JPH0648995B2 - Method for producing optically active indoline-2-carboxylic acid by immobilized enzyme or immobilized microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is an optically active indoline useful as a raw material for synthesizing a drug, which is obtained by optically resolving indoline-2-carboxylic acid ester using an immobilized enzyme or an immobilized microorganism. It relates to a method for producing a 2-carboxylic acid. More specifically, the general formula I (In the formula, R is an alkyl group or alkenyl group having 2 to 10 carbon atoms, an alkyl group or alkenyl group having 2 to 10 carbon atoms, which is independently or simultaneously substituted with a hydroxyl group and a halogen atom, an unsubstituted or substituted resin Cyclic hydrocarbon group,
It represents an unsubstituted or substituted phenyl group or a benzyl group. (R, S) -indoline-2-carboxylic acid ester I represented by the formula (I) and an enzyme or a microorganism having a stereoselective esterase activity immobilized on a hydrophobic carrier are brought into contact with each other to react with each other to give a compound of the formula (R ) − II And an optically active (R) -indoline-2-carboxylic acid represented by the formula (S) -I (In the formula, R is the same as above), an optically active indoline-2-carboxylic acid ester (S) -I is produced, and hydrophilicity is increased by utilizing the difference in affinity with the carrier used for immobilization. indoline-2-carboxylic acid (R) - II recovered with water or a buffer solution, after collection, then
Carrier adsorption, indoline-2-carboxylic acid ester, which is held (S) - I and alkaline hydrolysis, and the resulting ahead (R) - II and the optically active indoline-2-carboxylic acid having an opposite optical rotation (S) - II elution relates to a method for harvesting.

These optically active indoline-2-carboxylic acid compounds are important compounds that can be used as raw materials for various pharmaceuticals. For example, (S) -indoline-2-carboxylic acid is useful as an inhibitor of angiotensin I converting enzyme (S) -1-.
[(S) -3-Mercapto-2-oxopropyl] -indoline-2-carboxylic acid structural formula It can be used for [Journal of Medicinal
Chemistry (J. Med. Chem.), 26, 39
4 (1983)].

(Conventional technology and problems) In the conventional enzyme reaction, a free enzyme was added to the reactor and the reaction was performed by a batch method, and after the reaction was completed, the enzyme was used up, but the enzyme is generally expensive. Therefore, it is disadvantageous in terms of cost, and is unstable, so that the industrial use of the enzyme was limited. Furthermore, in the batch method, after the enzymatic reaction is completed, the reaction product is separated from the reaction solution by extraction and separation with an organic solvent, the reaction solution is once redissolved in an organic solvent, or the reaction solution is directly subjected to column chromatography treatment. The method of separating by, the method of separating the reaction solution by once being redissolved in an organic solvent, or by fractionating the reaction solution as it has been performed, but the operation is complicated and the yield is poor, There is a drawback that the cost is high because it takes time and a special device is required.
Therefore, in recent years, immobilization of enzymes and microorganisms has been studied, and it has become possible to repeatedly use the enzymes and microorganisms, and further to fill the column to carry out the reaction continuously. However, the reaction product is separated at the same time as the asymmetric hydrolysis from the racemate as the raw material using the immobilized enzyme, and then the unreacted ester adsorbed on the carrier is subjected to alkaline hydrolysis to elute and collect it. No success cases have been reported so far.

(Problem means and effect for solving) The present inventors have previously to act on indoline-2-carboxylic acid ester I, the optically active indoline-2-carboxylic acid (R) - II and the optically active An enzyme or a microorganism that stereoselectively resolves into indoline-2-carboxylic acid ester (S) -I has been found, and a method for producing indoline-2-carboxylic acid by optical resolution has been found and proposed (Japanese Patent Application Laid-Open Publication No. Sho. 6
1-92595, 61-192596, 61-227
796).

The present inventors have described the immobilization of these enzymes or microorganisms,
We have made diligent efforts to establish a simpler product separation technique. As a result, as a carrier, a carrier having a difference in affinity for the substrate and the product is selected, and the enzyme or microorganism is immobilized on the carrier, whereby the substrate indoline-
Indoline-2 produced by asymmetric hydrolysis of 2-carboxylic acid ester, separation of product indoline-2-carboxylic acid and unreacted ester, and further hydrolysis of acari while adsorbing unreacted ester on a carrier -The recovery of carboxylic acid was succeeded in succession, and the present invention was completed. The present invention will be described in detail below.

