JPS61282093A - Production of optically active indoline-2-carboxylic acid by immobilized enzyme or immobilized microorganism - Google Patents

Abstract

PURPOSE:To produce advantageously a synthetic raw material for medicines, by bringing an indoline-2-carboxylic acid ester into contact with an enzyme or microorganism, immobilized in a carrier and having stereoselective esterase activity to carry out optical resolution. CONSTITUTION:An (R,S)-indoline-2-carboxylic acid ester expressed by formula I is brought into contact with an immobilized enzyme or microorganism, immobi lized in a carrier with hydrophobicity and having stereoselective esterase activ ity. Examples of the above-mentioned microorganism or enzyme include microorganisms, having (R)-selective esterase activity and belonging to the genus Streptomyces, Saccharomyces, etc. microorganisms, having (S)-selective esterase activity and belonging to the genus Arthrinium, etc. or enzymes derived from the above-mentioned microorganisms. The (R,S)-indoline-2-carboxylic acid ester expressed by formula I is converted into a carboxylic acid expressed by formula I and a carboxylic acid ester expressed by formula III by the asym metric hydrolysis, which are separately eluted. The carboxylic acid ester expressed by formula II is further hydrolyzed to give a carboxylic acid.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention optically resolves indoline-2-carboxylic acid ester using immobilized enzymes or immobilized microorganisms to produce optically active indoline-2-carboxylic acid ester useful as a raw material for pharmaceutical synthesis. The present invention relates to a method for producing 2-carboxylic acid. More specifically, the general formula (wherein is an alkyl group or alkenyl group having 2 to 10 carbon atoms, an alkyl group having 2 to 10 carbon atoms substituted singly or simultaneously with a hydroxyl group and a halogen M, or alkenyl group, unsubstituted or substituted alicyclic hydrocarbon group,
Represents an unsubstituted or substituted 7enyl group or benzyl group.

)-indoline-2-carboxylic acid ester represented by (R,S)-indoline-2-carboxylic acid ester is brought into contact with an enzyme or microorganism having stereoselective esterase activity immobilized on a carrier to cause asymmetric hydrolysis, Optically active indoline-2-carboxylic acid I expressed by formula 9 (in the formula, the umbrella represents an asymmetric carbon) and optically active indoline-2 expressed by formula 11 (in the formula, the symbol is the same as above) -Produce carboxylic acid ester r, take advantage of the difference in affinity with the carrier used for immobilization, collect and collect hydrophilic indoline-2-carboxylic acid with water or a buffer solution, and then This invention relates to a method for alkaline hydrolysis of indoline-2-carboxylic acid ester adsorbed and retained on a substance, and elution and collection of optically active indoline-2-carboxylic acid P, which has an optical rotation opposite to that of the product r.

That is, an asymmetric hydrolysis reaction, separation of the product indoline-2-carboxylic acid and unreacted ester, and further alkali hydrolysis with the unreacted ester adsorbed on the carrier to produce indoline-2-carboxylic acid.
The present invention relates to a method for producing indoline-2-carboxylic acid, characterized in that indoline-2-carboxylic acids having different optical activities are simultaneously obtained in one operation by sequentially collecting 2-carboxylic acids.

These optically active indoline-2-carboxylic acid compounds are important compounds that can be used as raw materials for various pharmaceutical products. For example, (8)-indoline-2-carboxylic acid is useful as an angiotensin I-converting enzyme inhibitor.
(8)-8-mercapto-2-ochinpropyl]-indoline-2-carboxylic acid@) [Journal op Medicinal]
Ket Story (J, Med, Chem,
).

26.894 (1988)).

(Conventional technology and problems) In conventional enzymatic reactions, free enzymes were added to a reactor and the reaction was carried out in batches, and after the reaction was completed, the enzymes were discarded, but enzymes were generally expensive. Therefore, the industrial use of enzymes has been limited because they are disadvantageous in terms of cost and unstable. Furthermore, in the batch method, after the enzymatic reaction is complete, the reaction product can be separated from the reaction solution by: (1) extraction and separation with an organic solvent, (2) dissolving the reaction solution once in an organic solvent, or directly using column chromatography. Methods such as separation by graphic treatment, (1) dissolving the reaction solution with an organic solvent, or fractional distillation of the reaction solution as it is have been used, but the operations are complicated and the yield is low. The drawbacks were that it was slow, time consuming, and required special equipment, resulting in high costs.

