JPH02156892A - Production of optically active alpha-hydroxycarboxylic acid - Google Patents

Abstract

PURPOSE:To obtain the subject compound in remarkably high efficiency by treating a hydroxycarboxylic acid ester with a microorganism transformed with a recombinant plasmid containing a microorganism-originated esterase gene inserted into the plasmid. CONSTITUTION:A gene of an esterase performing asymmetric hydrolysis of a hydroxycarboxylic acid ester of formula I (R1 is alkyl, aryl, etc.; R2 is 1-4C alkyl) and originated from a microorganism (e.g. Pseudomonas fluorescens) is linked to a vector plasmid (e.g. pUC18 of Escherichia coli) to obtain a recombinant plasmid and a host microorganism is transformed with said plasmid. The objective compound of formula II is produced by treating the ester of formula I with cultured liquid or (treated) cell of the transformed microorganism.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention is based on the general formula % (wherein R is an alkyl group, an aryl group, an aralkyl group or an aryloxyl group, R2 is a lower alkyl group, CI
A recombinant plasmid in which esterase (abbreviated as esterase) gene DNA that asymmetrically hydrolyzes hydroxycarboxylic acid esters represented by ~4 (indicating methyl group, ethyl group, etc.) was linked to a vector plasmid was introduced into a host microorganism. A method for producing an optically active hydroxycarboxylic acid represented by the general formula % (II) RI-CH-COOH (wherein R1 represents an alkyl group, an aryl group, an aralkyl group, or an aryloxyl group) using a transformed microorganism. .

[Prior Art] As a simple method for producing optically active α-hydroxycarboxylic acid derivatives, a method is known in which they are produced from racemic esters using bacteria capable of hydrolyzing esters (Japanese Patent Application Laid-open No. 63397/1983). . However, there is no example of utilizing a similar method for producing optically active α-hydroxycarboxylic acid.

The characteristics of the present invention are that optically active α-hydroxycarboxylic acid is produced using a conversion reaction of microorganisms (and enzymes);
Moreover, it involves using bacteria whose activity has been amplified using genetic engineering techniques.

[Problem to be solved by the invention]

In the asymmetric hydrolysis of hydroxycarboxylic acid esters using an esterase gene cloned by genetic recombination, the catalytic ability of microorganisms can be greatly improved compared to conventional methods because a large number of genes can be present in the bacterial body. It can be expected that this will increase the Therefore, the present inventors conducted intensive research on Pseudomonas fluorescens (p3
The esterase gene of Idomonas f.
richia coli) vector plasmid pUc
18 and pUc19 to obtain a recombinant plasmid, which was introduced into Escherichia coli to express the esterase gene, thereby completing the present invention.

[Means to solve the problem]

of the present invention, the general formula % formula % (wherein R1 is an alkyl group, aryl group, aralkyl group or aryloxyl group, R2 is a lower alkyl group, 0
A recombinant plasmid is produced in which the gene DNA of an esterase derived from a microorganism that asymmetrically hydrolyzes a hydroxycarboxylic acid ester represented by 1 to 4 (methyl group, ethyl group, etc.) is linked to a vector plasmid. An optically active hydroxycarboxylic acid represented by the general formula (wherein R1 is an alkyl group, an aryl group, an aralkyl group, or an aryloxyl group) is produced by acting on cells of the transformed microorganism or a processed product of the cells. This is a method of manufacturing.

Hydroxycarboxylic acid of formula (2) is used as a raw material for synthesizing various physiologically active substances having optical activity.

The recombinant plasmid contains a DNA gene encoding Bebutide having esterase activity, which includes the amino acid sequence shown below or a partial sequence thereof.