The general formula used as the substrate of the present invention The indoline-2-carboxylic acid ester represented by
Substituent R is an alkyl group or alkenyl group having 2 to 10 carbon atoms, an alkyl group or alkenyl group having 2 to 10 carbon atoms which is independently or simultaneously substituted with a hydroxyl group and a halogen atom, an unsubstituted or substituted alicyclic group Compounds of hydrocarbon group, unsubstituted or substituted phenyl group or benzyl group, preferably ethanol, butanol, amyl alcohol, hexanol, glycerol, ethylene glycol, glycerol-α-monochlorohydrin, 2,3-dichloro-1- It is an ester with indoline-2-carboxylic acid such as propanol, 1,3,5-pentanetriol, cyclohexanol and benzyl alcohol.

Indoline-2-carboxylic acid ester I is obtained as follows. That is, (R, S) -indoline-2-carboxylic acid is added with an alcohol which also serves as a solvent and a reaction reagent,
Indoline-2-carboxylic acid concentration 5-20% (W /
V) under strong acidity in the range of 50 ° C. to reflux temperature of 1 to
The condensation reaction is performed 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, S). −
Indoline-2-carboxylic acid ester I is obtained.

Racemic I asymmetrically hydrolyzing (R) - The microorganisms having stereoselective esterase activity to generate II, belonging for example Arthrobacter (Arthrobacter) genus Brevibacterium (Brevibacterium) genus There are microorganisms, more specifically, Arthrobacter paraffineus ATCC 21317,
Arthrobacter nicotian
ae) IFO 14234, Brevibacterium protophormiae IFO 12
There are 128 etc. Also, the nutritional sources of these microorganisms are usually
An organic and inorganic carbon source, nitrogen source, vitamins and minerals that can be assimilated are appropriately mixed, and the culture temperature is 20.
The range of -40 ° C and pH 2-8 is used. It is also possible to promote the growth of microorganisms by aeration and stirring.

The cells obtained by culturing in this way can be used by directly immobilizing them with a hydrophobic resin, but after crushing the microbial cells, the crude enzyme obtained by ammonium sulfate fractionation or acetone treatment or purification It can be used after it is fixed.

As the carrier for immobilizing the enzyme in the present invention, various carriers having hydrophobicity are used. The carrier having a hydrophobic to be used in the present invention, water or a hydrophilic compound produced by the asymmetric solution reaction in buffer (R) - II and does not adsorb, ester compounds of unreacted (S) - I is It is a carrier that has the property of being adsorbed by hydrophobic interaction and has a pH of 8-10.
It is desirable that the carrier be stable even in the alkaline region. More specific carriers include, for example, hydrophobic synthetic adsorbents, hydrophobic chromatography resins, hydrophobic photocrosslinkable resins, hydrophobic urethane prepolymer resins,
Examples thereof include a polymer substance introduced by chemically bonding a hydrophobic group.

Immobilization of the enzyme on such a carrier can be carried out by various known methods. For example, a physical adsorption method, a covalent bond method, an ionic bond method, a crosslinking method, an encapsulation method and the like can be mentioned. In the case of microorganisms, the comprehensive method etc. can be mentioned [Fukui / Chihata / Suzuki, Enzyme Engineering, 157-243, Kodansha (198).
1); edited by Ichiro Chibata, immobilized enzyme, Kodansha) 197
5)].

Among these methods for preparing immobilized enzymes or immobilized microorganisms, in the case of an enzyme, the enzyme is physically adsorbed on a synthetic adsorbent having hydrophobicity due to the simplicity of the method, the physical strength of the carrier, and the low cost. In the case of microorganisms, a method of encapsulating microbial cells in a photo-crosslinking resin having hydrophobicity or a hydrophobic urethane prepolymer resin is industrially desirable.

The amount of the enzyme or microorganism to be loaded on the carrier is not necessarily unique because it depends on the loading capacity of the carrier, but for enzymes, about 0.1 mg to about 100 mg per 1 g wet weight of the carrier,
Usually, the amount may be about 1 mg to about 20 mg, and the microbial cells may be about 0.1 g to 1 g, usually about 0.15 g to about 0.5 g, per 1 g wet weight of the carrier.

The amount of substrate that can be loaded on the column packed with the immobilized enzyme or immobilized microorganism varies depending on the type of the immobilized carrier and the substrate ester, but it is not determined during the loading of the substrate or when the buffer solution starts to flow. If the reaction substrate is not discharged from the column, it can be discharged up to the limit amount. For example, when an immobilized enzyme having the synthetic adsorbent Amberlite XAD-7 as a carrier is packed and an ester of ethylene glycol is used as a substrate, it is possible to load the substrate up to 1/5 of the column volume. As for the method of charging the substrate, in the case of a column type, the substrate may simply be charged at the top of the column and the buffer solution may be allowed to flow. In the case of the batch method, the substrate may be mixed with the immobilized enzyme or the immobilized microorganism.