For this reason, in recent years, research has been conducted on the immobilization of enzymes and microorganisms, and it has become possible to use enzymes and microorganisms repeatedly, and even to perform continuous reactions by filling them in columns.

However, using an immobilized enzyme to separate the reaction product from the racemate as a raw material at the same time as asymmetric ice lysis, the unreacted ester adsorbed on the carrier is subsequently eluted by alkaline hydrolysis.
To date, there have been no reports of successful collection or continuous collection.

(Means and effects for solving the problems) The present inventors first discovered that indoline-2-carboxylic acid ester I
optically active indoline-2-carboxylic acid 1*
and optically active indoline-2-carboxylic acid ester 11
discovered an enzyme or microorganism that stereoselectively splits the two, and proposed a method for producing indoline-2-carboxylic acid by optical resolution (Japanese Patent Application No. 59-211).
986, 59-214725, 6O-69769)
.

The present inventors have investigated the immobilization of these enzymes or microorganisms,
Efforts have been made to establish a simple product separation technology. As a result, by selecting a carrier that has a different affinity for the substrate and the product and immobilizing the enzyme or microorganism on the carrier, the substrate indoline
Indoline is produced by asymmetric hydrolysis of 2-carboxylic acid ester, separation of the product indoline-2-carboxylic acid and unreacted ester, and further alkaline hydrolysis with the unreacted ester adsorbed on the carrier. -2-Carboxylic acid recovery was successfully carried out in order, and the present invention was completed. The present invention will be explained in detail below.

The indoline-2-carboxylic acid ester represented by the general formula H used as the substrate of the present invention is
An alkyl group or alkenyl group having a substituent hole of 2P-10 carbon atoms, an alkyl group or alkenyl group having 2 to 10 carbon atoms substituted singly or simultaneously with a hydroxyl group and a halogen atom, an unsubstituted or substituted alicyclic group hydrocarbon group,
Compounds with unsubstituted or substituted phenyl or benzyl groups, preferably ethanol, butanol, amyl alcohol, hexanol, glycerol, ethylene/IJ
Cole, glycerol-α-7sonochlorohydrin2.
These are esters of indoline-2-carboxylic acids such as 8-dichloro-1-propatol, 1,8.5-pentanetriol, cyclohexanol, and benzyl alcohol.

Indoline-2-carboxylic acid ester I is expressed as K
It can be obtained by That is, adding alcohol that serves as both a solvent and a reaction reagent to (R,8)-indoline-2-carboxylic acid,
Concentration of indoline-2-carboxylic acid 5-20% (W/v
) in the range of 1 to 50°C to reflux temperature under strong acidity.
The condensation reaction is carried out for 5 hours. Saturated sodium bicarbonate water was added to this reaction solution to adjust the pH to 7, followed by extraction with a hydrophobic organic solvent such as ethyl acetate, chloroform, methylene chloride, hexane, etc., and further concentration to obtain highly pure (R).

8) -Indoline-2-carboxylic acid ester (■) is obtained.

Examples of microorganisms that have stereoselective esterase activity that asymmetrically hydrolyze racemates to produce (R)-4 include Streptomyces (8treptomyces).
ces), genus Baccharo
myces) genus, Trichosporon (Tr 1chos)
poron), Aerosonas
), Genus A/I/ Arthrobacter
er ) genus, Acidiphi A
lium) Horse, Plebipacterium A (Breviba
cterium), Corynebacterium (Coryn
ebacterium), Pseudomonas (Ps.
There are microorganisms belonging to the genus (Streptomonas), and more specifically, Streptomyces griseus (Str+
eptomyces griseus) I FO
885g, Saccharomyces cerevisiae (Bacch
aromyces cerevisiae) HUT
7017.7018.7119, Trichosporon cutaneum
IFO1200, Aerosonus hydrophila (Ae
romonas hydropTlila) IFO
8820° Arthrobacter paraffineus
s) ATOO21817, Arthrobacter n1cotian
ae) IFO14284, Acidiphilium cryptum
I FO14242, Brevibacterium protophori
rmiae) I F 012128, Corynebacterium paulometabo, Fum (Oorynebacte
rium paurometabolum) IFO12
160, Corynebacterium acetacidoquim (
Oorynebacterium acetoacido
philum) AT 00 21476, Pseudomonas oxa2thecus (Pseudomonas
xalaticus) I Fo 18598. In addition, (R)-selective animal-derived enzymes include tl:t such as Steapunun and Pancreatea derived from pig pancreas.