Met Thr Glu Pro Ala Asp Leu Gly Ala Ala Glu Ala Arg　Phe　Val Val Thr l1e Trp Tyr Asp Ser　Ile　5er Lys Thr Val Arg Thr Gly Ala Gly Phe His Thr Ala Gly Gly Val Thr Phe Asp Gln Gln Arg Gin Tyr Asp 8rg Ser Ala Val Thr Val His Glu Val Gly Ala Trp Pro Leu l1e Ala Cys Val Asp Arg Tyr Leu　Gln　Glu Leu Pro Gln ^sn　Gly　Gly 11e Lys Ala Leu Glu Glu Thr　Asp　Leu 11e Asp Thr Ser　Gin　Gly Phe Lys Lys lie Ala Leu Asn Asp Leu lie Pro Thr Glu Val Val Tyr Glu His Thr　Trp　Gin Leu Pro Gln 1611 Ala Glu Leu　Gln　Pr.

11e Trp Leu Asp　Phe　Leu Thr　Leu　Leu Ala Pro Thr Tyr Glu Met Met Ser Pr.

Leu Glu Thr 11e Glu Thr Ser　Arg　l1e Gly Ala Val Trp Glu Gly Ser　Thr　Tyr Gln Leu 5er Leu Cys Leu Gin Asn Ala Leu Lys Gly Glu　Tyr　Pr.

Glu　Ile　His 8rg Leu Arg Ala Lys His Gly Pro Val Ser　Thr Arg　Pr.

Pro Ser ^1a　Arg Ser　Ala Gln　Gin Phe Leu Val　Phe Pro Leu 8Ia Pr.

8 la 5er His Gly Met Gly Arg Gly Met Gly Asp Ile Examples of DNA sequences encoding peptides with esterase activity include the following.

ATG ACCGAA CCCTTG ATT
CTT CAG CCCGCCAAGCCA
GCA GACGCCTGCGTT ATCTGG
CTG CAT GGCCTG GGCGC
CGAT CGCTAC GACTTCCTG CC
G GTGGCCGAA GCG CTG CAG
GAA ACCTTG CTG AGCACCCGCT
TT GTT TTG CCCCAG GCCCCCA
CG CGCCCGGTG ACG ATT A
AT GGCGGCTACGAG ATG CC
G AGCTGG TACGACATCAAG GC
CATG AGCCCG GCCCGTTCG AT
CAGCCTG GAA GAA CTG G
AA ACG TCG GC 8 to AA ACG
GTT ACG GACCTG ATCGA
G ACA CAA CAGAGA ACCGG
A ATA GACACT TCG CGA ATT
TTCCTCGCT GGT TTT TCT CAA
GGCGGCGCCGTG GTT TTTCACA
CCGCG TTCAAG AAA TGG
GAG GGG CCCTTGGGCGGT G
TG ATCGCCCTCTCCACCTAT G
CG CCCACCTTCGAT AAT GA
CCTG CAA TTA TCCGCCAGC
CAG CAA CGCATCCCT ACG
CTCTGCCTG CACGGCAG TA
CGACGAA GTG GTG CAG A
ACGCCATG GGCCGCAGCGCCTAC
GAG (, AT TTG to A^ GGCCG
TGGTGTCACCGTG ACA TGG
CAG GAA TACCCA ATG
GGCCACGAA GTG TTA CCCC
Hehe GAG ATA CACGAT ATCG
GCGCCTGG CTG GCT GAA
CGG CTG CGC Pseud in the present invention
omonas fluorescens I F030
Escherichia of 81 esterase genes
Cloning into E.coli is performed as follows.

