activity of enzyme, interpolation can reach the amount of above-mentioned cell concn.

Among the preparation method of the present invention, preferably add coenzyme NAD P ⁺Or NADPH or NAD ⁺Perhaps NADH about the concentration of coenzyme, adds 0.001mM-100mM usually, preferred 0.01-10mM.

When adding above-mentioned coenzyme, the NADP that will generate by NADPH (NADH) ⁺(NAD ⁺) being regenerated as NADPH (NADH), this can enhance productivity, thereby preferred, and renovation process has following several: the NADP that 1) utilizes host microorganism self ⁺(NAD ⁺) and the method for proper energy; 2) in reaction system, add and have by NADP ⁺(NAD ⁺) generate the method that the microorganism of ability of NADPH (NADH) or its handled thing or glucose dehydrogenase, hydrogenlyase, alcoholdehydrogenase, amino acid dehydrogenase, organic acid desaturase (malate dehydrogenase (malic acid dehydrogenase) etc.) etc. can be used for the regenerated enzyme (regeneration enzyme) of NADPH (NADH); (3) during transformant, will can be used for the regenerated enzyme of NADPH (NADH) in preparation---the gene of above-mentioned regeneration enzyme and DNA of the present invention import the host simultaneously and make the method etc. of its expression.

Wherein, in the method for above-mentioned (1), preferably in reaction system, add glucose or ethanol, formic acid etc.

In the method for above-mentioned (2), can use the microorganism that contains above-mentioned regeneration enzyme, with this microbial cells after acetone treatment, freeze-drying handles the bacterial disposing thing that back, physics or enzyme fragmentation etc. make, this enzyme component is extracted with the form of thick purifying thing or purifying thing, and they are fixed in polyacrylamide gel, carrageenin gel etc. is to use or the like on the carrier of representative, also can use the enzyme of market sale.

Specifically, in this case, the usage quantity of above-mentioned regeneration enzyme and carbonyl reductase of the present invention relatively in enzymic activity, add 0.01-100 doubly usually, preferred about 0.5-20 times.

Also must add compound as the substrate of above-mentioned regeneration enzyme, glucose when for example utilizing glucose dehydrogenase, the formic acid when utilizing hydrogenlyase, the ethanol when utilizing alcoholdehydrogenase or Virahol etc., its addition is with respect to reaction raw materials 2 pentanone or methyl-n-butyl ketone, usually add 0.1-20 times of molar equivalent, preferred 1-5 times of molar equivalent.

For the method for above-mentioned (3), can adopt following method: the DNA of the carbonyl reductase of will encoding and the DNA of above-mentioned regeneration enzyme are incorporated into the method in the karyomit(e); In single carrier, import two kinds of DNA, transform host's method; And two kinds of DNA are imported to respectively in the different carriers, transform host's method then; When adopting two kinds of DNA are imported different carriers respectively, when transforming host's method then, must consider the uncompatibility between two kinds of carriers, suitably select carrier.

When importing a plurality of gene in single carrier, can adopt the method that the relevant range is connected with each gene is respectively controlled in expression such as promotor and terminator, perhaps the form that contains polycistronic operon with lactose operon etc. makes its expression.

Preparation method of the present invention be contain reaction substrate and express the thick purifying thing of transformant, this cell handled thing, this cell culture fluid of DNA of coding carbonyl reductase and/or the carbonyl reductase component that obtains by this cell or the aqueous medium of purifying thing and various coenzyme that add as required and regeneration system rapidly thereof in or carry out in the mixture of this aqueous medium and organic solvent.

Preparation method of the present invention is containing reaction substrate and Brettanomyces genus, mycocandida, Hortaea, the Yi Shi yeast belong, the rood yeast belong, Pichia, Rhodotorula, genus arthrobacter, brevibacterium sp, Curtobacterium, ground bacillus belongs to, Microbacterium, pale Bacillaceae, paracoccus, rhizobium, Rhod have an active microorganism of carbonyl reductase, this microbiological treatment thing, this microbial culture medium, and/or the thick purifying thing or the purifying thing of the carbonyl reductase component that obtains by this microorganism, and carry out in the aqueous medium of various coenzyme that add as required and regeneration system rapidly thereof or in the mixture of this aqueous medium and organic solvent.

Aqueous medium has water or damping fluid.Damping fluid has: sodium phosphate or potassiumphosphate, Tris, sodium acetate, Trisodium Citrate etc., organic solvent can use the high solvents of reaction substrate solubleness such as ethyl acetate, butylacetate, toluene, chloroform, normal hexane, dimethyl sulfoxide (DMSO).

Method of the present invention under 4-60 ℃, preferred 10-45 ℃ temperature of reaction, is carried out among pH3-11, the preferred pH 5-8 usually usually.Reaction times was generally about 1-72 hour.Also can utilize membrane reactor to carry out.

The optical activity alcohol that the method according to this invention generates can following purifying: behind the reaction terminating, separate thalline or protein in the reaction solution by centrifugal, film processing etc., will distill with organic solvent extractions such as ethyl acetate, toluene then, column chromatography, appropriate combination such as partial crystallization are carried out purifying.

2. the preparation method of optical activity 3-methyl carboxylic acids

Among the preparation method of the present invention, optical activity 3-methyl carboxylic acids shown in the following general formula (5) is at high polar solvent and/or promotes to prepare by the optical activity 1-methyl alkyl propanedioic acid shown in the above-mentioned general formula (1) is carried out decarboxylation in the presence of the additive of decarboxylation.

In the above-mentioned general formula (1), R ¹Be straight chain, side chain or cyclic alkyls such as the n-propyl of carbonatoms 3-5, normal-butyl, n-pentyl, sec.-propyl, isobutyl-, isopentyl, cyclopentyl.Wherein preferred n-propyl, normal-butyl, n-pentyl, sec.-propyl, isobutyl-, further preferred n-propyl or normal-butyl.

* represent unsymmetrical carbon, it can be any configuration of R configuration and S configuration, preferred R configuration, its optical purity be generally 80%e.e. or more than, preferred 90%e.e. or more than, during in particular as medical material or intermediate, require high-optical-purity, therefore further preferred 95%e.e. or more than, preferred especially 99%e.e. or more than.

Above-mentioned decarboxylic reaction can heat in the presence of the additive of high polar solvent and/or promotion decarboxylation, also can carry out heat in the presence of the low polar solvent or under solvent-free, consider from the easy degree of industrializing implementation, more preferably in the presence of the additive of high polar solvent and/or promotion decarboxylation, temperature of reaction is controlled at lower.Usually decarboxylic reaction must carry out under 150 ℃ of-200 ℃ of hot conditionss, and the operating limit temperature near common glass lined reaction vessel heats up, cools off also spended time, and a large amount of heats must be arranged, and therefore makes the advantage of temperature of reaction low temperatureization very big.In addition, usually optical activity 1-methyl alkyl propanedioic acid is solid at normal temperatures, when reacting under solvent-free, must heat fusion after joining reactive tank.But only adding under the solid state, in common reactive tank, can't stir, promptly not heat and do not stir, heat transfer efficiency extreme difference then, the part danger out of control that responds that heats up in addition therefore can't practical application.Therefore the preferred solvent that uses is handled optical activity 1-methyl alkyl propanedioic acid with solution or slurry state.

On the other hand, reaction produces great amount of carbon dioxide, and therefore when industrially scalable was implemented, for guaranteeing security, the generation speed of control carbonic acid gas was very important.

Consider from the generation speed angle of pressing the system carbonic acid gas, preferably will dissolve the solution of optical activity 1-methyl alkyl propanedioic acid or continuous dropping of optical activity 1-methyl alkyl propanedioic acid of heating fusion and react.Dripping method is controlled reaction easily, and can use the popular response device.In addition, also the optical activity 3-methyl carboxylic acids that generates in the reaction can be used as solvent, at this moment, owing to do not contain the solvent that must remove after responding etc., the purifying burden is little, and is therefore preferred.

Can also use than the high solvent of optical activity 3-methyl carboxylic acids boiling point that generates.By using than the high solvent of the boiling point of product as reaction solvent, during distillation reaction liquid, optical activity 3-methyl carboxylic acids distillates to become to fractionate out as first, the problem of sneaking into of the low boiling point component that occurs in the time of therefore can avoiding using low boiling point solvent, and by high boiling solvent is remained in the distil container as the high boiling point thinner, can distillate target product more, improve distillation efficiency.

Can also carry out reaction distillation, that is, under reduced pressure, to reaction vessel in drip optical activity 1-methyl alkyl propanedioic acid solution and make its reaction on one side, heat up in a steamer the optical activity 3-methyl carboxylic acids that generates on one side continuously.The reaction distillation method is controlled reaction easily, and heat-up time is short, can suppress the impurity that produces because of long-time heating, so preferably.

Flow method by in the reaction unit that optical activity 1-methyl alkyl propanedioic acid solution is passed into heating can be transformed into optical activity 3-methyl carboxylic acids.In the flow method, the solution of reaction unit is supplied with, can be controlled carbonic acid gas generation speed by control.In addition heat-up time short, therefore can suppress the impurity that produces because of long-time heating, so preferably.The reaction unit of flowing reactive preferably uses tubular reactor, film distillator or multi-stage type grooved flow reactor.When adopting film distillator, can under reduced pressure react.

The solvent that uses in the above-mentioned decarboxylic reaction has: butyl ether, tetrahydrofuran (THF), 1, ether series solvents such as 2-glycol dimethyl ether, two  alkane, polyoxyethylene glycol, Polyethylene glycol dimethyl ether, polytetrahydrofuran; Halogen such as tetracol phenixin, dichlorobenzene series solvent; Pure series solvent such as butanols, ethylene glycol; Ester series solvents such as ethyl acetate, dioctyl phthalate (DOP), diisononyl phthalate, phthalic acid two (tridecyl) ester, trioctyl trimellitate; Nitrile such as acetonitrile, propionitrile series solvent; Hydrocarbon system solvents such as toluene, dimethylbenzene, the tetradecane, tridecane, whiteruss, monomethyl naphthalene, isopropyl biphenyl, dibenzyl toluene, hydrogenation triphen, silicone oil; Acid amides such as dimethyl formamide, N-Methyl pyrrolidone series solvent; Organic acid such as formic acid, acetate series solvent; Pyridine, 2, basic solvents such as 6-lutidine, triethylamine; Dimethyl sulfoxide (DMSO); Water etc.Also the multiple solvent that is selected from wherein can be mixed use with arbitrary proportion.In order to dissolve high polarity substrate or additive, preferred high polar solvent acetonitrile, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), pyridine, acetate, water etc., special big non-protonic solvent dimethyl sulfoxide (DMSO), the pyridine of preferred reaction acceleration effect.