In addition, examples of microorganisms having stereoselective esterase activity that asymmetrically hydrolyze racemic body (1) to produce (8)-i include, for example, Arthrinium (4rthrinium).
Genus, Aspergillus,
Cephalospori
um ) genus, Echinopodo
spora), Emerica psi, <(Emeric
el 1opsis) genus, Hybocrea (HypOCr)
ea) genus, Isaria (5aria) genus, Lepista (
[,epista) genus, Nectria
Genus, Pestalotiopsis
s) genus, phyalophor 7 aura (, phialophor
a ) genus, Podosbo, 71odospora ) genus, Botryoascus spp., Candida spp., Sitelomyces (C
i teromyces), Debaryomyces (D
ebaryomyces), Honimorescus ([O
rmOaSCuS ), Moniliella (ldoni 1
11ella), Kluyperomyce, r, (xtu
yveromyces), Nadsonia (Nadso
nia), genus Rhodotorula
) genus, Schizosatucharomyces (8chi zosac
haromyces) genus, Torul
opsis) genus, Wicke no mia (Wicke
rhamia), Alcaligenes
gene), Arthrobacter (Arthrob
acter) genus, Brevibacterium (Brevib
genus acterium, protaminobacter
aminobacter ) genus, Pseudomonas (Ps
Microorganisms belonging to the genus eudomonas9, more specifically, Arthrinium phaeospermum (Ar
thrinium phaeospermum) I
F 05708, Aspergillus fiku A (Asp
ergi llusficum) IFO4280, Cephal
osporium mycophilum) I F 0
8580. Echinopodospora jamaisii, x
, (Echinopodosporajamaice
nsis) I F O80406, Emerythelopsis grub? (Ernericellopsis g.
labra) IFO9081, Hypocrea 1actea I F 0
8484, Isaria-7 Chibikola (ENG 5aria a
typicola) IFO9206, Lepista nuda (I, epista nuda) IFO8104, Nectria flammea (Nectria flammea)
IFO80806, Pestalotiopsis distinctta
) IFO9981, phialophora fastigiata
I FO6850, Bodosbora carbonaria (p
odoapora carbonaria ) I F
0 80294, notryoascusaynnaedendru
s) I FO1604, Candeida Deipe hf
(Qandida diversa) Engineering FO109G
, Candeida Sinidoto Blasphemous Charis ((3andi
da pseudotropicalis) xλM
484G, Cytelomyces matritensis (ai t
eromycesmatri tensis) I
F 0 0651, Debaryomyces hansenii
) IFO0015, Hormoascus platypodis
I FO1471, Moniliella tomentosa (goni
CB 8 220
e 82, Thalyveromyces-7 lagiris (zl
uyveromyces fragilis) I Fo
0288, Nadsonia elongap (Hadso
nia elongata) IFO0665, Rhodotorula glutinis (Rhodotorula glutinis)
inis) IAM 4642, Bchizosaccharomyces
pombe) IFO0847, IFO0862, Torulopsis gropengietsse! j (Torulops
iggropeng, 1esseri) I F 0
0659, Wikerhamia fluorescens x (
yickerhamia fluorescens) I
FO1116, Alcaligenes, 7 aecalis (Al
caligenes faecalis) IFO126
69, Furthro / (/ Tar Cristaloboetes (
Arthrobacter crystallopoie
tes) I FO14285, Brevibacterium
7 rapa, 14 (Brevibacterium fla
vum) ATOO21269, Grotaminova/fi-
・7kyt (75 buses)
ralboflavus) I F 0 8707,
Pseudomonas acidoborys
nasacidovorans) I F O185
There is 82 mag.

The nutritional source for these microorganisms is usually an appropriate combination of assimilable organic and inorganic carbon sources, nitrogen sources, vitamins and minerals, and the culture temperature is 20-40°C, I) H2-8
range is used. Furthermore, the growth of microorganisms can be promoted by aeration and stirring.

The microbial cells obtained by culturing in this way can be used as is by immobilizing them with a hydrophobic resin, but after crushing the microbial cells, the crude enzyme obtained by ammonium sulfate fractionation or acetone treatment or purified enzymes can be used. It can also be used after fixation.

As the carrier for enzyme immobilization in the present invention, various carriers having water-repellent properties can be used. The hydrophobic carrier used in the present invention does not adsorb hydrophilic compounds generated by an asymmetric ice-breaking reaction in water or a buffer solution, but adsorbs unreacted ester compounds through hydrophobic interaction. 1) It is desirable that the carrier is stable even in the alkaline range of H8-10. More specific carriers include, for example, hydrophobic synthetic adsorbents,
Examples include resins for hydrophobic chromatography, hydrophobic photocrosslinkable resins, hydrophobic urethane prepolymer resins, and polymeric substances into which hydrophobic groups are chemically bonded.