The asymmetric hydrolytic reaction in the present invention can be carried out usually in the range of 10 ° C to 60 ° C, but is preferably carried out at 20 ° C to 40 ° C. This asymmetric hydrolysis reaction can be carried out in the pH range of 4.5 to 10, but is preferably carried out in the pH range of 6 to 7.5, which has a high reaction rate. In addition, in this reaction, as the asymmetric hydrolysis progresses, indoline-2-carboxylic acid is produced and the pH is lowered.
Therefore, when the loading amount of the substrate compound is large, it is desirable to control the pH within a certain range by using a buffer solution or the like. Suitable buffers for this purpose include inorganic acid salts,
Any organic acid salt buffer can be used.

Further, the alkaline hydrolysis of the unreacted ester adsorbed on the carrier of the immobilized enzyme or the immobilized microorganism is such that the immobilized enzyme or microorganism is not inactivated, and the pH is such that the ester is hydrolyzed. It is sufficient that the pH is usually 8 to 10, though it depends on the enzyme or the microorganism.

From the viewpoint of keeping the pH constant when the reaction is carried out using a column, it is preferable to use a buffer solution, after packing the immobilized enzyme or the immobilized microorganism in the column, then flowing a buffer solution of pH 7.0, and then Substrate ester compound (R, S)-
Loaded with I, tighten to perform the the asymmetric solution reaction in a column by flowing again buffer When finished loading, and resulting hydrophilic compounds (R) - II is discharging from the column was dissolved in buffer. This buffer fraction was subjected to high performance liquid chromatography (Finepak SIL C ₁₈ , developing solution; acetonitrile-water = 1.5: 1 (v / v), detection; UV21.
Were analyzed by 5 nm), the compound in fractions (R) - II is flushed by changing the buffer to buffer pH8~10 when almost ceased, adsorbed to the immobilized enzyme or immobilized microorganisms in a column compounds of to have unreacted (S) - and I is hydrolyzed, it has the optical rotation of the previous opposite (S) - desorption as II, eluted. This buffer fraction was analyzed by liquid chromatography (conditions, the same as above), and the compound (S) -I
When I is almost not observed, the buffer solution having a pH of 8 to 10 is changed to a buffer solution having a pH of 7.0, and the mixture is allowed to flow, and the substrate ester compound (R, S) -I is loaded on the column again. By repeating these series of operations, the compound (R,
It is possible to continuously carry out the asymmetric hydrolyzation of S) -I , the separation of reaction products, and the alkaline hydrolysis in a pulsed manner.

When performing the asymmetric solution of the immobilized enzyme or immobilized microbial racemic compound in a batch method using I is produced optically active hydrophilic compound (R) - optical water layer and unreacted containing II The active enzyme adsorbing the hydrophobic compound (S) -I is separated from the immobilized enzyme or the immobilized microorganism by filtration or gentle centrifugation, and further, the pH is adjusted to a level at which the immobilized enzyme or the immobilized microorganism is not inactivated. by desorbed while adjusting the alkali solution, or a buffer solution in an immobilized enzyme or an ester adsorbed on the immobilized microorganisms pH8~10 hydrolyzed to 10, the previous (R) - II opposite optically active compound with a optical rotation of (S) - II can be obtained, immobilized enzymes or immobilized microorganisms can be used to re-reaction.

The fraction containing the indoline-2-carboxylic acid obtained by this method is adjusted to a pH of around 5.0 and precipitated to precipitate and precipitate by concentration, and then collected by filtration or saturated with ammonium sulfate and then adjusted to a pH of 5.0.
It can be obtained by adjusting to the vicinity, extracting with an organic solvent such as ethyl acetate and methylene chloride, and concentrating. If necessary, crystallization may be performed in an organic solvent such as acetone.

(Examples) Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1 Synthesis of substrate Synthesis of (R, S) -indoline-2-carboxylic acid ethylene glycol ester Ia 50 g of (R, S) -indoline-2-carboxylic acid II and 250 g of ethylene glycol were mixed, and 20 ml of concentrated sulfuric acid was added. In addition, the condensation reaction was performed in the range of 95 to 100 ° C. for 3 hours. After the reaction, the reaction mixture was once cooled, and sodium bicarbonate and saturated sodium bicarbonate water were added to adjust the pH to 7.0. Next, extraction with methylene chloride (500 ml) three times (total 1.5) was performed, the methylene chloride layer was washed with 100 ml of water, dehydrated with anhydrous sodium sulfate, and further concentrated under reduced pressure (R, S) -Ia.
Was obtained in a yield of 50.2 g and 79%.