In addition, high boiling solvents such as the tetradecane, tridecane, polyoxyethylene glycol, Polyethylene glycol dimethyl ether, polytetrahydrofuran, dioctyl phthalate (DOP), phthalic acid diisononyl esters, phthalic acid two (tridecyl) ester, trioctyl trimellitate, whiteruss, monomethyl naphthalene, isopropyl biphenyl, dibenzyl toluene, hydrogenation triphen, silicone oil can be used as the use of high boiling point thinner when distillation.

The usage quantity of solvent can be used the solvent of any amount, is 1-20 times of volume with respect to the raw material substrate usually, preferred 1-5 times of volume.

Also can use the additive that is used to promote to react as solvent.At this moment, for making the dissolving of optical activity 1-methyl alkyl propanedioic acid or suspending, usually, use 0.5-20 times of volume with respect to substrate.Heat up in a steamer in order to alleviate solvent, burden such as recovery, preferred 0.5-3 times of volume.

The additive that uses in the above-mentioned decarboxylic reaction has: mineral acids such as sulfuric acid, hydrochloric acid; Inorganic salt such as sodium-chlor, lithium chloride; Organic salt such as sodium acetate, ammonium formiate; Prussiate such as sodium cyanide, cupric cyanide; Heavy metallic salt such as cupric chloride, iron(ic) chloride; Heavy metal oxide such as cupric oxide, silver suboxide; Pyridine, 2,6-lutidine, triethylamine, benzylamine, 1,8-diazabicyclo [5.4.0]-7-undecylene, 1, organic basess such as 4-diazabicyclo [2.2.2] octane; Mineral alkalis such as sodium hydroxide, calcium hydroxide, salt of wormwood; Acid anhydrides such as diacetyl oxide, fumaric acid anhydride; Deng.They can be with mixed use arbitrarily.Preferred heavy metallic salt, heavy metal oxide, organic bases, acid anhydrides and their mixture, further preferred cupric oxide, pyridine, 2,6-lutidine, diacetyl oxide and their mixture, preferred especially cheap and the reaction acceleration effect is big, can be by distillation and the isolating pyridine of target compound.

The amount of employed additive is 0.01-50wt% with respect to substrate usually, for fear of the violent generation of carbonic acid gas, and alleviates the purifying burden, and preferred the inhibition is necessary bottom line, preferred 0.01-5wt%.

Temperature of reaction is generally 30-200 ℃, according to the reaction conditionss such as type of the having or not of additive, employed additive, temperature required having nothing in common with each other.Consider that from the industrialization angle over-drastic pyroreaction is restricted on device, be difficult to carry out that simultaneously, intensification, cooling need long-time, thus not preferred, preferred 30-150 ℃, further preferred 30-110 ℃.

The optical activity 3-methyl carboxylic acids that makes in the above-mentioned reaction is preferably by method purifying such as distillation and/or extractions.

3. the preparation method of optical activity 1-methyl alkyl propanedioic acid

Optical activity 1-methyl alkyl propanedioic acid of the present invention is that the optical activity alcohol shown in the following general formula (2) is transformed to alkylsulfonyl oxygen base, obtains the optically active compound shown in the following general formula (3),

React with the carbon nucleophile shown in the following general formula (9) then,

To make again that the optically active compound hydrolysis prepares shown in the following general formula (4).

R in above-mentioned general formula (2), (3) and (4) ¹Aforementioned identical with this specification sheets.

In above-mentioned general formula (4) and (9), R ²And R ³Be ester group, carboxyl or cyano group independently of one another, preferred ester group.Here, R ²And R ³Can form 5-(1-methyl alkyl)-2 together, 2-dimethyl-1,3-two  alkane-4, ring texturees such as 6-diketone.

The pure composition of this ester group is not particularly limited straight chain, side chain or cyclic alkyl alcohol such as particular methanol, ethanol, propyl carbinol, Virahol, hexalin so long as reaction is not produced dysgenic group and do not get final product; Aryl alcohol such as phenol, naphthols, further particular methanol or ethanol.

In the above-mentioned general formula (3), X is sulfonyloxies such as mesyloxy, tosyloxy, oil of mirbane sulfonyloxy, chloromethane sulfonyloxy, trifluoro-methanesulfonyl oxy, preferred mesyloxy, tosyloxy, oil of mirbane sulfonyloxy, chloromethane sulfonyloxy, trifluoro-methanesulfonyl oxy, further preferred mesyloxy or tosyloxy.

In above-mentioned general formula (1)-(5), * represents unsymmetrical carbon, its optical purity be generally 80%e.e. or more than, preferred 90%e.e. or more than, further preferred 95%e.e. or more than, preferred especially 99%e.e. or more than.Absolute steric configuration can be any configuration of R configuration and S configuration, is the S configuration in preferred above-mentioned general formula (2) and (3), is the R configuration in above-mentioned general formula (1), (4) and (5).

The optical activity alcohol that uses as raw material can be by comprising corresponding ketone asymmetric reduction, the asymmetric reaction of cutting apart etc. by lipase, it is synthetic arbitrarily to adopt known method to carry out, but cutting method must be given up alcohol or its ester of unwanted steric configuration, and is therefore preferred by utilizing the asymmetric reaction of the ketone of raw material to synthesize fully.Further preferably make have reducing carbonyl, with the protein of the ability of higher optical purity synthesis of optically active alcohol or express the microorganism of this protein DNA of coding or the synthetic method of the aliphatic ketone effect that transformant etc. and structure are simple.

The method that optical activity alcohol shown in the above-mentioned general formula (2) is transformed to sulfonyloxy has the sulfonylation of hydroxyl.

The sulfonylation method of hydroxyl has methylsulfonyl agent such as the methylsulfonyl chloride of use, methylsulfonic acid acid anhydride; Tosylation agent such as toluene sulfonyl chloride, toluenesulphonic acids acid anhydride; The method of trifluoromethanesulfonic acid agent such as trifluoromethanesulfanhydride anhydride etc.

Preferred sulfonyloxy has methylsulfonyl, tosyl group, oil of mirbane alkylsulfonyl, chloromethane sulfonyloxy, trifyl, further preferably can low-cost industrialized methylsulfonyl or tosyl group.

The amount of the sulfonyl agent that uses in the above-mentioned reaction is the 1-10 equivalent with respect to substrate, preferred 1-2 equivalent.

Employed solvent has: ether series solvents such as ether, propyl ether, butyl methyl ether, tetrahydrofuran (THF); Halogen series solvents such as methylene dichloride, chloroform, ethylene dichloride, chlorobenzene; Ester such as ethyl acetate, butylacetate series solvent; Hydrocarbon system solvents such as hexane, benzene, toluene; Acid amides such as dimethyl formamide, N-Methyl pyrrolidone series solvent; Nitrile series solvents such as acetonitrile etc.The multiple solvent that is selected from wherein can be mixed use with arbitrary proportion.Preferred solvent has methylene dichloride, ethyl acetate, the toluene of the low and easy recovery of cost.

The usage quantity of solvent can be used the solvent of any amount, is 2-50 times of volume with respect to the raw material substrate usually, preferred 3-10 times of volume.

In the above-mentioned reaction, preferably coexist with alkali.Employed alkali has: organic basess such as triethylamine or pyridine; Mineral alkalis such as sodium hydroxide, salt of wormwood, sodium bicarbonate, preferred organic bases, further preferred triethylamine, pyridine.

The equivalent of the employed alkali secondary sour required amount of giving birth to that normally is used to neutralize is the 1-10 equivalent with respect to substrate, and preferably the 1-2 equivalent also can use alkali as solvent.

Temperature of reaction is generally-20 to 100 ℃, and according to leavings group that is imported and/or reaction conditions, its optimum temps is also different.When using particularly preferred methylsulfonyl or tosyl group, preferred 0-40 ℃.

Reaction times can be set arbitrarily, considers from reducing the manufacturing cost angle, preferably at 10 hours or carry out with interior.

Carbon nucleophile shown in the general formula that uses in the above-mentioned reaction (9) has: malonic ester, propanedioic acid, propane dinitrile, malonic acid monoester, cyanoacetic acid, cyan-acetic ester, propanedioic acid ring (Asia) isopropyl ester (Meldrum ' s acid), preferred malonic ester, propane dinitrile, cyan-acetic ester, the malonic ester of the low and easy hydrolysis of further preferred industrialization cost.

The pure composition of this ester is not particularly limited straight chain, side chain or cyclic alkyl alcohols such as particular methanol, ethanol, Virahol, propyl carbinol, hexalin so long as do not have dysgenic group to get final product to reaction; Aryl alcohol such as phenol, naphthols, further particular methanol or ethanol.

The amount of employed carbon nucleophile is the 1-10 equivalent with respect to substrate usually, generate dialkyl compound in order to suppress in the same carbon nucleophile bimolecular substrate reactions to be arranged,, be the 1-3 equivalent preferably with respect to substrate than the excessive use of substrate, further preferred 1.2-2.0 equivalent.

In the above-mentioned reaction, employed alkali has: metal hydride compounds such as sodium hydride, lithium hydride; Metal amide compounds such as lithium diisopropylamine, potassium hexamethydisilazide; Organometallic compounds such as n-Butyl Lithium, bromination isopropyl-magnesium; Basic metal such as sodium, potassium, lithium; Alkaline-earth metal such as calcium, magnesium; Metal alcoholates such as sodium methylate, sodium ethylate, potassium tert.-butoxide; Mineral alkali such as sodium hydroxide, salt of wormwood etc., preferred metal hydride compounds, basic metal and metal alcoholate, further preferred sodium hydride, sodium and sodium methylate.

The equivalent of employed alkali is the 1-10 equivalent with respect to substrate, preferred 1-3 equivalent.

Employed solvent has: butyl methyl ether, tetrahydrofuran (THF), 1, ether series solvents such as 2-glycol dimethyl ether, two  alkane; Halogen series solvents such as methylene dichloride, ethylene dichloride, chlorobenzene; Pure series solvents such as methyl alcohol, ethanol, Virahol; Hydrocarbon system such as hexane, toluene solvent; Acid amides such as dimethyl formamide, N-Methyl pyrrolidone series solvent; Dimethyl sulfoxide (DMSO) etc.The multiple solvent that is selected from wherein can be mixed use with arbitrary proportion.Preferred solvent has: methyl alcohol, ethanol, dimethyl formamide, tetrahydrofuran (THF), toluene, and further preferred tetrahydrofuran (THF), it can make smooth and easy the carrying out of reaction with high polarity, and can separate with water layer during extraction, even sneak into, next step can not have problems yet.

The usage quantity of solvent can be used any amount, is 0.5-20 times of volume with respect to the raw material substrate usually, as long as can smooth and easyly react, can improve speed of response more more at least, therefore preferred 1-8 times of volume, further preferred 2-4 times of volume.