Enzymes can be immobilized on such carriers by various known methods. Examples include physical adsorption methods, covalent bonding methods, ionic bonding methods, crosslinking methods, and entrapment methods. In the case of microorganisms, comprehensive methods are available [edited by Fukui, Chibata, and Suzuki, Enzyme Engineering.

pp. 157-248, Kodansha (1981); Chibata, ed., Immobilized Enzyme, Kodansha (1975)].

Among these methods for preparing immobilized enzymes or immobilized microorganisms, in the case of enzymes, it is preferable to physically transfer the enzyme to a hydrophobic synthetic adsorbent due to the simplicity of the method, the physical strength and low cost of the carrier, etc. In the case of microorganisms, a method of enclosing microbial cells in a hydrophobic photocrosslinkable resin or a hydrophobic urethane prepolymer resin is industrially desirable.

The amount of enzymes or microorganisms supported on the carrier is not necessarily unambiguous as it depends on the supporting capacity of the carrier, but for enzymes it is about 0.1 to about 100" per 1f of wet weight of the carrier.
9. Normally, it should be about 1 to about 201 F, and for microbial cells, the wet weight of the carrier should be 11 kg, and about 0.1 to 1 g of wet bacterial cells, usually about 0.151 to about 0.5 f. good.

The amount of substrate that can be loaded onto a column filled with this immobilized enzyme or immobilized microorganism will vary depending on the type of immobilized carrier and substrate ester, but if the amount is If the substrate of the reaction is not discharged from the column, it is possible up to a limit amount to be discharged. For example, when an immobilized enzyme is packed using the synthetic adsorbent Amberlite XAD-7 as a carrier and ethylene glycol ester is used as a substrate, it is possible to load up to 115 of the column volume. As for the method of charging the substrate, in the case of a column type, it is sufficient to simply charge the substrate at the top of the column and flow the buffer solution. In the case of a batch method, the substrate and immobilized enzyme or immobilized microorganism may be mixed.

The asymmetric ice-melting reaction in the present invention is usually carried out at 10°C to 60°C.
Although possible within the range of 20°C to 40°C, it is preferable. This asymmetric ice-melting reaction is possible at a p T of 14.5 to 10, but at a pH of 5 to 7, where the reaction rate is high.
It is desirable to do this within the range of 5. In addition, in this reaction, indoline-2-carboxylic acid is produced as asymmetric ice melting progresses, and p
H decreases. Therefore, when a large amount of substrate compound is loaded, it is desirable to use a buffer to control the pH within a certain range.Buffers suitable for this purpose include either inorganic acid salt buffers or organic acid salt buffers. can also be used.

In addition, alkaline thawing of unreacted esters adsorbed on the carrier of immobilized enzymes or immobilized microorganisms can be carried out at a pH level that does not deactivate the immobilized enzymes or microorganisms, and at a pH lower than that at which the esters are hydrolyzed. The pH may vary depending on the enzyme or microorganism, but it is usually desirable to have a pH of 8 to 10.

When conducting a reaction using a column, it is desirable to use a buffer solution from the viewpoint of keeping the pH constant. After filling the column with the immobilized enzyme or immobilized microorganism,
, and then the substrate ester compound (R, 8)
-I is loaded, and after loading is completed, the buffer solution is flowed again to cause an asymmetric ice-breaking reaction within the column, and the generated hydrophilic compound P is dissolved in the buffer solution and discharged from the column. This buffer fraction was analyzed by high performance liquid chromatography (Finepak 8IL Cu v developing solution; acetonitrile-water = 1.5:1 (v/v) + detection; Uv215 nm), and compound When pH 9 is almost no longer observed, adjust the buffer solution to pH 9.
The unreacted compound f adsorbed to the immobilized enzyme or immobilized microorganism in the column is changed to a buffer solution of 8 to 10.
is hydrolyzed and desorbed and eluted as C, which has the opposite optical rotation. Analyze this buffer fraction by liquid chromatography (same conditions as above), and if Compound 9 is hardly observed in both fractions, change from pH 8-10 buffer to pH 7.0 buffer again. Change the run and reload the column with substrate ester compound (R,8)-4.