From Pseudomonas fluorescens, the method of Thomas et al. (Cantoni and Da
vies ed, ”Proceduresin
Nucleic Ac1d Re5earch
” Vol, 1 p535.Harperand
Chromosomal DNA is prepared according to Row, New York (1966). This is completely digested with the restriction enzyme EcoRI to obtain a DNA fragment. On the other hand, vector plasmid pUc18 is cut with the same enzyme. pUc18 has only one EcoRI cleavage site in its multiple cloning site. pU linearized by EcoRI cleavage
, c18 and Pseudomonas fluoresc
ens I F 03081 and the ends are joined with T4 DNA ligase to form a hybrid DNA. Add this in advance to CaCl2
The foreign DNA is introduced into the JM109 strain, which is one of the Escherichia coli K-12 strains, which is a host microorganism that has been treated to make it easier to accept foreign DNA. When the JM109 strain containing ptJc18 is grown on an LB agar medium containing ampicillin and IPTGSXGa1, XGa1 is cleaved by the β-galactosidase activity expressed in the JM109 strain, forming blue colonies. When a foreign DNA fragment is inserted into the multi-cloning site of pUc18, β-galactosidase activity is not expressed and the colonies become colorless. Therefore, Pseudomonas f l
Ampicillin and IP
Colorless colonies are selected on LB agar medium containing TC;, XGa 1, and strains expressing esterase activity are screened among them. The search for Escherichia coli J M2O3 strain having the esterase gene is performed as follows. 10mM) Lis-HC1
(pH 7.5), 0.01% bromocresol purple, and 1% (±)-β-acetylthio-α-methylpropionate methyl. The colony was transferred to a paper and left at room temperature for several hours. Colonies with esterase activity produce acid and the pH in the vicinity of the colony decreases. Since bromocresol purple, which is a pH indicator, changes from blue-purple to yellow, it is possible to obtain strains containing the esterase gene by visual observation.

Plasmid DNA was extracted again from this transformed strain,
This is cut with various restriction enzymes to create plasmids containing smaller NA fragments. The location of the esterase gene can be determined by the presence or absence of esterase activity in strains transformed with these plasmids. In the present invention, the esterase gene is transduced into 1ac on pUc19.
By placing the UV5 promoter under the control, the esterase activity of the transformed strain is reduced to that of the parent strain Pseudomonas.
It can dramatically increase the activity of S fluorescens I F 03081.

In addition to microorganisms of the genus Pseudomonas, the donor microorganisms of the esterase gene of the present invention include microorganisms of the genus Nigellysia, genus Staphylococcus, alkaline earth metals, genus Streptomyces, genus Nocardia, genus Mycobacteria, genus Torulopsis, genus Bacillus, and genus Aspergillus. , Candida , Potulitis , Ophyllorus , Chaetomium , or Talatosporium , specifically N. coli I F 013500, respectively.
, Staphylococcus aureus I F 0127
32, Streptomyces griseus I F 03
355, Nocardia Elthropolis T F 012
538, Mycobacterium freyi I F 013
160, Streptomyces talabrigels IF
013307, Torulopsis globengieseri I
F 00659, Bacillus subtilis pearl niger IF○3108, Aspergillus sojae T A
M2703, Candida Rugoza I F 007
50. Potulitis cinerea AM5126, Ophyllorus myabianus IAM8053, Chaetomium semispirale I F 08363, and Thallatosborium recinae furapellaneum HU T5050.

Vectors used in the present invention include, for example, p U C1B+ p
The restriction enzyme maps of UCl3 and M13m p 18 are shown in FIGS. 1, 2, and 3. All of these can be purchased from Takara Shuzo Co., Ltd. The microorganism to be transformed is Esche-ri
chia colt J M2O3 strain, which can also be purchased from Takara Shuzo Co., Ltd. In addition, examples of microorganisms that can be transformed include the above-mentioned Escheric
In addition to the h 1 acoli J M2O3 strain, yeast, Bacillus subtilis, actinomycetes, and the like can be mentioned.

Examples of the alkyl group for the substituent R1 of the compounds of formulas (1) and (H) include methyl group and ethyl group, examples of the aralkyl group include benzyl group, examples of the aryl group include phenyl group, and examples of the aryloxyl group include Examples include phenoxydyl groups, and examples of the hydroxycarboxylic acid ester (1) include methyl mandelate, methyl lactate, and the like.

The transformed microorganism of the present invention is used as a culture solution containing the same, isolated bacterial cells, or a treated bacterial cell.

Cultivation of these transformed microorganisms is usually carried out by liquid culture, but it can also be carried out by solid culture. As the medium, for example, LB medium is used. Culture is 10-5
It is carried out at a temperature of 0°C and a pH range of 2-11. Aeration and stirring may be performed to promote the growth of microorganisms.