Temperature of reaction is generally 0-100 ℃, and according to the kind or the reaction conditions of leavings group, carbon nucleophile, its optimum temps is also different.In order to suppress racemization, preferably carry out in not oversize scope of reaction times and at low temperature, during with the malonic ester reaction, be preferably 30-100 ℃, further preferred 50-80 ℃ at above-mentioned general formula (3) with particularly preferred methylsulfonyl or tosyl group.

The kind of reaction times and leavings group, carbon nucleophile or reaction conditions have very big relevant, consider from reducing the manufacturing cost angle, preferably at 10 hours or carry out with interior.If but adopt too drastic reaction conditions such as high temperature in order to shorten the reaction times, then the optical purity of product reduces, therefore must the selective reaction time, suitable conditions such as temperature, solvent.

When the optical compounds shown in the general formula that above-mentioned reaction is made (4) is transformed to optical activity 1-methyl alkyl propanedioic acid shown in the above-mentioned general formula (1), also can be without purifying, directly use reaction soln, but by method purifying such as distillation and/or extractions, can obtain the higher optical activity 1-methyl alkyl propanedioic acid of purity, thus preferred.

The method that is transformed into optical activity 1-methyl alkyl propanedioic acid has: the method that ester group and/or cyano group is transformed into carboxyl by acid treatment, alkaline purification etc., also can be after cyano group be transformed to ester group again hydrolysis etc. undertaken by stepped approach, but in water-containing solvent to carry out in the single stage, this can reduce number of steps, thereby preferred.

In the above-mentioned reaction there be employed reagent: mineral acids such as sulfuric acid, hydrochloric acid; Mineral alkalis such as sodium hydroxide, potassium hydroxide, salt of wormwood; 1, organic basess such as 8-diazabicyclo [5.4.0]-7-undecylene, sodium methylate etc.Low sodium hydroxide, potassium hydroxide, sulfuric acid, the hydrochloric acid of preferred industrial cost.

Employed solvent has: propyl ether, tetrahydrofuran (THF), 1, ether series solvents such as 2-glycol dimethyl ether, two  alkane; Halogen series solvents such as methylene dichloride, ethylene dichloride, chlorobenzene; Pure series solvents such as methyl alcohol, ethanol, ethylene glycol; Hydrocarbon system such as hexane, toluene solvent; Acid amides such as dimethyl formamide, N-Methyl pyrrolidone series solvent; Organic acid such as formic acid, acetate series solvent; Dimethyl sulfoxide (DMSO); Water etc.The multiple solvent that is selected from wherein can be used with mixed arbitrarily.Owing to be hydrolyzed, thus preferably water or with the mixed solvent system of water blended solvent and water, have with water blended solvent: tetrahydrofuran (THF), methyl alcohol, ethanol, acetate, further preferably water.

The usage quantity of solvent can be used the solvent of any amount, is 1-20 times of volume with respect to the raw material substrate usually, preferred 2-8 times of volume.

Optical activity 1-methyl alkyl propanedioic acid shown in the above-mentioned general formula (1) is the compound of good crystallinity, when optical purity is not enough, preferably improves optical purity by partial crystallization.Optical purity behind the partial crystallization be preferably 90%e.e. or more than, when particularly using as medical material or intermediate, demanding optical purity, therefore further preferred 95%e.e. or more than, preferred especially 99%e.e. or more than.

Partial crystallization is except separating out the conventional crystallization of crystalline from the extraction liquid equal solvent, also comprise by concentrating, cool off, add operations such as solvent and separate out crystal, the crystal that this is separated out is by adding dissolvings such as solvent, heating again, therefrom separate out the crystalline recrystallization again, then with the crystal that produces with methods such as solvent cleaning.

Employed solvent has: propyl ether, methyl butyl ether, tetrahydrofuran (THF), 1, ether series solvents such as 2-glycol dimethyl ether, two  alkane; Halogen series solvents such as methylene dichloride, chloroform, ethylene dichloride, chlorobenzene; Pure series solvents such as methyl alcohol, ethanol, Virahol, ethylene glycol; Ester such as ethyl acetate, butylacetate series solvent; Nitrile such as acetonitrile, propionitrile series solvent; Hydrocarbon system solvents such as hexane, heptane, benzene,toluene,xylene; Acid amides such as dimethyl formamide, N-Methyl pyrrolidone series solvent; Dimethyl sulfoxide (DMSO); Water etc.The multiple solvent that is selected from wherein can be used with mixed arbitrarily.Preferred cost is low and make crystal exsiccant propyl ether, hexane, heptane, benzene, toluene, ethyl acetate easily, further preferred flash-point is higher, the heptane of easy industrial treatment, toluene, dimethylbenzene, ethyl acetate, and is especially preferably higher and can separate out crystalline toluene under single solvent in proper range, to the solubleness of the impurity of sneaking into to the solubleness of target compound.

Solvent can use any amount, usually be 1-50 times of volume with respect to the raw material substrate, but quantity of solvent is relevant with crystalline size or solvent cost, therefore few as far as possible in the scope that can realize the crystallization purpose, preferred 1-20 times of volume, further preferred 1-10 times of volume.

4. optical activity 1-methyl alkyl propanedioic acid

Optical activity 1-methyl alkyl propanedioic acid of the present invention is a compound shown in the following general formula (1).

In the above-mentioned general formula (1), R ¹Be straight chain, side chain or cyclic alkyl such as the n-propyl of carbonatoms 3-5, normal-butyl, n-pentyl, sec.-propyl, isobutyl-, isopentyl, cyclopentyl.Wherein preferred n-propyl, normal-butyl, n-pentyl, sec.-propyl, isobutyl-, further preferred n-propyl or normal-butyl.

* represent unsymmetrical carbon, it can be any configuration of R configuration and S configuration, preferred R configuration, its optical purity be generally 80%e.e. or more than, preferred 90%e.e. or more than, when particularly using, require high-optical-purity as medical material or intermediate, therefore further preferred 95%e.e. or more than, preferred especially 99%e.e. or more than.

Further specify the present invention by following embodiment, but scope of the present invention is not limited by following examples.

Embodiment

Embodiment A (using the microorganism preparation to have optically active alcohol)

(1) generates the microorganism of (S)-2-amylalcohol and the separation that generates the microorganism of (S)-2-hexanol by the generation methyl-n-butyl ketone by 2 pentanone

Contain the various bacterial strains shown in the inoculation table 1 in the liquid nutrient medium that the poly-peptone (Japanese pharmacy manufacturing) of 5g/L yeast extract (Difco manufacturing), 5g/L, 3g/L malt extract (Difco manufacturings), 20g/L glucose (japanese food processing and manufacturing) form at 2.5mL, 30 ℃ of aerobics cultivations 24-72 hour.From each nutrient solution of gained, get the 1mL nutrient solution respectively, by centrifugal collection thalline.In this thalline, add 0.04mL Tris-HCl damping fluid (pH 7.0), 9.028mL de-salted water, thalline is fully suspended, add glucose, the 9.02mL 12g/L NADP of 0.05mL 100g/L then ⁺(manufacturing of Oriental yeast) adds 0.01mL again reaction substrate 2 pentanone or methyl-n-butyl ketone is dissolved in the solution that forms 100g/L in the Virahol, 30 ℃ of reactions 20 hours.

With the reaction solution ethyl acetate extraction behind the reaction terminating, to (S)-2-amylalcohol of generating or (S)-the 2-hexanol carries out quantitatively.Resultant quantitatively be to use gas chromatograph (GC), ethyl acetate extraction solution is measured.The condition of GC is as described below.

Post: β-DEX 120 (SUPELCO manufacturing, 30m * 0.25mm ID, 0.25 μ m film)

Carrier gas: He 1.5mL/ minute, splitting ratio 1/50

Column temperature: to carry out (S)-2-amylalcohol be 50 ℃ when quantitative, be 65 ℃ when carrying out that (S)-2-hexanol is quantitative.

Annotate the sample temperature: 250 ℃

Detect: 250 ℃ of FID

GC: Tianjin, island GC-14A

(S)-quantitative result of 2-amylalcohol is as shown in table 1, (S)-quantitative result of 2-hexanol is as shown in table 2.

Table 1

Generate the yeast of (S)-2-amylalcohol by 2 pentanone

Bacterial strain	Production concentration (g/L)	Product optical purity (%e.e.)
Bacterial strain	Production concentration (g/L)	Product optical purity (%e.e.)	Brussels brettanomyce NBRC 0629 Brussels brettanomyce NBRC 0797 candida tropicalis NBRC 0006 candida zeylanoides CBS 6408 candida zeylanoides JCM 1627 Hortaea werneckii NBRC 4875 Lodderomyces elongisporus NBRC 1676 Pichia segobiensis JCM 10740 Spartacus Pichia pastoris JCM 10741	1.07 1.18 1.17 1.36 1.45 1.34 1.25 1.16 1.48	100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s

Generate the bacterium of (S)-2-amylalcohol by 2 pentanone

Bacterial strain	Production concentration (g/L)	Product optical purity (%e.e.)
Bacterial strain	Production concentration (g/L)	Product optical purity (%e.e.)	Certain (ovum shape pseudomonad) NBRC 12953 radiation rhizobium IAM 12048 of Arthrobacter globiformis NBRC 12137 oxidation arthrobacterium DSM 20120 polychrome arthrobacterium DSM 342 wilting bacillus pumilis ATCC 12813 stearothermophilus ground bacillus NBRC 12550 stearothermophilus ground bacillus IAM 11002 stearothermophilus ground bacillus IAM 11004 stearothermophilus ground bacillus IAM 12043 certain (ovum shape pseudomonad) NBRC 12952 Ochrobactrums of certain (ovum shape pseudomonad) NBRC 12950 Ochrobactrums of brick-red microbacterium JCM 1353 human pallid bacillus ATCC 49237 Ochrobactrums	1.36 1.25 2.36 1.94 1.54 1.39 1.48 1.06 1.2 1.47 1.02 1.49 1.57 1.1	99.1 s 100 s 100 s 99.7 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s