By repeating these series of operations, the compound (
It is possible to continuously perform the asymmetric ice melting of R,8)-I, the separation of the reaction product, and the alkali ice melting in a pulsed manner.

When the present immobilized enzyme or immobilized microorganism is used to perform asymmetric deicing of racemic compound l in a batch method, the aqueous layer containing the optically active hydrophilic compound r generated and the unreacted optically active hydrophobic compound The immobilized enzyme or immobilized microorganism adsorbing compound r is separated by filtration or gentle centrifugation, and the alkaline solution is adjusted to a pH of 8 to 10 to the extent that the immobilized enzyme or immobilized microorganism is not inactivated. Alternatively, by hydrolyzing and desorbing the ester adsorbed to the immobilized enzyme or the immobilized microorganism using a pusSlo buffer, an optically active compound (9) with optical rotation opposite to (1) above is obtained. The immobilized enzyme or immobilized microorganism can be used again in the reaction.

The fraction containing indoline-2-carboxylic acid obtained by this method is either precipitated by adjusting the pH to around 5.0 and condensing for 1 s, and collected, or after being saturated with ammonium sulfate, the pH is reduced. The color can be obtained by adjusting the concentration to around 5.0, extracting with an organic solvent such as ethyl acetate or methylene chloride, and adding +1 to 1 m. If necessary, it may be further crystallized in an organic solvent such as acetone.

(5j! Examples) Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Synthesis of substrate (R,8)-(indoline-2-carboxylic acid ethylene glycol ester) Synthesis of a (R,8)-indoline-2-carboxylic acid 150F and ethylene glycol 250F were mixed, and concentrated sulfuric acid 20- was added, and a condensation reaction was carried out for 3 hours in the range of 95 to 100°C.After the reaction, once cooled, sodium bicarbonate and saturated sodium bicarbonate water were added to adjust the pH to 7.0.Next, methylene chloride The methylene chloride layer was washed with 10 ml of water, dehydrated with anhydrous sodium sulfate, and further concentrated under reduced pressure to obtain (R, 8)-I.
a was obtained with a yield of 50.2F and 79%.

In a similar manner, by adding 5 times the amount of alcohol to (R,8)-indoline-2-carboxylic acid 50F, the following substrate (R,8)-indoline-2-carboxylic acid glycerol α-monochrome Ruhydrin ester (I b) 45.49
．． Yield 58.0% (R#8)-Indoline-2-carboxylic acid butyl ester (Ic) 5B, 8f, yield 80% (R,S)
-indoline-2-carboxylic acid glycerol ester (
Id) 57.7! i, yield 79% (R,8)-indoline-2-carboxylic acid cyclohexanol ester (Ie
) 52J t, yield 69% (R,8)-Indoline-2-carboxylic acid benzyl alcohol ester (If) 49.7 f, yield 4164
．． 0% (R,8)-indoline-2-carboxylic acid 1,8°5-
59.5 pentanetriol esters (!J) were obtained in a yield of 78.8%. In addition, as an extraction solvent, (R,8)-indoline-2-carboxylic acid glycerol ester (Id) was used in place of methylene chloride, ethyl acetate was used in place of methylene chloride, (R,8)-indoline-2-carboxylic acid cyclohexanol ester (Ie) used hexane.

Example 2 Actinase E (derived from Streptomyces grisecus) (Kaken Pharmaceutical @lli8 ffpH7.0 0, 1 M
The mixture was added to 60% of phosphate buffer and mixed, and insoluble materials were removed by filtration. The methacrylate porous adsorbent Amberlite
%) and stirred overnight in a cold room (4°C).
The enzyme was adsorbed and immobilized. The immobilized enzyme suspension was suction-filtered using a glass filter, and further filtered at pH 7.0.
1M! 8 times with 100% acid buffer (total aOOg)
After washing, the wet immobilized enzyme was obtained by suction filtration. This immobilized lipase was packed into a column with an inner diameter of 2.2c11 to a height of 161cm, and kept at 88°C to release racemic indoline-2.
-/j Rubonic acid ethylene glycol ester (R9S)
-Ia5f was loaded and reacted by flowing 0.1 M phosphate buffer with p EI 7.0 at a flow rate of 20 -/hour.