In the same manner, by adding 5 times the amount of alcohol to 50 g of (R, S) -indoline-2-carboxylic acid, the following substrate (R, S) -indoline-2-carboxylic acid glycerol α-monochrome Luhydrin ester ( Ib ) 45.4 g, yield
58.0% (R, S) -Indoline-2-carboxylic acid butyl ester ( Ic ) 53.8 g, yield 80% (R, S) -Indoline-2-carboxylic acid glycerol ester ( Id ) 57.7 g, yield 79% (R, S) -Indoline-2-carboxylic acid cyclohexanol ester ( Ie ) 52.3 g, yield 69% (R, S) -Indoline-2-carboxylic acid benzyl alcohol ester ( If ) 49.7 g, yield 64.0% 59.5 g of (R, S) -indoline-2-carboxylic acid 1,3,5-pentatriol ester ( Ig ) was obtained at a yield of 73.3%. As the extraction solvent, instead of methylene chloride, (R, S) -indoline-2-carboxylic acid glycerol ester ( Id ) was ethyl acetate, and (R, S) -indoline-2-carboxylic acid cyclohexanol ester ( Ie). ) Used hexane.

Reference Example 1 3 g of actinase E (derived from Streptomyces griseus) (manufactured by Kaken Pharmaceutical Co., Ltd.) was added to 60 ml of 0.1 M phosphate buffer of pH 7.0 and mixed, and the insoluble matter was removed by filtration. The filtrate was washed with a methacrylate-based porous adsorbent, Amberlite XAD-7, manufactured by Rohm and Haas Co., with methanol and water, then 60 g wet weight (water content 71%) was added, and the mixture was slowly stirred overnight in a low temperature room (4 ° C). Then, the enzyme was adsorbed and immobilized. The immobilized enzyme suspension was suction filtered using a glass filter, and 0.1 M phosphate buffer solution of pH 7.0 1
After washing with 00 ml three times (300 ml in total), suction filtration was performed to obtain a wet immobilized enzyme. The immobilized lipase was packed to a height 15cm in a column having an inner diameter of 2.2 cm, and kept at 33 ° C. indoline-2-carboxylic acid ethylene glycol esters of racemic (R, S) - and Ia 5 g loaded, pH 7. 0 of 0.1M
The phosphate buffer was reacted at a flow rate of 20 ml per hour.
20 ml of the discharged liquid from the column was collected by a fraction collector and analyzed by liquid chromatography. This phosphate buffer fraction contained only the hydrophilic indoline-2-carboxylic acid produced by asymmetric hydrolysis. Ammonium sulfate was added to 360 ml of the phosphate buffer fraction until saturation, and the pH was adjusted to 5.0. Next, an equal amount of ethyl acetate was added, and the indoline-2-carboxylic acid was extracted three times. After dehydration, the mixture was concentrated under reduced pressure, and the dried solid was reconstituted with acetone-hexane (8 ml-2 ml). After drying in vacuum, the specific rotation ▲ [α] ²⁵ _D ▼ -30.5 ° (c = 1.0, dimethylformamide) [reference value, Journal of Medicinal Chemistry, 26 , 394 (1983),
[Α] _{^{25 D + 34.5 ° (c =}} 0.91, dimethylformamide) white powder having] (R) - indoline-2-carboxylic acid (R) - II is 1.51g ((R, S) - than Ia Of 77%) was obtained. When 400 ml of phosphate buffer was flowed, the pH of the phosphate buffer was changed to 0.1 MK ₂ H of pH 10.0.
The PO ₄ -NaOH solution was flowed at a flow rate of 40 ml per hour, and the unreacted indoline-2-carboxylic acid ethylene glycol ester Ia adsorbed on the carrier of the immobilized enzyme in the column.
Was hydrolyzed and desorbed and eluted as II . 300 ml of the alkaline buffer fraction containing II was adjusted to pH 5.0 with 2N hydrochloric acid, extracted 3 times with an equal amount of ethyl acetate, dehydrated, and concentrated under reduced pressure. When the dried solid is reconstituted with acetone-hexane (6 ml-1.5 ml), the specific light intensity is ▲ [α] ²⁵ _D ▼ + 32.7 ° (c = 1.0,
White powder with dimethylformamide) (S) - indoline-2-carboxylic acid (S) - II is 1.34 g ((R,
S) -Ia yield 68%) was obtained.

Desorption of the enzyme was not observed in a series of operations with the above pH 7.0 and pH 10.0 buffer solutions.