Table 2

Generate the yeast of (S)-2-hexanol by methyl-n-butyl ketone

Bacterial strain	Production concentration (g/g)	Product optical purity (%e.e.)
Bacterial strain	Production concentration (g/g)	Product optical purity (%e.e.)	Different brettanomyce NBRC 0627 candida famata ATCC 10539 Ke Lusishi Candida JCM 2284 Ke Lusishi Candida JCM 2341 Ke Lusishi Candida NBRC 1664 maltose Candida NBRC 1977 candida zeylanoides CBS 6408 Issatchenkia scutulata var.scutulata JCM 1828 Lodderomyces elongisporus NBRC 1676 Angus Pichia pastoris NBRC 1024 Angus Pichia pastoris NBRC 1071 Pichia cactophila JCM 1830 Pichia segobiensis JCM 10740 happiness trehalose Pichia pastoris JCM 3651 little rhodotorula NBRC 0879	1.23 1.20 0.68 0.88 1.10 1.48 1.51 1.07 1.58 1.16 0.94 1.30 1.48 1.16 0.92	100 s 93.0 s 100 s 100 s 100 s 100 s 100 s 91.8 s 100 s 100 s 100 s 93.9 s 100 s 100 s 100 s

Generate the bacterium of (S)-2-hexanol by methyl-n-butyl ketone

Bacterial strain	Production concentration (g/L)	Product optical purity (%e.e.)
Bacterial strain	Production concentration (g/L)	Product optical purity (%e.e.)	Certain DSM 20407 sulphur look arthrobacterium JCM1 338 (Sulfur brevibacterium) Brevibacterium butanicum ATCC 21196 Sulfur brevibacterium JCM 1485 wilting bacillus pumilis ATCC 12813 of Arthrobacter separate certain ATCC 15960 (corynebacterium hydrocarboclastus) of certain (ovum shape pseudomonad) NBRC 12953 Paracoccus denitrificans NBRC 12442 radiation rhizobium IAM 12048 radiation rhizobium IAM 13129 Rhods of certain (ovum shape pseudomonad) NBRC 12952 Ochrobactrums of certain NBRC 15615 human pallid bacillus ATCC 49237 Ochrobactrums of cutin microbacterium NBRC 13309 longicorn microbacterium JCM 1352 Microbacteriums	1.04 1.26 1.12 0.5 1.29 1.16 1.25 0.68 0.91 1.58 1.73 1.91 0.91 0.79 1.22	100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s 100 s 99.3 s 100 s

(2) from the separation of the carbonyl reductase of Issatchankia scutulata var.scutulata JCM 1828 bacterial strains

Issatchankia scutulata var.scutulata JCM 1828 bacterial strains are cultivated in 2L substratum (80g glucose, 20g yeast extract (Difco manufacturing), 40g/L peptone (extremely eastern pharmacy manufacturing)), by centrifugal preparation thalline.150g gained wet thallus is suspended in 10mM potassium phosphate buffer (pH 7), 0.1mM DTT (being designated hereinafter simply as " damping fluid "), by DYNO-MILL KDL (Shimaru Enterprises manufacturing) fragmentation, by the centrifugal thalline residue of removing, obtain cell-free extract then.Add PEG6000 in this cell-free extract, making concentration is 90g/L, leaves standstill 1 hour at 4 ℃, then by the centrifugal precipitation of removing.Then the anion-exchange chromatography by using DEAE Sepharose Fast Flow (Amersham Biosciences manufacturings) to carry out, use gel filtration chromatography that hydrophobic interaction chromatogram that Butyl Sepharose4 Fast Flow (AmershamBiosciences manufacturings) carries out, anion-exchange chromatography that use MonoQ (AmershamBiosciences manufacturing) carries out and use Superdex 200 (Amersham Biosciences manufacturing) carry out etc., purification of target carbonyl reductase from this supernatant presents single band until electrophoresis.

During purifying, the activity of carbonyl reductase can followingly be measured: make reaction solution (100mM Tris-HCl pH 7.5,0.32mM NADPH, the 2mM 1-acetoxy-3-chloro-2-acetone) reaction that contains enzyme liquid at 37 ℃, calculated the consumption of NADPH by the minimizing of 340nm absorbancy.The mensuration of absorbancy is used SPECTRAmax 190 (Molecular Devices manufacturing).In the above-mentioned reaction, the activity that consumed 1nmol NADPH with 1 minute is 1U.

Purification result from the carbonyl reductase of Issatchankia scutulata var.scutulataJCM 1828 bacterial strains is as shown in table 3.

Table 3

Purification phase	Gross activity (U)	Total protein concentration (mg)	Specific activity (U/mg)	The purifying multiplying power	Yield (%)
Purification phase	Gross activity (U)	Total protein concentration (mg)	Specific activity (U/mg)	The purifying multiplying power	Yield (%)	Cell-free extract PEG6000 supernatant DEAE Sepharose FF Butyl Sepharose 4 FF MonoQ Superdex200	1360 693 857 488 592 348	16800 4680 266.0 10.6 3.11 1.038	0.0810 0.148 3.22 46.0 190 335	1 1.83 39.8 569 2351 4141	100.0 51.0 63.0 35.9 43.5 25.6

By polyacrylamide gel electrophoresis (SDS-PAGE) the Superdex200 active ingredient in the purification phase of above-mentioned table 3 is analyzed, the result, the protein of purifying is almost single band, and its molecular weight is about 40,000Da.

(3) Substratspezifitaet of IsADH1

Preparation contains the reaction solution (100mM Tris-HCl pH 7.5,0.32mM NADPH, 2mM substrate) of the carbonyl reductase liquid of purifying in above-mentioned (2), reacts with various carbonyl compound at 37 ℃.By the consumption of the NADPH in the 340nm absorbancy monitoring reaction solution, measure the activity of carbonyl reductase thus to all cpds.The mensuration of absorbancy is used SPECTRAmax 190 (Molecular Devices manufacturing).Is 100 with carbonyl reductase to the activity of 1-acetoxy-3-chloro-2-acetone, and this moment, carbonyl reductase was as shown in table 4 to the relative reactivity of each substrate compounds.

Table 4

Substrate	Relative reactivity
Substrate	Relative reactivity	1-acetoxy-3-chloro-2-acetone 4-chloro-ethyl 3-oxobutanoate ethyl 3-oxobutanoate 3-hydroxy-2-butanone diacetyl 2; 3-pentanedione ethyl pyruvate 3-methyl-2-Oxobutyric acid ethyl ester acetophenone propiophenone phenacetol 3-propiono pyridine 2; 2,2-trifluoroacetophenone pyruvic acid phenyl ester ethyl benzoylacetate nicotinoyl guanidine-acetic acid ethyl ester parahydroxyben-zaldehyde 4-chloro-benzaldehyde 2-norborneol ketone 3-quinuclidone	100 212 25.5 - - trace trace trace - trace - 11.2 53.8 12.2 trace 20.7 - 58.0 - trace

-: do not detect activity

Trace: faint activity is arranged

(4) amino acid sequence analysis of IsADH1

The carbonyl reductase component that contains of Superdex200 in the purification phase of the above-mentioned table 3 of gained in above-mentioned (2) is carried out desalination, concentrated, by Ai Demanfa N-terminal amino acid is analyzed then, determined the N-terminal aminoacid sequence of 18 residues.The result is shown in SEQ ID NO.4.

Digestion method by using the lysyl endopeptidase (under the experimental protein handbook, sheep Tu She publish), carbonyl reductase to purifying digests, and uses reversed-phase HPLC (AmershamBiosciences makes μ RPC C2/C18 PC3.2/3) the gained peptide to be carried out the separation of peptide.By Ai Demanfa isolating a kind of peptide peak is carried out amino acid sequence analysis, aminoacid sequence is shown in SEQID NO.5.

(5) sequential analysis of the DNA of coding IsADH1 and the preparation of transformant

Issatchankia scutulata var.scutulata JCM 1828 bacterial strains are cultivated the preparation thalline in the substratum shown in above-mentioned (2).

Use Dneasy tissue kit (Qiagen manufacturing) from thalline, to extract genomic dna and purifying.According to the gained genomic dna, use reversed transcriptive enzyme Super Script II reversed transcriptive enzyme (Invitrogen manufacturing), according to synthesizing of enzyme appended explanation carrying out cDNA.

According to the N-terminal aminoacid sequence of above-mentioned (4) gained SEQ ID NO.4, synthetic have an adopted degenerated primer, and according to the internal amino acid sequence of SEQ ID NO.5, the degenerated primer of synthetic antisense, synthetic two kinds altogether.Their base sequence is respectively shown in SEQ ID NO.6,7.Use this two kinds of primers, the cDNA of Issatchankia scutulata var.scutulata JCM 1828 bacterial strains is carried out degenerate pcr, the amplified fragments of the 350bp that can confirm to have an appointment.

This dna fragmentation is carried out agarose gel electrophoresis, cut the segmental band of about 350bp, carry out purifying and recovery by MinElute gel extraction kit (Qiagen manufacturing).The gained dna fragmentation is connected transformed into escherichia coli DH5 α bacterial strain (Japan's spinning is made) with pGEM-Teasy Vector (Promega manufacturing).To transform bacterial strain and in the LB nutrient agar that contains penbritin (100 μ g/mL), cultivate, get some bacterium colonies, and use T7 primer (Promega manufacturing) and SP6 primer (Promega manufacturing) to carry out the direct PCR of bacterium colony, confirm to insert segmental size.To think that having inserted the segmental bacterium colony of target dna cultivates on the LB substratum that contains 100 μ g/mL penbritins, by QIAPrep Spin Mini Prep test kit (Qiagen manufacturing) plasmid purification.

Use the plasmid of purifying, the DNA base sequence that inserts is analyzed by dyestuff terminator method.The base sequence of determining is shown in SEQ ID NO.8.

Then, genomic dna according to Issatchankia scutulata var.scutulata JCM 1828 bacterial strains, according to the method for record in " molecular cloning ", the cDNA of synthetic RACE reaction usefulness carries out 5 '-and 3 '-RACE reaction according to the method for putting down in writing in the document.Used in the reaction according to two kinds of gene-specific primers shown in the SEQ ID NO.9 and 10 of above-mentioned base sequence design.

To carrying out sequential analysis by the gene fragment of RACE reaction amplification, the result, carbonyl reductase of the present invention infer the cDNA sequence shown in SEQ ID NO.11, the coded aminoacid sequence of this DNA is shown in SEQ ID NO.1.The base sequence of the aminoacid sequence of coding SEQ ID NO.1 is shown in SEQ ID NO.2.

Then, sequence according to above-mentioned SEQ ID NO.11, the base sequence of synthetic SEQ ID NO.12 and the base sequence of SEQ ID NO.13, use primer with this as the clone, use 100 μ L to contain the above-mentioned primer of each 50pmol, each 1000nmol dNTP, the cDNA of 250ng Issatchankiascutulata var.scutulata JCM 1828 bacterial strains, 10 μ L ExTaq archaeal dna polymerases are with 10 * damping fluid (precious biological make), the reaction solution of 5 ExTaq of unit archaeal dna polymerases (precious biological the manufacturing), use PTC-200 (MJ Research manufacturing), carry out (95 ℃ of sex change, 1 minute), anneal (58 ℃, 1 minute), extend (72 ℃, 1 minute) totally 30 circulations.The part of PCR reaction solution is analyzed by agarose gel electrophoresis, and the result can detect specific band.