When carrying out the hydrolysis reaction, the hydroxycarboxylic acid ester (I) may be added to the medium at the beginning or during the culture, or the hydroxycarboxylic acid ester (1) may be added to the culture solution after culturing the microorganism in advance. You can.

In addition, the cells of the grown microorganisms are collected by centrifugation, etc.
This may be added to the reaction medium containing the hydroxycarboxylic ester. In this case, for convenience of handling, the bacterial cells are
Dried microbial cells, such as freeze-dried microbial cells, spray-dried microbial cells, microbial cells treated with organic solvents such as acetone, toluene, etc., or microbial cell-treated products such as microbial cell destruction products and microbial cell extracts can also be used. For example, ion-exchanged water or a buffer solution is used as the reaction medium. The concentration of hydroxycarboxylic acid ester in the reaction medium or culture solution is preferably 0.01 to 50% by weight. The hydroxycarboxylic acid ester can also be added in suspension in water. The solubility of the ester can also be improved by adding an organic solvent such as methanol or acetone to the reaction solution. The pH of the reaction solution is in the range of 2-11, preferably 5-8. As the reaction progresses, the pH of the reaction solution decreases due to the hydroxycarboxylic acid produced; in this case, it is preferable to maintain the pH at an optimum level using a suitable neutralizing agent. The reaction temperature is 5-50°C.

Separation and purification of the product from the reaction solution or culture solution can be carried out by conventional methods such as extraction, recrystallization, column chromatography, etc.

Reference Example 1 (Preparation of recombinant plasmid) 1) Preparation of chromosomal DNA: 200 ml of LB medium (1% Batato tryptone, 0.5
% Bacto yeast extract, 0.5% NaCl) and Pse
ud.

monas fluorescens I F 03
081 strain was inoculated and cultured overnight. After culturing, the bacterial cells are collected by centrifugation. Bacterial cells were added to 30ml of SSC
(0,15M NaCl, 15mM sodium citrate), suspended in 25% sucrose, 10mM EDTA, 12m
After adding g of lysozyme, leave it for a few minutes and add 10% of 3d.
Add SDS and stir. After incubating for 37°CIO minutes, add 24μ of pronase E and continue to incubate at 37°C.
The cells were incubated at C for 90 minutes. This was combined with an equal volume of water and two volumes of phenol (10mM TRI 5-HCI (
pH 8), saturated with 1 mM EDTA) was added and slowly stirred. The mixture was centrifuged at room temperature, the aqueous layer was removed, and the mixture was extracted again with the same phenol. An equal volume of chloroform (containing 0.4% isoamyl alcohol) was added to the obtained aqueous layer and slowly stirred. The centrifugal condensate layer was taken and extracted again with the same chloroform. An equal volume of ether was added to the aqueous layer for extraction. After removing the aqueous layer and removing the remaining ether with nitrogen gas, RNaseA was added at a concentration of 50 μg/d and incubated at 37°C for 30 minutes, and the above pronase E was added at a concentration of 100 μg/d.
Incubate at 7°C for 30 minutes. Then, as mentioned above, phenol extraction was performed twice and chloroform extraction was performed twice.
Ether extraction was performed once, and the ether was removed by bubbling nitrogen gas. A 2.5-fold volume of ethanol was added to the aqueous layer, and the DNA was collected by centrifugation, dried, and then dissolved in water.

2) Creation of a recombinant plasmid containing the esterase gene: - The chromosomal DNA obtained above was treated with the restriction enzyme EcoRI to obtain a chromosomal DNA fragment. On the other hand, plasmid pUc18
was treated with the same restriction enzyme to obtain a linear plasmid.

By reacting at 4°C overnight using T4 ligase,
Both DNA fragments were ligated.