The effluent from the column was collected in 20-unit fractions using a 7-lux collector and analyzed by liquid chromatography. This phosphate buffer fraction contained only hydrophilic indoline-2-carboxylic acid produced by asymmetric hydrolysis. Ammonium sulfate was added to both portions of the phosphate buffer until saturation, and the pH was further adjusted to 5.0. Next, an equal amount of ethyl acetate was added to extract the indoline-2-carboxylic acid eight times, and after dehydration, the indoline-2-carboxylic acid was extracted under reduced pressure.
Reconsolidation was performed with hexane (8m-2m'). After drying in vacuum,
The specific optical rotation [α] is also '-80.5° (C=1.0, dimethylformamide) [literature value, Journal Op.
Medicinal Chemistry, 26°894 (19
88), (α], +84.5° (c=0.91, dimethylformamide)].
R, 8)-1a was obtained in a yield of 77%. At the point when the phosphate buffer solution was flowed at 400 °C, a 0.1M K2HPO4-NaOH solution with a pH of 10.0 was flowed at a flow rate of 40 °C per hour instead of the phosphate buffer solution, so that the immobilized enzyme in the column was adsorbed onto the carrier. The unreacted indoline-2-carboxylic acid ethylene glycol ester Ia was hydrolyzed, and was desorbed and eluted as ■. The alkaline buffer fraction containing summer 80
0- was adjusted to pH 5.0 with 2N hydrochloric acid, extracted 8 times with an equal amount of ethyl acetate, dehydrated, and concentrated under reduced pressure. When the dried product is reconstituted with 77 tons of hexane (6d-1,5-), a white powder (S) having a specific rotation [α)”+82.7° (C = i, o, dimethylformamide) is obtained. -indoline-2-carboxylic acid (S)-■ is 1.84 f ((R,
8) Yield of -1a and -1
-68%) was obtained.

No enzyme desorption was observed in the series of operations using the above pH 7.0 and pH 10.0 buffers.

Example 8 After flowing 50 g of 0.1M phosphate buffer at pH 7.0 into the column packed with immobilized actinase E used in Example 2, racemic indoline was prepared in the same manner as in Example 2.
2-carboxylic acid ethylene glycol ester (R,5)
Loading 5 g of -Ia, asymmetric ice-thawing reaction with a phosphate buffer at pH 7.0, elution of the generated carboxylic acid, and pE
[Alkaline ice thawing and carboxylic acid desorption and elution were performed using a 10.0 buffer solution. Furthermore, this series of reaction and elution operations was repeated 10 times in succession, and each time the pH 7.0 buffer fraction and the pH 10 buffer fraction were treated in the same manner as in Example 2. As a result, the specific optical rotation [α) D-29,15° to -80,
9° (C=1.0, dimethylformamide) (
Yield of R)-indoline-2-carboxylic acid (R) 71
~79%). In addition, the specific optical rotation [α] 0 + 80.8° ~ +82
．． (8)-Indoline-2-carboxylic acid (S)-11 with 2° (C=1.0, dimethylformamide) from 1.211 to 1.851 ((Ill.

8)-Ia was obtained in a yield range of 65 to 69%).

Examples 4 to 9 In Example 2, the same operations as in Example 2 were performed except that the ester of the substrate and the carrier for immobilizing the enzyme were changed, and the results shown in Table 1 were obtained. All substrate loadings are 6g.

(The following is a blank space) Examples 10 to 14 In Example 2, the same operations as in Example 2 were performed by changing the enzyme, and the results shown in Table 2 were obtained. However, Example 12.11
In , a pH 9.0 buffer was used for the hydrolysis of the ester. The enzymes in Examples 10 to 18 hydrolyzed (R)-form esters, and the enzymes in Example 14 hydrolyzed (8)-form esters.

(Left below) Table 2 *1 Origin: Bacillus subtilis, Nagashio Sangyo ■Umbrella 2
Origin: Aspergillus meleus, Ohno Pharmaceutical @ Bookbinding 8
Amano Pharmaceutical Co., Ltd. Bookbinding 4 Pig Pancreas, Wako Pure Chemical Co., Ltd. "5 Ki&s Aspergillus niger, manufactured by Ohno Pharmaceutical Co., Ltd. Example 15 A nutrient liquid medium with the following composition was prepared, and after dispensing 400 liters into a 21-saka Lof flask, , sterilized at 120°C for 115 minutes.

[Medium composition]

Glucose 4%, yeast extract 0.3%, meat extract 0
．． 8%, peptone 0.8%, ammonium phosphate 0.
2%, potassium phosphate 0.1% (pH 6,8) Separately, Pseudomonas acitoporans (Pseudomonas acitoporans) was precultured in a medium with the same composition.
vorans) IFO18582 seed solution lO
- was seeded in the flask and incubated at 80°C for 124 hours. A total of 5 cells were cultured, and a culture solution needle 21 was obtained. This culture solution was centrifuged and the bacterial cells were collected.