With above-mentioned reaction solution MinElute PCR purification kit (Qiagen manufacturing) purifying.The dna fragmentation of purifying is digested with limiting enzyme EcoRI and XbaI, carry out agarose gel electrophoresis, cut the target stripe part, carry out purifying, reclaim then by Qiagen gel extraction kit (Qiagen manufacturing).Use pUC118 to be connected transformed into escherichia coli JM109 bacterial strain the gained dna fragmentation with Ligation high (Japan's weaving is made) through EcoRI and XbaI digestion.

Transformant is cultivated on the LB nutrient agar that contains penbritin (50 μ g/mL), carry out the direct PCR of bacterium colony, confirm to insert segmental size.

Cultivate on the LB substratum that contains 50 μ g/mL penbritins being considered to have inserted the segmental transformant of target dna, carry out plasmid purification, make pUCIsADH1 with QIAPrepSpin Mini Prep test kit (Qiagen manufacturing).

By dyestuff terminator method the base sequence that is inserted into the DNA in the plasmid is analyzed, the result, the dna fragmentation that is inserted is consistent with the base sequence of SEQ ID NO.2.

(6) use the intestinal bacteria of the DNA conversion of encoded IsADH1 to carry out the synthetic of (S)-2-amylalcohol

With above-mentioned (5) gained transformant in 100mL contains the Circle Grow substratum of penbritin (50 μ g/mL) (DIO 101 makes), 30 ℃ cultivated 30 minutes, 10 grades (series) cultivates.By centrifugal collection gained thalline,, be that substrate carries out the synthetic of (S)-2-amylalcohol with the 2 pentanone then according to method shown below.

In the above-mentioned thalline of 10g, add 20mg 0.6g/L NADP ⁺(amano pharmaceutical manufacturing, 76 units/mg), 1g 2 pentanone (Tokyo changes into manufacturing, Neat) are then 30 ℃ of reactions 8 hours for (manufacturing of Oriental yeast), 10mL 1M Tris-HCl damping fluid (pH 7.0), 40mL 100g/L glucose, 20mg Hexose phosphate dehydrogenase.PH when making reaction by 2M yellow soda ash remains on 7.0.With the reaction solution ethyl acetate extraction behind the reaction terminating, (the S)-2-amylalcohol that generates is carried out quantitatively.

Quantitatively use gas-chromatography (GC) that ethyl acetate solution is measured.The condition of GC is as follows.

Post: β-DEX120 (SUPELCO manufacturing, 30m * 0.25mm ID, 0.25 μ m film)

Carrier gas: He 1.5mL/ minute, splitting ratio 1/50

Column temperature: 50 ℃

Annotate the sample temperature: 250 ℃

Detect: 250 ℃ of FID

GC: Tianjin, island GC-14A

As a result, (S)-the receipts amount of 2-amylalcohol is 0.99g, optical purity＞99.0%e.e..

(7) use the intestinal bacteria of the DNA conversion of encoded IsADH1 to carry out the synthetic of (S)-2-hexanol

Using the transformant of above-mentioned (6) gained, according to method shown below, is substrate with the methyl-n-butyl ketone, carries out the synthetic of (S)-2-hexanol.

In the above-mentioned thalline of 10g, add 20mg 0.6g/LNADP ⁺(amano pharmaceutical manufacturing, 76 units/mg), 1g methyl-n-butyl ketone (Tokyo changes into manufacturing, Neat) are then 30 ℃ of reactions 6 hours for (manufacturing of Oriental yeast), 10mL 1M Tris-HCl damping fluid (pH 7.0), 40mL 100g/L glucose, 20mg Hexose phosphate dehydrogenase.PH in the time of will reacting by 2M yellow soda ash remains on 7.0.Reaction solution ethyl acetate extraction behind the reaction terminating carries out quantitatively (the S)-2-hexanol that generates.

Quantitatively use gas-chromatography (GC), ethyl acetate solution is measured.The condition of GC is as follows.

Post: β-DEX120 (SUPELCO manufacturing, 30m * 0.25mm ID, 0.25 μ m film)

Carrier gas: He 1.5mL/ minute, splitting ratio 1/50

Column temperature: 65 ℃

Annotate the sample temperature: 250 ℃

Detect: 250 ℃ of FID

GC: Tianjin, island GC-14A

As a result, (S)-the receipts amount of 2-hexanol is 0.99g, optical purity＞99.0%e.e..

(8) use the intestinal bacteria of the DNA conversion of encoded IsADH1 to carry out synthetic (amplification) of (S)-2-amylalcohol

Using above-mentioned (6) gained transformant, according to method shown below, is substrate with the 2 pentanone, carries out the synthetic of (S)-2-amylalcohol.

In the above-mentioned thalline of 140g (being equivalent to about 42g dry mycelium weight), add 84mg NADP ⁺(manufacturing of Oriental yeast), 140mL 1M Tris-HCl damping fluid (pH 7.0), 118g glucose, 40mg Hexose phosphate dehydrogenase (amano pharmaceutical manufacturing, 76 units/mg), 28g 2 pentanone (Tokyo changes into manufacturing, Neat), add de-salted water then, making reaction capacity was 1.4L, 30 ℃ of reactions 16 hours.PH when making reaction with 2M yellow soda ash remains on 7.0.Reaction solution ethyl acetate extraction behind the reaction terminating carries out quantitatively (the S)-2-amylalcohol that generates, (S)-and the receipts amount of 2-amylalcohol is 17.2g, optical purity＞99.0%e.e..

(9) use the intestinal bacteria of the DNA conversion of encoded IsADH1 to carry out synthetic (amplification) of (S)-2-hexanol

Using above-mentioned (6) gained transformant, according to method shown below, is substrate with the methyl-n-butyl ketone, carries out the synthetic of (S)-2-hexanol.

In the above-mentioned thalline of 50g (being equivalent to about 15g dry mycelium weight), add 30mg NADP ⁺(manufacturing of Oriental yeast), 50mL 1M Tris-HCl damping fluid (pH7.0), 54g glucose, 20mg Hexose phosphate dehydrogenase (amano pharmaceutical manufacturing, 76 units/mg), 15g methyl-n-butyl ketone (Tokyo changes into manufacturing, Neat), add de-salted water then, making reaction capacity was 500mL, 30 ℃ of reactions 7.5 hours.PH when making reaction with 2M yellow soda ash keeps 7.0.Reaction solution ethyl acetate extraction behind the reaction terminating carries out quantitatively (the S)-2-hexanol that generates, and the receipts amount of result (S)-2-hexanol is 15.1g, optical purity＞99.0%e.e..

Embodiment B (preparing optically active carboxylic acid) by chemosynthesis

(embodiment 1) (S)-2-mesyloxy pentane synthetic

Add 4.14g (47.0mmol, 99.1%e.e.) (S)-2-amylalcohol and 9.8mL (71mmol) triethylamine, 41mL methylene dichloride to the 200mL three-necked flask.This mixed solution is ice-cooled, drip 4.36mL (56.4mmol) methylsulfonyl chloride.Stirred then 30 minutes, and added 40mL saturated aqueous ammonium chloride and 20mL water, stopped reaction.Mixture with the extraction of 80mL diethyl ether, with 20mL saturated aqueous ammonium chloride and the water washing of 20mL saturated common salt, is used dried over mgso with organic layer.Heat up in a steamer and desolvate, obtain rough (the S)-2-of 8.5g mesyloxy pentane, need not once more purifying and promptly can be used for following reaction.

¹H-NMR(400MHz，CDCl ₃)δ0.95(t，J＝7.2Hz，3H)，1.42(d，J＝6.3Hz，3H)，1.34-1.50(m，2H)，1.50-1.63(m，1H)，1.67-1.77(m，1H)，3.00(s，3H)，4.77-4.86(m，1H).

(embodiment 2) are synthesizing of diethyl malonate (R)-(1-methyl butyl)

Rough (S)-2-mesyloxy pentane of gained among the 8.5g embodiment 1 and 14.2g (88mmol) diethyl malonate, 22mL DMF are packed in the eggplant type flask of 200mL, this mixed solution is ice-cooled, add 3.5g (88mmol) 60% sodium hydride (oiliness).60 ℃ of reactions 5 hours,, at room temperature add 40mL saturated aqueous ammonium chloride and 10mL water then, stopped reaction then 80 ℃ of reactions 3 hours.With mixture 100mL ethyl acetate extraction, organic layer with 20mL saturated aqueous ammonium chloride, 20mL water, the water washing of 20mL saturated common salt, is used dried over mgso.Heat up in a steamer and desolvate,, obtain 7.77g (R)-(1-methyl butyl) diethyl malonate (colourless oily mater, 33.8mmol, yield 72%) by purification by silica gel column chromatography.

¹H-NMR(400MHz，CDCl ₃)δ0.89(t，J＝6.9Hz，3H)，0.98(d，J＝6.6Hz，3H)，1.27(t，J＝7.1Hz，6H)，1.15-1.46(m，4H)，2.20-2.31(m，1H)，3.22(d，J＝8.1Hz，1H)，4.19(q，J＝7.1Hz，4H).

(embodiment 3) are synthesizing of propanedioic acid (R)-(1-methyl butyl)

(R)-(1-methyl butyl) diethyl malonate of 7.77g (33.5mmol) embodiment 2 gained and 16.1g (100mmol) 25% aqueous sodium hydroxide solution, 3.9mL ethanol, 15.4mL water are packed in the eggplant type flask of 200mL.This mixed solution 60 ℃ of reactions 1.5 hours, is at room temperature added the 9mL concentrated hydrochloric acid then, be acid.In mixed solution, add salt, make it saturated, use the 100mL ethyl acetate extraction then.With 50mL ethyl acetate aqueous layer extracted once more, use the dried over sodium sulfate organic layer.Heat up in a steamer desolvate after, from 58mL ethane and 5.8mL ethyl acetate, separate out crystal, obtain 4.73g (R)-(1-methyl butyl) propanedioic acid (white plate crystal, 27.1mmol, yield 81%).Chiral analysis result, optical purity are 99.3%e.e. (inj.300 ℃, FID250 ℃, Det.250 ℃, 130 ℃ of Oven analyze the 3-methylhexanoic acid that the decarboxylation of when injection produces for Supelco, β-DEX120).

¹H-NMR(400MHz，CDCl ₃)δ0.91(t，J＝6.8Hz，3H)，1.06(d，J＝6.8Hz，3H)，1.22-1.40(m，2H)，1.40-1.58(m，2H)，2.25-2.37(m，1H)，3.37(d，J＝7.6Hz，1H)，10.67(brs，2H).