3) Recombinant plasmid Escherichia
Introduction into E.coli: Escherichia colt J M2O3 strain was cultured in LB medium at 37°C for 2-3 hours, then collected and suspended in a water-cooled 50mM CaC1z solution. After centrifugation, the cells were resuspended in an ice-cold 50mM CaC1z solution and left standing in water for 30 minutes. About 100-200μ! The recombinant plasmid obtained in 2) was added to a test tube, and the mixture was left standing in water for 30 minutes. Heat LB medium at 37°C for 2 minutes to 1111
/ and shaken for 60 minutes at 37'C.

100uf was added to LB agar medium (100μg/Inl ampicillin, 0.5mM IPTG, 0.2% XGa
1) was expanded and kept overnight at 37°C to form colonies.

From colorless colonies that did not exhibit blue color, transformants into which a recombinant plasmid containing an esterase gene had been introduced were selected using the method described below.

10mM) Lis-HC1 (pH 7,5), 0.01%
Colonies are transferred to paper that has been impregnated with bromocresol purple and 1% (±)-β-acetylthio-α-methylpropionate, and left at room temperature for several hours.

Colonies with esterase activity produce acid and the pH in the vicinity of the colony decreases. Since bromocresol purple, which is a pH indicator, changes from blue-purple to yellow, it is possible to obtain strains containing the esterase gene by visual observation.

The transformed microorganism thus obtained (Feikoken Joyori No. 146
8) and the method of Birnboim and Dory (Nuc Ac1d Res, 7.1513-15).
23 (1979)), the plasmid was treated with various restriction enzymes to determine the cleavage site (No. 4).
figure). This plasmid DNA was named pFY501.

Reference Example 2 Expression of esterase by Escherichia coli transformed strain 1) Asymmetric hydrolysis of methyl (±)-β-acetylthio-α-methylpropionate: The above transformed strain was treated with 50 μg/− of ampicillin.
After overnight shaking culture at 37°C in 00-LB medium, bacterial cells were obtained by centrifugation. The total amount of bacterial cells was reduced to 2% (±)-
Methyl β-acetylthyl-α-methylpropionate 15
Suspended in 0d, 0. The reaction was carried out at 30°C for 5 hours while controlling the pH to 7.0 with IN NaOH. After the reaction is complete, the bacterial cells are removed by centrifugation, and the unreacted β is extracted from the supernatant.
Methyl -acetylthio-α-methylpropionate was extracted and removed with ethyl acetate.

Next, the pH of the aqueous layer of the extraction residue was lowered to 2.0 or less with dilute sulfuric acid, and then β-acetylthio-α-methylpropionic acid was extracted with ethyl acetate. Then, after adding anhydrous sodium sulfate to the extract and dehydrating it, the solvent was removed by evaporation.
An oil was obtained. After diluting a portion with water, β was determined by HPLC.
-Acetylthio-α-methylpropionic acid was quantified.

A part of the solution was dissolved in chloroform, and the optical rotation was measured using a digital automatic polarimeter (Model DIP-360, manufactured by JASCO Corporation).

As a result, 270 μ of the desired product was obtained, and the specific optical rotation was [α]6°=-48.6 (C=1.10CHct:+
The parent strain (Pseudomonas fluoresce)
Specific rotation of the product by ns IF03081 strain -4
It was almost equivalent to 8.1.

In addition, as a control experiment, vector plasmid (pUc
18) was used, but there was no consumption of NaOH during the reaction, and the desired product was not obtained.

2) Location of the esterase gene on plasmid pFY501, construction of a new plasmid, and amplification of esterase activity in the transformed strain: In order to clarify the location of the esterase gene on pFY501, we first
1 was cut with each restriction enzyme, the obtained DNA fragment derived from the parent strain was ligated to pUC18, and Escherichia
coli J M2O3 strain. Among them, 4.
Plasmid pFY50 containing the 9Kb HindI [[ fragment
Three introduced transformants retained esterase activity (Fig. 5A).