This wet bacterial cell 201 was dissolved in a 20mM phosphoric acid solution (l(7,0
) 40-, and the urethane prepolymer PU-
8 (Toyo Rubber Industries - [) 2 (l) was added, stirred quickly at 40°C, immediately cooled to 4°C, and left for 80 minutes. The immobilized microorganism thus obtained was cut into approximately 2ff square pieces. A column with an inner diameter of 2.2 cIt was packed to a height of 151 cm, kept at 38 °C, and 0.1 M l with a pE of 7 °C.
After flowing the phosphate buffer for 50 m, the substrate indoline-2-
Carboxylic acid glycerol ester (R,5)-Id 4
F was loaded. 0.1 M at pH 7.0! An asymmetric ice-melting reaction is carried out by flowing a phosphate buffer solution at a flow rate of 15 hours per hour.
The effluent from the column was collected in 20-unit fractions using a 7-lux collector and analyzed by liquid chromatography. This phosphate buffer fraction contained only hydrophilic indoline-2-carboxylic acid produced by asymmetric hydrolysis.

Ammonium sulfate was added to the 800m fraction of the phosphate buffer until saturation, and the pH was further adjusted to 5.0'v! 4 Adjustment,
The 8 homonuclear indoline-2-carboxylic acids were extracted with an equal volume of ethyl acetate. Separate the acetic acid layer, dehydrate it, and compress it under reduced pressure.
The dried product was reconsolidated with acetone-hexane (5 mg-1 m). After drying in vacuum, specific rotation [α], +28.7° (c
=1. 1.05f of (8)-indoline-2-carboxylic acid (8)-1 with (8)-dimethylformamide) was obtained.

At the point when the phosphate buffer solution was flowing for 840 minutes, 0.1 M K2HPO4NaOH at pH 9.5 was extracted from the phosphate buffer solution.
The unreacted indoline that had been adsorbed on the immobilized biological carrier in the column was replaced with a buffer solution and flowed at a flow rate of 40 mm per minute.
2-Carboxylic acid glycerol ester Id was hydrolyzed and desorbed as ■ and bathed out.

300 pieces of the alkaline buffer fraction containing 300 ml of aluminum were treated in the same manner as in Example 2, and the specific optical rotation [α)''-29.2° ('''
White powder (R)-indoline-2-carboxylic acid (R)-11 with 0
．． I got 941.

Examples 16 to 19 The same procedure as in Example 15 was carried out by changing the bacterial strain and culturing.
Immobilization, asymmetric hydrolysis and separation were performed, and the results shown in Table 8 were obtained. The substrate was loaded with 41 (R,8)-Ia.

The strains of Examples 16 and 17 stereospecifically hydrolyzed only the (R)-form ester, and the strains of Examples 18 and 19 stereospecifically hydrolyzed only the (S)-form ester.

Table 8 Patent applicant Kanekabuchi Chemical Co., Ltd. agent Patent attorney Makoto Asano - (C12P
17/10 C12R1:645)

Claims (9)