(embodiment 4) (S)-2-mesyloxy hexane synthetic

Add 6.3g (62.0mmol, 99.7%e.e.) (S)-2-hexanol and 13mL (93mmol) triethylamine, 126mL ethyl acetate to the 300mL three-necked flask.This mixed solution is ice-cooled, Dropwise 5 .7mL (74mmol) methylsulfonyl chloride.Stirred then 30 minutes, and added 40mL saturated aqueous ammonium chloride and 20mL water, make the stopping of reaction, separate water layer.Organic layer with 20mL saturated aqueous ammonium chloride and the water washing of 20mL saturated common salt, is used dried over mgso.Heat up in a steamer and desolvate, obtain rough (the S)-2-of 10.8g mesyloxy hexane, need not to be further purified and promptly can be used for following reaction.

¹H-NMR(400MHz，CDCl ₃)δ0.92(t，J＝7.1Hz，3H)，1.25-1.45(m，4H)，1.42(d，J＝6.3Hz，3H)，1.55-1.65(m，1H)，1.68-1.79(m，1H)，3.00(s，3H)，4.75-4.84(m，1H).

(embodiment 5) are synthesizing of diethyl malonate (R)-(1-methyl amyl)

Rough (S)-2-mesyloxy hexane and 19.2g (120mmol) diethyl malonate, the 32mL DMF of gained among the 10.8g embodiment 4 are joined in the eggplant type flask of 200mL.This mixed solution is ice-cooled, add 4.8g (120mmol) 60% sodium hydride (oiliness).Then 60 ℃ the reaction 1 hour, 80 ℃ the reaction 3 hours, at room temperature add 50mL saturated aqueous ammonium chloride and 10mL water then, make the stopping of reaction.With mixture 100mL ethyl acetate extraction, organic layer with 30mL water washing twice, is used the water washing of 30mL saturated common salt again, use dried over mgso.Heat up in a steamer desolvate after, by purification by silica gel column chromatography, obtain 12.0g (R)-(1-methyl amyl) diethyl malonate (colourless oily mater, 49mmol, yield 82%).

¹H-NMR(400MNz，CDCl ₃)δ0.891(t，J＝6.9Hz，3H)，0.98(d，J＝6.6Hz，3H)，1.27(t，J＝7.1Hz，6H)，1.15-1.45(m，6H)，2.17-2.29(m，1H)，3.22(d，J＝8.1Hz，1H)，4.19(q，J＝7.1Hz，4H).

(embodiment 6) are synthesizing of propanedioic acid (R)-(1-methyl amyl)

(R)-(1-methyl amyl) diethyl malonate of gained among 11.7g (48mmol) embodiment 5 and 23g (144mmol) 25% aqueous sodium hydroxide solution, 5.9mL ethanol, 24mL water are packed in the eggplant type flask of 100mL.This mixed solution 60 ℃ of reactions 2 hours, is at room temperature added the 13mL concentrated hydrochloric acid then, be acid.In mixed solution, add salt, make it saturated, use the 120mL ethyl acetate extraction then.With 40mL ethyl acetate aqueous layer extracted once more, use the dried over sodium sulfate organic layer.Heat up in a steamer desolvate after, partial crystallization from 90mL ethane and 18mL ethyl acetate obtains 6.5g (R)-(1-methyl amyl) propanedioic acid (white column crystallization, 35mmol, yield 72%).

¹H-NMR(400MHz，CDCl ₃)δ0.90(t，J＝6.7Hz，3H)，1.07(d，J＝6.8Hz，3H)，1.21-1.41(m，5H)，1.46-1.56(m，1H)，2.22-2.33(m，1H)，3.39(d，J＝7.3Hz，1H).

(embodiment 7) (R)-3-methylhexanoic acid synthetic

(R)-(1-methyl butyl) propanedioic acid of gained among 4.56g (26.2mmol) embodiment 3 is packed in the 50mL eggplant type flask, is warming up to 180 ℃, treat the stopping of gas after, restir 10 minutes is cooled to room temperature then.With the underpressure distillation of gained raw product, obtain 2.05g (R)-3-methylhexanoic acid (colourless oily mater, 77 ℃/3mmHg of boiling point, 15.7mmol, yield 60%).Chiral analysis result, optical purity are 99.2%e.e. (Supelco, β-DEX120, inj.250 ℃, FID250 ℃, Det.250 ℃, 130 ℃ of Oven).

¹H-NMR(400MHz，CDCl ₃)δ0.90(t，J＝7.1Hz，3H)，0.96(d，J＝6.6Hz，3H)，1.16-1.44(m，4H)，1.91-2.05(m，1H)，2.15(dd，J＝14.9，8.1Hz，1H)，2.35(dd，J＝14.9，5.8Hz，1H)，11.00(brs，1H).

(embodiment 8) (R)-3-methyl enanthic acid synthetic

(R)-(1-methyl amyl) propanedioic acid of 3.00g (15.9mmol) embodiment 6 gained is encased in the 20mL eggplant type flask, is warming up to 180 ℃.After treating the stopping of gas, restir 10 minutes is cooled to room temperature then.With the underpressure distillation of gained raw product, obtain 1.69g (R)-3-methyl enanthic acid (colourless oily mater, 86 ℃/4mmHg of boiling point, 11.7mmol, yield 74%).

¹H-NMR(400MHz，CDCl ₃)δ0.89(t，J＝6.8Hz，3H)，0.97(d，J＝6.8Hz，3H)，1.17-1.39(m，6H)，1.88-2.03(m，1H)，2.14(dd，J＝14.9，8.1Hz，1H)，2.35(dd，J＝14.9，5.9Hz，1H)11.36(brs，1H).

(embodiment 9) (R)-synthetic (pyridine solvent) of 3-methylhexanoic acid

Under nitrogen atmosphere,, and heat up (R)-(1-methyl butyl) propanedioic acid, the 2mL pyridine 15mL frosted oral examination pipe of packing into of 1.00g (5.8mmol).From 90 ℃ of initial actions, be warming up to 130 ℃ with 115 minutes, restir 30 minutes is cooled to room temperature then.Analyze by HPLC, transformation efficiency is 100.0%.

(embodiment 10) (R)-synthetic (the DMSO solvent) of 3-methylhexanoic acid

Under nitrogen atmosphere, (R)-(1-methyl butyl) propanedioic acid, 4mLDMSO join in the 15mL frosted oral examination pipe and heat up with 2.00g (11.5mmol).From 100 ℃ of initial actions, be warming up to 140 ℃ with 75 minutes, restir 20 minutes is cooled to room temperature then.Analyze by HPLC, transformation efficiency is 100.0%.

(embodiment 11) (R)-synthetic (the interpolation pyridine) of 3-methylhexanoic acid

Under nitrogen atmosphere, (R)-(1-methyl butyl) propanedioic acid, 182mg (2.3mmol) pyridine join in the 15mL frosted oral examination pipe and heat up with 2.00g (11.5mmol).From 100 ℃ of initial actions, be warming up to 150 ℃ with 95 minutes, restir 15 minutes is cooled to room temperature then.Analyze by HPLC, transformation efficiency is 100.0%.

(embodiment 12) (R)-synthetic (the adding DABCO) of 3-methylhexanoic acid

Under nitrogen atmosphere, with 2.00g (11.5mmol) (R)-(1-methyl butyl) propanedioic acid, 258mg (2.3mmol) 1,4-diazabicyclo [2.2.2] octane (DABCO) joins in the 15mL frosted oral examination pipe and heats up.From 100 ℃ of initial actions, be warming up to 140 ℃ with 95 minutes, restir 15 minutes is cooled to room temperature then.Analyze by HPLC, transformation efficiency is 100.0%.

(embodiment 13) (R)-synthetic (the adding DBU) of 3-methylhexanoic acid

Under nitrogen atmosphere, with 2.00g (11.5mmol) (R)-(1-methyl butyl) propanedioic acid, 350mg (2.3mmol) 1,8-diazabicyclo [5.4.0]-7-undecylene (DBU) joins in the 15mL frosted oral examination pipe, heats up.From 110 ℃ of initial actions, be warming up to 140 ℃ with 50 minutes, restir 20 minutes is cooled to room temperature then.Analyze by HPLC, transformation efficiency is 100.0%.

(embodiment 14) (R)-synthetic (the diacetyl oxide catalyzer pyridine solvent) of 3-methylhexanoic acid

Under nitrogen atmosphere, (R)-(1-methyl butyl) propanedioic acid, 2mL pyridine join in the 15mL frosted oral examination pipe interpolation 294mg (2.88mmol) diacetyl oxide with 1.00g (5.8mmol).At room temperature stirred 2 hours, and stirred 1 hour, be cooled to room temperature then at 40 ℃.Analyze with HPLC, transformation efficiency is 99.8%.

(embodiment 15) (R)-synthetic (the interpolation sulfuric acid) of 3-methylhexanoic acid

Under nitrogen atmosphere, (R)-(1-methyl butyl) propanedioic acid, 113mg (1.15mmol) sulfuric acid join in the 15mL frosted oral examination pipe and heat up with 2.00g (11.5mmol).From 130 ℃ of initial actions, be warming up to 180 ℃ with 100 minutes, restir 20 minutes is cooled to room temperature then.Analyze by HPLC, transformation efficiency is 100.0%.

(embodiment 16) (R)-the synthetic of 3-methylhexanoic acid (add Fe ₃O ₄)

Under nitrogen atmosphere, with 2.00g (11.5mmol) (R)-(1-methyl butyl) propanedioic acid, 40mg (2wt%) ferric oxide (Fe ₃O ₄) join in the 15mL frosted oral examination pipe and intensification.From 130 ℃ of initial actions, be warming up to 180 ℃ with 100 minutes, restir 20 minutes is cooled to room temperature then.Analyze by HPLC, transformation efficiency is 100.0%.

(embodiment 17) (R)-synthetic (the adding cupric oxide (I)) of 3-methylhexanoic acid

Under nitrogen atmosphere, (R)-(1-methyl butyl) propanedioic acid, 82.8mg (0.58mmol) cupric oxide (I), 20mL acetonitrile join in the flask of 100mL reflux with 2.00g (11.5mmol).React after 6 hours, be cooled to room temperature.Analyze by HPLC, transformation efficiency is 64.3%.

(embodiment 18) (R)-synthetic (additive-free) of 3-methylhexanoic acid

Under nitrogen atmosphere, with 3.00g (17.2mmol) (R)-(1-methyl butyl) propanedioic acid be encased in the eggplant type flask and heat up.From 130 ℃ of initial actions, be warming up to 180 ℃ with 75 minutes, restir 15 minutes is cooled to room temperature then.Analyze by HPLC, transformation efficiency is 100.0%.