As a result of further detailed examination, it was confirmed that the esterase gene was present in the 2.2 Kb SmaI fragment of the 4.9 Kb HindII fragment (Fig. 5B). This 2.2K
Since the esterase activity of plasmid pFY513 containing the bSmal fragment is induced by isopropyl β-D-thiogalactopyranoside (manufactured by Wako Pure Chemical Industries, Ltd.; abbreviated as IPTG), the esterase gene is located in the Ia portion of the vector.
It became clear that it was under the control of the cUV5 promoter. This also confirmed the direction of the esterase gene.

Furthermore, Exonucleas from one end of the Sma I fragment
e III +Mung Bean Nuclease
(using Takara Shuzo's TAKARA Kilo Sequencing Precision Kit)
The ranking region was removed to create pFY520 (Figure 6). The transformed strain containing this plasmid pFY520 (Feikoken Joyori No. 1469) also had an esterase gene and was under the control of the 1acUV5 promoter.

For transformants containing ρFY520, 0.2mM
The esterase activity was quantified by the same method as in 1) above, except that the culturing was performed in the presence of PTG, and as shown below, it was revealed that the pFY501 transformed strain had a specific activity about 19 times that of the transformed strain.

Transformed strain Specific activity [uni7)/g dry bacterial cells] E. coli JM109/pFY501
260E, coli JM109/pFY520
4900 However, under the above reaction conditions, the amount of enzyme that produces 1 mg of the target acid in the initial 1 hour is 1
defined as a unit.

In FIG. 5, the presence or absence of (A) activity was determined by observing the change in color of bromocresol purple on the paper as described in Example 1.

(A) pFY503 (see Figure 6) and pFY508 have 4.9 K b and 2.7 K b from pFY501, respectively.
K bHindII [fragment was converted into HindI of pUc18]
[Created by inserting into the I cleavage site. pFY5
06 and ρFY509 are Hind of pFY501, respectively.
I[[, was created by self-ligation after cutting with XhoI.

(B) pFY513 is 2.2 K b of pFY503
It was created by inserting the Sma ■ fragment into the Sma I cleavage site of pUc18. pFY510 and pFY5
No. 12 was created by self-ligation after cutting pFY503 with 5ail and PstI (see Figure 6). Reference Example 3 Determination of base sequence of esterase gene and predicted amino acid sequence From plasmid pFY520, HincII-3ma
A plasmid pFY525 lacking the r region was created (Fig. 6), and a transformed strain of this pFY525 was also used in Reference Example 2.
When tested using the method shown in 1), it was found that it retained esterase activity. Therefore, the entire base sequence of the DNA fragment derived from the parent strain contained in pFY525 was extracted using the Chain terminator method (S
angel, F, 5science 214.1205-
1210 (1981)). the result,
The base sequence of the approximately IKb DNA fragment derived from this parent strain is
It was as shown in the previous section. This base sequence contains Openre, which is assumed to encode the esterase gene.
There is only one adding frame, and pFY512 lacks the region upstream of the Pstl cleavage site.
This Ope has no esterase activity (Fig. 5B) and the orientation of the esterase gene described above
It was revealed that the n reading frame encodes an esterase gene, and that the amino acid sequence predicted from this or a portion thereof is the amino acid sequence of the esterase of interest.

Example 1 Creation of pcFOO5 Plasmid pcFOO5 was obtained as follows (
Figure 7). Cut pFY520 with restriction enzyme NaeI,
A restriction enzyme old ndIII linker was inserted into the cut site. The thus obtained plasmid pcFOO1 was cut with the restriction enzyme old ndIII, and the drug a containing the esterase gene was prepared.
A DNA fragment of oobp was obtained. This fragment was
It was inserted into the plasmid vector PUC18 cut with I. In the thus obtained plasmid pCF005, the esterase gene is 1a derived from vector pUc18.
It is under the control of the cUV5 promoter.

Example 2 Escherich of recombinant plasmid pcFOO5
Introduction to Escherichia coli J M2O3 strain was cultured in LB medium at 37°C for 2-3 hours, then harvested and suspended in ice-cold 50mM CaC1z solution. After microbial collection by centrifugation, the cells were resuspended in a water-cooled 50 mM CaCIz solution and left standing in water for 30 minutes. Approximately 100-200 μl was placed in a test tube, and the recombinant plasmid pcFOO5 obtained in Example 1 was added thereto, and the tube was left standing in water for 30 minutes.