[Claims] (1) General formula I ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ I (In the formula, R is a carbon substituted singly or simultaneously with an alkyl group or alkenyl group having 2 to 10 carbon atoms, a hydroxyl group, and a halogen atom. Several 2 to 10 alkyl groups or alkenyl groups, unsubstituted or substituted alicyclic hydrocarbon groups,
Represents an unsubstituted or substituted phenyl group or benzyl group. )-(R,S)-indoline-2-carboxylic acid ester represented by (R,S)-indoline-2-carboxylic acid ester and an immobilized enzyme or immobilized microorganism having stereoselective esterase activity immobilized on a hydrophobic carrier are brought into contact and reacted, Structural formula II^* ▲There are mathematical formulas, chemical formulas, tables, etc.▼II^* Optically active indoline-2-carboxylic acid represented by general formula I ^* ▲There are mathematical formulas, chemical formulas, tables, etc.▼ I ^* (wherein R is the same as above) is asymmetrically hydrolyzed into an optically active indoline-2-carboxylic acid ester, and the hydrophilic indoline-2-carboxylic acid II^* is eluted with water or a buffer solution. After collection, the indoline-2-carboxylic acid ester I^* adsorbed and retained on the immobilization carrier is hydrolyzed with alkali to produce optically active indoline-2-2-, which has an optical rotation opposite to that of II^*. A method for producing optically active indoline-2-carboxylic acid, which comprises eluting and collecting carboxylic acid II^*. (2) A patent claim in which the carrier for immobilizing hydrophobic enzymes or microorganisms is a synthetic adsorbent, a hydrophobic chromatography resin, a hydrophobic photocrosslinkable resin, or a polymer substance into which hydrophobic groups are chemically bonded. The manufacturing method according to item 1. (3) The production method according to claim 1 or 2, in which the asymmetric hydrolysis is carried out using an immobilized enzyme or an immobilized microorganism packed in a column. (4) Claim 1 in which asymmetric hydrolysis is carried out in a batch manner
The manufacturing method according to item 1 or 2. (5) The microorganism or enzyme has (R)-selective esterase activity.
yces), Saccharomyces (Saccharomyces)
yces), Trichosporon (Trichosporon)
on) genus, Aeromonas genus, Arthrobacter genus, Acidiphilium genus, Brevibacterium genus,
Corynebacterium
) or a microorganism belonging to the genus Pseudomonas, or an enzyme derived from the microorganism. (6) The microorganism is Streptomyces griseus (S
treptomyces griseus), Saccharomyces cerevisiae (Saccharomyces
cerevisiae), Trichosporon cutaneum, Aeromonas h.
hydrophila), Arthrobacter paraffinus (Arthrobacter paraffinus)
eus), Arthrobacter nicotianae (Arthr
obacter nicotianae), Acidiphilium cryptum (Acidiphilium c
ryptum), Brevibacterium protophori
miae), Corynebacterium paulometaborum (
Corynebacterium paurometa
Corynebacterium acetoa
cidophilum), Pseudomonas oxalaticus
), and the enzyme is an enzyme derived from the microorganism, the manufacturing method according to any one of claims 1 to 5. (7) The production method according to any one of claims 1 to 4, wherein the enzyme is an enzyme derived from a mammalian organ. (8) The microorganism or enzyme has (S)-selective esterase activity in the genus Arthrinium, Aspergillus (Asp.
ergillus), Cephalosporium (Ceph)
allosporium), Echinopodospora (Ech
inopodospora), Emericelopsis (E.
genus mericellopsis, Hypoclea
ocrea) genus, Isaria genus, Lepista genus, Nectria
), Pestalotiopsis
is) genus, Phialophora
), Podospora genus, Botryoascus spp., Candida spp., Citeromyces spp.
myces), Debaryomyces
yces), Hormoascus
), Monilliella, Kluyveromyces, Nadsonia, Rho
dotorula), Schizosacromyces (Sch
izosaccharomyces, Torulopsis, Wic
kerhamia), Alcaligenes
genus), Arthrobacter (Arthroba
cter) genus, Brevibacterium (Brevibac
terium), Protaminobacter (Protam)
inobacter), Pseudomonas (Pseud)
5. The production method according to any one of claims 1 to 4, which is a microorganism belonging to the genus P. omonas or an enzyme derived from the microorganism. (9) The microorganism is Arthrinium phaeospermum.
, Aspergillus ficum
ficuum), Cephalosporium mycophilum
um), Echinopodospora jamaicensis (Ech
inopodospora jamaicensis)
, Emericellopsis glabra
psis glabra), Hypocrea lacta (Hy
pocrea lactea), Isaria atypicola, Lepista nuda, Nectria flammea, Pestalotiops
is distinctta), Phialophora fastigiata (Phialophora fastigata)
iata), Podospora carbonaria (Podosp
ora carbonaria), Botryoascus
Botryoascus syn
naedendrus), Candida diversa (
Candida diversa, Candida pseudotropicalis
ropicalis), Citeromyces matritensis
), Debaryomyces hansenii (Debaryomyces
yces hansenii), Hormoascus platypodis
is), Moniliella tomentosa (Moniliell)
a tomentosa), Kluyveromyces fragilis
s), Nadsonia elongata (Nadsonia e
longata), Rhodotorula glutinis (Rho
dotorula glutinis), Schizosaccharomyces pombe (Schizosaccharomyces
ces pombe), Torulopsis gropengiess
eri), Wickerhamia fluorescens (Wic)
kerhamia fluorescens), Alcaligenes faecalis (Alcaligenes f.
aecalis), Arthrobacter crystallopoietes
lopoietes), Brevibacterium flavum, Protaminobacter alboflavas (Protamin)
obacter alboflavus), Pseudomonas ac
idovorans), and the enzyme is an enzyme derived from the microorganism, or the manufacturing method according to claim 8.