The result of the foregoing description 9-18 as shown in the following Table 5.

Table 5

Embodiment	Additive	Amount	Solvent	Amount	Temperature of reaction/℃	Time	Transformation efficiency
Embodiment	Additive	Amount	Solvent	Amount	Temperature of reaction/℃	Time	Transformation efficiency	9 10 11 12 13 14 15 16 17 18	--pyridine DABCO DBU Ac ₂O H ₂SO ₄ Fe ₃O ₄ Cu ₂O -	- - 0.2MR 0.2MR 0.2MR 0.5MR 0.1MR 2wt％ 0.05MR -	Pyridine DMSO---pyridine--CH₃CN -	2VR 2VR - - - 2VR - - 10VR -	90～130 100～140 100～150 100～140 100～140 rt～40 130～180 130～180 80 130～180	2.4h 1.6h 1.8h 1.6h 1.2h 3h 2h 2h 6h 1.5h	100％ 100％ 100％ 100％ 100％ 100％ 100％ 100％ 64％ 100％

(embodiment 19) (R)-2-mesyloxy pentane synthetic

In 2L separate type flask, add 417g and contain 100g (1.13mol, 100%e.e.) ethyl acetate solution of (S)-2-amylalcohol and 149g (1.47mol) triethylamine.This solution is cooled to 5 ℃, drips 156g (1.36mol) methylsulfonyl chloride.At room temperature stirred then 1 hour, and added 440g water, stopped reaction.Remove water layer, add 267g 10% salt solution in organic layer, it is neutral making the pH of water layer with 5% sodium bicarbonate aqueous solution then.Remove water layer, heat up in a steamer and desolvate, then interpolation toluene, spissated step are repeated twice, obtain rough (the S)-2-of 191g mesyloxy pentane (filbert oily material, chemical purity 93.5%, 1.07mol, yield 94.6%).

(embodiment 20) are synthesizing of diethyl malonate (R)-(1-methyl butyl)

In 4000mL separate type flask, add 71.7g (1.79mol) 60% sodium hydride (oiliness), 605g THF.287g diethyl malonate (1.79mol) and 28.4g THF are added drop-wise in the reaction solution at 25-40 ℃, with 28.4g THF flushing.Be warming up to 65-70 ℃, add rough (S)-2-mesyloxy pentane of 266g (purity 80.1%, 1.28mol) and 28.4g THF then.Under refluxing, reacted 6 hours then, at room temperature add 638mL water, add concentrated hydrochloric acid then, make the pH value be adjusted to 6-7.Remove water layer, heat up in a steamer and desolvate, obtain 443g rough (R)-(1-methyl butyl) diethyl malonate (colourless oily mater, purity 59.9%, 1.15mol, yield 90.5%).

(embodiment 21) are synthesizing of propanedioic acid (R)-(1-methyl butyl)

439g (purity 59.9%, 1.14mmol) rough (R)-(1-methyl butyl) diethyl malonate and 6650g (4.04mol) 25% aqueous sodium hydroxide solution, 797mL water are joined in the 4000mL separate type flask.This mixed solution 60-70 ℃ of reaction 6 hours, is cooled off then.Reaction solution is poured out, and (4.05mol) joins in the flask with the 421g concentrated hydrochloric acid, then dropwise reaction liquid.The pH of mixed solution is 1.1.With mixed solution 717g ethyl acetate extraction.With 532mL 0.26M salt acid elution organic layer, heat up in a steamer and desolvate, replace with toluene.Partial crystallization from 974g toluene obtains 170g (R)-(1-methyl butyl) propanedioic acid (white platelike crystal, purity 95.3%, 0.931mmol, yield 81.7%).The result of chiral analysis, optical purity are 99.7%e.e..

(embodiment 22) (R)-3-methylhexanoic acid synthetic

Under nitrogen atmosphere, the 89g pyridine is joined in the eggplant type flask of 1L, be warming up to 110 ℃, with dripped in 5 hours make 140g (766mmol, purity 95.4%) (R)-(1-methyl butyl) propanedioic acid be dissolved in the solution of 147g pyridine gained.Restir 1 hour is cooled to room temperature then, obtains the pyrrole shallow lake solution of 329g (R)-3-methylhexanoic acid.Analyze by HPLC, transformation efficiency is 100.0%, and yield is quantitative.

The pyridine solution that 754g is contained (R)-3-methylhexanoic acid of 169g (1.29mmol) reaction gained carries out underpressure distillation under about 50Torr, remove pyridine, obtains rough (the R)-3-of 197g methylhexanoic acid.With this rough (R)-3-methylhexanoic acid rectifying under about 10Torr of 190g, obtain 152g (R)-3-methylhexanoic acid (colourless oily mater, 1.17mol, yield 90%, purity 99.3%).The result of chiral analysis, optical purity are 99.5%e.e..

Industrial applicability

The method according to this invention can obtain to can be used as the industrial useful compound (S) of the intermediate feed of medicine, agricultural chemicals etc.-2-amylalcohol or (S)-2-hexanol with high-optical-purity and high concentration.

According to the present invention, can be low by cost and industrialized process for preparing that efficient is high, obtain can be used as optical activity 1-methyl alkyl malonic acid and the optical activity 3-methyl carboxylic acids of medicine, pesticide intermediate with high-optical-purity.