Heat at 37°C for 2 minutes and add 1rn1 LB medium to 37°C.
Shake for 60 minutes. Transfer 100μffi to LB agar medium (l
(contains oug/ml ampicillin) at 37°C.
The cells were kept overnight to form colonies.

Microorganism Escherichia coli J M1
09/p CF 005 has been deposited as FEIKEN Article No. 2166.

Example 3 J M109/ p F Y525 and J M 10
9/p Asymmetric hydrolysis of methyl mandelate by CF 005 Escherichia coli J M1
09/p F Y525 and JM109/p C
F005 was added to 100/d LB medium (0.1mM IP
Contains TG (isopropyl-β-thiogalactoside)
After culturing at 37°C for 115 hours, the bacterial cells were collected by centrifugation. Pseudomonas fl, a gene-donor bacterium
uorescens IF 03081 was cultured in a 100 d meat extract medium at 25°C for 140 hours, and then the bacterial cells were collected in the same manner. These were suspended in cold water, and a portion thereof was used for the reaction.

1 relative 0.1 M sodium phosphate buffer (pH 7.0
) 1% D, L-methyl mandelate with bacterial cells? A suspension was added and the reaction was carried out at 30°C. The bacterial cell concentration of the reaction solution is E, c
oli J M109/ pF Y525 and J
0.35111g for M109/pCF005
/mL 5.0mg/mL for IF 03081
m (all dry weight).

On the way, we sampled 50μ2 several times and found 450μg of 0.
．． The reaction was stopped by addition to 2N hydrochloric acid.

After stopping the reaction, the substrate and product were analyzed by high performance liquid chromatography (HPLC), and the conversion rate of methyl mandelate to mandelic acid was calculated.

(Conditions) Column: ODS-80TM (Tosoh ■) Solvent: Methanol; Water; Phosphoric acid; (5015010,025)
Temperature: 40°C Flow rate: 1 relative/min Detection: 254 nm In addition, the stereoisomerism of the product (mandelic acid) is determined by CHIRALP.
H using an ACK-WH column (Daicel Chemical Industries ■)
Analyzed by PLC. As a result, it was found that methyl L-mandelate was selectively hydrolyzed.

(Conditions) Solvent: 0.25mM copper sulfate Flow rate: lIn1/min Detection: 254nm Table 1 shows changes over time in the conversion rate and the optical purity of the produced mandelic acid.

Furthermore, if the activity of producing 1 μmo+ mandelic acid in − minutes is 1 unit, then J M109/ p F Y5
25 and J M109/p CFOO5, IF
The specific activities of 03081 are 1.1.1.4.0.0, respectively.
08 units/■ dry bacterial cells.

Table 1 Table 2 Example 4 Asymmetric hydrolysis of lactic acid ester using J M109/p CFOO5 The bacterial cells obtained in the same manner as in Example 2 were used for the reaction. 1
In1. 0. IM sodium phosphate buffer (pH
7,0) - Add the bacterial cell suspension to 1% D, L methyl lactate,
The reaction was carried out for 1 hour at 30"C. After the reaction, analysis was carried out in the same manner as in Example 3. For detection with the ODS column, 210"
nm was used.

Example 5 Purification of enzyme Escherichia colt J M109/
pCF 005 was added to 3L of LB medium (0.1mM IP
After culturing for 115 hours at 37°C (containing TG), the bacterial cells were collected by centrifugation. After washing twice with cold water, about 20 g of wet bacterial cells were obtained. The bacterial cells were suspended in 20mM Tris hydrochloric acid buffer (pH 8), and cellumil (Edmund Bj.
Glass beads (manufactured by IHLER) with a diameter of 0.1 mrn were used to destroy the beads. The membrane fraction was removed by centrifugation, and the soluble fraction was dialyzed against 20mM Tris-HCl buffer (pH 8) and fractionated by ion exchange chromatography. DEAE Cellulofine (Seikagaku Kogyo ■) equilibrated with the same buffer was used as the ion exchanger, and a concentration of O-0.8M sodium chloride was used for elution of the esterase.

The active fraction was concentrated and gel filtration column (Tosoh ■, G300
Further purification was performed by HPLC using 0SW). As a solvent, 50mM sodium phosphate buffer (pH 7.0
)It was used.

When the esterase thus obtained was analyzed by SDS gel electrophoresis, it showed a single band. Purified esterase was used in the following experiments.

Example 6 Asymmetric hydrolysis of lactic acid ester by purified esterase ld 0.1 M) Lis-HCl buffer (pH 7,5) -
1Mg in 1% D, L methyl lactate (or ethyl lactate)
of purified esterase was added and reacted at 30°C for 130 hours. After the reaction, analysis was performed in the same manner as in Example 2. O
A wavelength of 210 nm was used for detection with the DS column.

L-lactic acid ester was selectively hydrolyzed to produce L-lactic acid. Table 3 shows the conversion rate after 30 hours of reaction and the optical purity of the produced lactic acid.

Table 3 Example 7 Asymmetric hydrolysis of methyl lactate by purified esterase 0.1M sodium phosphate buffer (pH 7.0)
-1 Mg of purified esterase was added to 1% D, L methyl lactate and reacted at 30°C for 130 hours. After the reaction, analysis was performed in the same manner as in Example 5. The results are shown in Table 4.

Table 4 Example 8 Asymmetric hydrolysis of methyl mandelate by purified esterase 1 ml of 0.1 M sodium phosphate buffer (pH 7)
, 0)-1% D, L 1Mg of purified esterase was added to methyl mandelate, and the reaction was carried out at 30"C.
μ! Sampling was carried out several times and 450 μl of 0.2 N
The reaction was stopped by adding to hydrochloric acid. The analysis was carried out in the same manner as in Example 3. Table 5 shows the change over time in the conversion rate and the optical purity of the produced mandelic acid.

Table 5 [Effects of the invention] Recombinant plasmid pFY501 obtained by the present invention
, pFY520, pF Y525, pCFOO5 and Escherichia transformed therewith.
The col, i transformed strain contains an esterase gene that asymmetrically hydrolyzes hydroxycarboxylic acid ester (1). Furthermore, the esterase activity of transformed strains such as pcFO05 was able to be dramatically increased compared to that of its parent strain. Furthermore, it was possible to efficiently obtain an optically active carboxylic acid by allowing the cells of this transformed strain or a treated product thereof to act on the hydroxycarboxylic acid ester (1).

[Brief explanation of the drawing]

Figures 1, 2 and 3 are vector pU
Restriction enzyme maps of C18, pU C19 and M13m p18 are shown. FIG. 4 shows a restriction enzyme map of plasmid pFY501 containing the esterase gene. Figure 5 shows the deletion plasmid constructed from pFY501 and esterase activity. FIG. 6 shows the construction process of the main plasmid used in the present invention. Figure 7 shows the process of creating plasmid pcFO05. Figure 8 shows the entire base sequence of the parent strain-derived DNA in pFY525 and the 0 pen reading fra contained therein.
The predicted amino acid sequence of esterase is shown.

Claims (1)

[Claims] 1. General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I) (In the formula, R_2 is an alkyl group, aryl group, aralkyl group, or aryloxyl group, R_2 is a lower alkyl group C
A recombinant in which gene DNA of an esterase derived from a microorganism that asymmetrically hydrolyzes a hydroxycarboxylic ester represented by _1_ to _4 (indicating a methyl group, an ethyl group, etc.) is linked to a vector plasmid. There are general formulas ▲ mathematical formulas, chemical formulas, tables, etc. ▼ (II) (in the formula, R_1 is alkyl A method for producing an optically active hydroxycarboxylic acid represented by a group, an aryl group, an aralkyl group, or an aryloxyl group.