Sequence table

<110>API Corporation

＜120〉has the preparation method of optically active alcohol and carboxylic acid

<130>A51051A

<160>13

<210>1

<211>345

<212>PRT

<213>Issatchenkia scutulata

<400>1

Met Ser Asn Lys Thr Val Leu Val Thr Gly Ala Thr Gly Phe Ile Ala

1 5 10 15

Leu His Ile Ile Asp Asn Leu Leu Ser Lys Gly Tyr Ser Val Ile Gly

20 25 30

Thr Ala Arg Ser Gln Ser Lys Tyr Gln Pro Ile Leu Asp Ala Phe Lys

35 40 45

Lys Lys Tyr Pro Asp Ala Asn Leu Thr Phe Glu Val Val Pro Asp Ile

50 55 60

Ser Thr Glu Asn Ala Phe Asp Asp Val Leu Lys Lys His Pro Glu Ile

65 70 75 80

Thr Ala Val Leu His Thr Ala Ser Pro Phe Ser Phe Gly Leu Asn Lys

85 90 95

Asp Leu Lys Glu Ala Tyr Leu Lys Pro Ala Val Asp Gly Thr Leu Asn

100 105 110

Ile Leu Lys Ala Ile Glu Lys Tyr Ala Pro Gln Val Thr Lys Val Val

115 120 125

Ile Thr Ser Ser Tyr Ala Ala Ile Met Thr Gly Asn Pro Ser His Val

130 135 140

His Thr Ser Glu Thr Trp Asn Pro Ile Asn Trp Glu Asn Asp Val Lys

145 150 155 160

Asn Glu Tyr Phe Ala Tyr Ile Ala Ser Lys Thr Tyr Ala Glu Lys Ala

165 170 175

Ala Arg Asp Phe Val Lys Glu His Lys Val Asn Phe Lys Leu Ala Thr

180 185 190

Val Asn Pro Pro Tyr Val Leu Gly Pro Gln Leu Phe Asp Phe Ser Val

195 200 205

Gly Pro Val Leu Asn Thr Ser Asn Gln Leu Ile Thr Asp Ala Thr Lys

210 215 220

Ile Asp Lys Asn Ser Thr Lys Pro Glu Leu Gly Thr Pro Ala Leu Ala

225 230 235 240

Val Asp Val Arg Asp Val Ala Ala Phe His Val Leu Pro Leu Glu Asp

245 250 255

Asp Lys Val Ala Ser Glu Arg Leu Phe Ile Val Ala Gly Pro Ala Val

260 265 270

Val Gln Thr Phe Leu Asn Ile Ile Asn Glu Asn Ile Pro G1u Leu Lys

275 280 285

Gly Lys Val Ala Leu Gly Asp Pro Ala Ser Glu Lys Glu Leu Ile Glu

290 295 300

Lys His Thr Asp Lys Tyr Asp Leu Thr Asn Leu His Asn Val Ile Gly

305 310 315 320

Lys Tyr Asp Phe Ile Pro Val Glu Lys Ser Val Val Asp Val Leu Glu

325 330 335

Gln Tyr Tyr Lys Ile Asn Lys Ile Asp

340 345

<210>2

<211>1038

<212>DNA

<213>Issatchenkia scutulata

<400>2

atgtcgaaca aaacagttct agtcaccggg gctaccggtt ttattgcact acacatcatt 60

gataatttat tgtctaaggg ttattccgtt attggtacag ctagatccca atctaaatat 120

caaccaatcc ttgatgcttt caagaaaaaa taccctgatg caaatttgac ttttgaagtt 180

gtccctgaca tctccactga aaacgcattc gatgatgttt tgaagaagca tccagaaatt 240

actgctgtcc ttcacacagc atctccattc tcttttggtt tgaacaagga tctgaaggaa 300

gcatatttga agcctgccgt tgatggtact ttgaatattc tcaaggcaat tgagaagtat 360

gcaccacagg ttactaaagt tgttatcaca tcttcttatg ctgcaattat gacaggtaat 420

ccaagtcatg tccacaccag tgaaacctgg aacccaatta attgggaaaa cgatgtgaag 480

aatgaatact ttgcatatat tgcctccaag acgtatgctg aaaaagctgc gagagatttt 540

gtcaaggagc ataaggtcaa tttcaagtta gcaactgtta acccaccata cgttctgggt 600

ccacaattat ttgacttctc agttggtcca gtcttgaaca cttccaacca attgatcacg 660

gatgcgacta aaattgataa gaactctact aagccggaat taggtacacc agctttagca 720

gtcgatgtta gagatgttgc tgcgttccat gttttaccat tggaagatga taaagttgca 780

agtgaaagat tatttattgt tgctggtcca gcagttgttc aaacattctt aaacatcatc 840

aacgagaaca ttccagaact taaaggtaag gttgccctag gagatccagc ttcagagaag 900

gagttgattg aaaagcacac agataagtat gatttgacaa atcttcacaa cgttattggt 960

aaatatgatt tcattccagt tgaaaagtcc gttgtcgacg tcttagaaca atattacaaa 1020

atcaataaaa ttgattag 1038

<210>3

<211>344

<212>PRT

＜213〉Saccharomyces cerevisiae

<400>3

Met Ser Asn Thr Val Leu Val Ser Gly Ala Ser Gly Phe Ile Ala Leu

1 5 10 15

His Ile Leu Ser Gln Leu Leu Lys Gln Asp Tyr Lys Val Ile Gly Thr

20 25 30

Val Arg Ser His Glu Lys Glu Ala Lys Leu Leu Arg Gln Phe Gln His

35 40 45

Asn Pro Asn Leu Thr Leu Glu Ile Val Pro Asp Ile Ser His Pro Asn

50 55 60

Ala Phe Asp Lys Val Leu Gln Lys Arg Gly Arg Glu Ile Arg Tyr Val

65 70 75 80

Leu His Thr Ala Ser Pro Phe His Tyr Asp Thr Thr Glu Tyr Glu Lys

85 90 95

Asp Leu Leu Ile Pro Ala Leu Glu Gly Thr Lys Asn Ile Leu Asn Ser

100 105 110

Ile Lys Lys Tyr Ala Ala Asp Thr Val Glu Arg Val Val Val Thr Ser

115 120 125

Ser Cys Thr Ala Ile Ile Thr Leu Ala Lys Met Asp Asp Pro Ser Val

130 135 140

Val Phe Thr Glu Glu Ser Trp Asn Glu Ala Thr Trp Glu Ser Cys Gln

145 150 155 160

Ile Asp Gly Ile Asn Ala Tyr Phe Ala Ser Lys Lys Phe Ala Glu Lys

165 170 175

Ala Ala Trp Glu Phe Thr Lys Glu Asn Glu Asp His Ile Lys Phe Lys

180 185 190

Leu Thr Thr Val Asn Pro Ser Leu Leu Phe Gly Pro Gln Leu Phe Asp

195 200 205

Glu Asp Val His Gly His Leu Asn Thr Ser Cys Glu Met Ile Asn Gly

210 215 220

Leu Ile His Thr Pro Val Asn Ala Ser Val Pro Asp Phe His Ser Ile

225 230 235 240

Phe Ile Asp Val Arg Asp Val Ala Leu Ala His Leu Tyr Ala Phe Gln

245 250 255

Lys Glu Asn Thr Ala Gly Lys Arg Leu Val Val Thr Asn Gly Lys Phe

260 265 270

Gly Asn Gln Asp Ile Leu Asp Ile Leu Asn Glu Asp Phe Pro Gln Leu

275 280 285

Arg Gly Leu Ile Pro Leu Gly Lys Pro Gly Thr Gly Asp Gln Val Ile

290 295 300

Asp Arg Gly Ser Thr Thr Asp Asn Ser Ala Thr Arg Lys Ile Leu Gly

305 310 315 320

Phe Glu Phe Arg Ser Leu His Glu Ser Val His Asp Thr Ala Ala Gln

325 330 335

Ile Leu Lys Lys Glu Asn Arg Leu

340

<210>4

<211>18

<212>PRT

<213>Issatchenkia scutulata

<400>4

Arg Asn Lys Thr Val Leu Val Thr Gly Ala Thr Gly Phe Ile Ala Leu

1 5 10 15

Asp Ile

<210>5

<211>15

<212>PRT

<213>Issatchenkia scutulata

<400>5

Val Val Ile Thr Ser Ser Tyr Ala Ala Ile Met Thr Gly Asn Pro

1 5 10 15

<210>6

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉PCR primer

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉n; Inosine

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉n; Inosine

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉n; Inosine

<220>

<221>misc_feature

<222>(18)..(18)

＜223〉n; Inosine

<400>6

acnggnttya thgcnytnga yat 23

<210>7

<211>26

<212>DNA

＜213〉artificial

<220>

＜223〉PCR primer

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉n; Inosine

<220>

<221>misc_feature

＜222〉(9) .. (9)＜223〉n; Inosine

<220>

<221>misc_feature

<222>(18)..(18)

＜223〉n; Inosine

<220><221>misc_feature

<222>(21)..(21)

＜223〉n; Inosine

<400>7

ggrttnccng tcatdatngc ngcrta 26

<210>8

<211>341

<2l2>DNA

<213>Issatchenkia scutulata

<400>8

cattgataat ttattgtcta agggttattc cgttattggt acagctagat cccaatctaa 60

atatcaacca atccttgatg ctttcaagaa aaaataccct gatgcaaatt tgacttttga 120

agttgtccct gacatctcca ctgaaaacgc attcgatgat gttttgaaga agcatccaga 180

aattactgct gtccttcaca cagcatctcc attctctttt ggtttgaaca aggatctgaa 240

ggaagcatat ttgaagcctg ccgttgatgg tactttgaat attctcaagg caattgagaa 300

gtatgcacca caggttacta aagttgttat cacatcttctt 341

<210>9

<211>28

<212>DNA

＜213〉artificial

<220>

＜223〉PCR primer

<400>9

agggttattc cgttattggt acagctag 28

<210>10

<211>31

<212>DNA

＜213〉artificial

<220>

＜223〉PCR primer

<400>10

gagaatggag atgctgtgtg aaggacagcag 31

<210>11

<211>1212

<212>DNA

<213>Issatchenkia scutulata

<220>

<221>CDS

<222>(55)..(1092)

<223>

<400>11

tcatgacctg tccactgata gcatcatacc aaacatattc agtatattgt aaca atg 57

Met

1

tcg aac aaa aca gtt cta gtc acc ggg gct acc ggt ttt att gca cta 105

Ser Asn Lys Thr Val Leu Val Thr Gly Ala Thr Gly Phe Ile Ala Leu

5 10 15

cac atc att gat aat tta ttg tct aag ggt tat tcc gtt att ggt aca 153

His Ile Ile Asp Asn Leu Leu Ser Lys Gly Tyr Ser Val Ile Gly Thr

20 25 30

gct aga tcc caa tct aaa tat caa cca atc ctt gat gct ttc aag aaa 201

Ala Arg Ser Gln Ser Lys Tyr Gln Pro Ile Leu Asp Ala Phe Lys Lys

35 40 45

aaa tac cct gat gca aat ttg act ttt gaa gtt gtc cct gac atc tcc 249

Lys Tyr Pro Asp Ala Asn Leu Thr Phe Glu Val Val Pro Asp Ile Ser

50 55 60 65

act gaa aac gca ttc gat gat gtt ttg aag aag cat cca gaa att act 297

Thr Glu Asn Ala Phe Asp Asp Val Leu Lys Lys His Pro Glu Ile Thr

70 75 80

gct gtc ctt cac aca gca tct cca ttc tct ttt ggt ttg aac aag gat 345

Ala Val Leu His Thr Ala Ser Pro Phe Ser Phe Gly Leu Asn Lys Asp

85 90 95

ctg aag gaa gca tat ttg aag cct gcc gtt gat ggt act ttg aat att 393

Leu Lys Glu Ala Tyr Leu Lys Pro Ala Val Asp Gly Thr Leu Asn Ile

100 105 110

ctc aag gca att gag aag tat gca cca cag gtt act aaa gtt gtt atc 441

Leu Lys Ala Ile Glu Lys Tyr Ala Pro Gln Val Thr Lys Val Val Ile

115 120 125

aca tct tct tat gct gca att atg aca ggt aat cca agt cat gtc cac 489

Thr Ser Ser Tyr Ala Ala Ile Met Thr Gly Asn Pro Ser His Val His

130 135 140 145

acc agt gaa acc tgg aac cca att aat tgg gaa aac gat gtg aag aat 537

Thr Ser Glu Thr Trp Asn Pro Ile Asn Trp Glu Asn Asp Val Lys Asn

150 155 160

gaa tac ttt gca tat att gcc tcc aag acg tat gct gaa aaa gct gcg 585

Glu Tyr Phe Ala Tyr Ile Ala Ser Lys Thr Tyr Ala Glu Lys Ala Ala

165 170 175

aga gat ttt gtc aag gag cat aag gtc aat ttc aag tta gca act gtt 633

Arg Asp Phe Val Lys Glu His Lys Val Asn Phe Lys Leu Ala Thr Val

180 185 190

aac cca cca tac gtt ctg ggt cca caa tta ttt gac ttc tca gtt ggt 681

Asn Pro Pro Tyr Val Leu Gly Pro Gln Leu Phe Asp Phe Ser Val Gly

195 200 205

cca gtc ttg aac act tcc aac caa ttg atc acg gat gcg act aaa att 729

Pro Val Leu Asn Thr Ser Asn Gln Leu Ile Thr Asp Ala Thr Lys Ile

210 215 220 225

gat aag aac tct act aag ccg gaa tta ggt aca cca gct tta gca gtc 777

Asp Lys Asn Ser Thr Lys Pro Glu Leu Gly Thr Pro Ala Leu Ala Val

230 235 240

gat gtt aga gat gtt gct gcg ttc cat gtt tta cca ttg gaa gat gat 825

Asp Val Arg Asp Val Ala Ala Phe His Val Leu Pro Leu Glu Asp Asp

245 250 255

aaa gtt gca agt gaa aga tta ttt att gtt gct ggt cca gca gtt gtt 873

Lys Val Ala Ser Glu Arg Leu Phe Ile Val Ala Gly Pro Ala Val Val

260 265 270

caa aca ttc tta aac atc atc aac gag aac att cca gaa ctt aaa ggt 921

Gln Thr Phe Leu Asa Ile Ile Asn Glu Asa Ile Pro Glu Leu Lys Gly

275 280 285

aag gtt gcc cta gga gat cca gct tca gag aag gag ttg att gaa aag 969

Lys Val A1a Leu Gly Asp Pro Ala Ser Glu Lys Glu Lau Ile Glu Lys

290 295 300 305

cac aca gat aag tat gat ttg aca aat ctt cac aac gtt att ggt aaa 1017

His Thr Asp Lys Tyr Asp Leu Thr Asn Leu His Asn Val Ile Gly Lys

310 315 320

tat gat ttc att cca gtt gaa aag tcc gtt gtc gac gtc tta gaa caa 1065

Tyr Asp Phe Ile Pro Val Glu Lys Ser Val Val Asp Val Leu Glu Gln

325 330 335

tat tac aaa atc aat aaa att gat tag tttatataga aaattttata 1112

Tyr Tyr Lys Ile Asn Lys Ile Asp

340 345

gctaaaggcc gaatcaactt ctttcttcct cttcaaaaaa aaaaaaaaaa aaaaaaaaaa 1172

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1212

<210>12

<211>35

<212>DNA

＜213〉artificial

<220>

＜223〉PCR primer

<400>12

cggaattcat gtcgaacaaa acagttctag tcacc 35

<210>13

<211>38

<212>DNA

＜213〉artificial

<220>

＜223〉PCR primer

<400>13

gctctagatt aatcaatttt attgattttg taatattg 38

Claims

(A) coding has the protein DNA of aminoacid sequence shown in the SEQ ID NO.1;

(D) has the DNA of base sequence shown in the SEQ ID NO.2;

In the formula (8), R ⁴Identical with aforementioned implication.

In the formula (8), R ⁴Identical with aforementioned implication.