Reference Example 2 In the same manner as in Reference Example 1, 50 ml of 0.1 M phosphate buffer of pH 7.0 was passed through the immobilized actinase E packed column used in Reference Example 1, and then racemic indoline-2-carboxylic acid ethylene glycol ester was prepared. (R, S) -Ia 5
g was added to carry out asymmetric hydrolytic reaction with a pH 7.0 phosphate buffer solution and elution of the carboxylic acid produced, and alcal hydrolysis with a pH 10.0 buffer solution and desorption and elution of the carboxylic acid.
Further, this series of reaction and elution operations were repeated 10 times continuously, and the buffer solution fraction of pH 7.0 and the buffer solution fraction of pH 10 were treated in the same manner as in Reference Example 1 each time. as a result,
Specific rotation ▲ [α] ²⁵ _D
▼ -29.5 ° ～30.9 DEG (c = 1.0, dimethylformamide) with a (R) - indoline-2-carboxylic acid (R) - II and 1.40g~1.56g ((R, S) - yield from Ia 71 to 79%). In addition, pH1 of each time
Specific rotation from buffer solution fraction of 0.0 ▲ [α] ²⁵ _D ▼ + 30.3 ° ~
Tasu32.2 DEG (c = 1.0, dimethylformamide) with a (S) - indoline-2-carboxylic acid (S) - II 1.28
g~1.35g ((R, S) - yield than Ia 65 to 69
%).

Reference Examples 3 to 8 In Reference Example 1, the same procedure as in Reference Example 1 was performed except that the substrate ester and the carrier for immobilizing the enzyme were changed, and the results in Table 1 were obtained. The substrate loadings are all 5 g.

Reference Examples 9 to 10 In Reference Example 1, the same operation as in Reference Example 1 was performed by changing the enzyme, and the results in Table 2 were obtained. However, in Examples 9 and 10, a buffer solution having a pH of 9.0 was used for hydrolysis of the ester.

Comparative Example 1 A nutrient liquid medium having the following composition was prepared, 400 ml each was dispensed into a 2 Sakaguchi flask, and sterilized at 120 ° C. for 15 minutes.

[Media composition]

Glucose 4%, yeast extract 0.3%, meat extract 0.3
%, Peptone 0.3%, diammonium phosphate 0.2%, monopotassium phosphate 0.1% (pH 6.8) Pseudomonas acidovorans I precultured separately in a medium of the same composition
10 ml of the inoculum solution of FO 13582 was inoculated into the flask and shaken at 30 ° C. for 24 hours. A total of 5 cultures were performed to obtain a total culture solution 2. The culture was centrifuged to collect the bacterial cells. 20 g of this wet microbial cell was added to a 20 mM phosphate buffer solution (p
H7.0) suspended in 40 ml, and urethane prepolymer PU-3 (manufactured by Toyo Tire & Rubber Co., Ltd.) 20 g was added thereto to obtain 40
After stirring rapidly at 0 ° C, the mixture was immediately cooled to 4 ° C and left for 30 minutes. The thus-obtained immobilized microorganism was cut into pieces of about 2 mm square, packed in a column having an inner diameter of 2.2 cm to a height of 15 cm, kept at 33 ° C., and 50 ml of 0.1 M phosphate buffer of pH 7.0 was flowed. The substrate indoline-2-carboxylic acid glycerol ester (R, S) -Id 4 g was loaded from
An asymmetric hydrolytic reaction was carried out by flowing 0.1 M phosphate buffer of pH 7.0 at a flow rate of 15 ml per hour, and 20 ml of the discharged liquid from the column was fractionated by a fraction collector and analyzed by liquid chromatography. This phosphate buffer fraction contained only the hydrophilic indoline-2-carboxylic acid produced by asymmetric hydrolysis. Ammonium sulfate was added to 300 ml of the phosphate buffer solution until it was saturated, and the pH was adjusted to 5.0.
And indolin-2-fold three times with an equal volume of ethyl acetate.
The carboxylic acid was extracted. The ethyl acetate layer was separated, dehydrated, and then concentrated under reduced pressure, and the dried solid was acetone-hexane (5 ml-1 ml).
Reunited with. After drying in vacuum, specific rotation ▲ [α] ²⁵ _D ▼ +2
3.7 ° (c = 1.0, dimethylformamide) with a (S) - indoline-2-carboxylic acid (S) - II 1.05
g was obtained.

When 340 ml of phosphate buffer was flowed, the phosphate buffer was changed to 0.1 MK ₂ HPO ₄ -NaOH buffer of pH 9.5 and flowed at a flow rate of 40 ml per minute to be used as a carrier for the immobilized microorganisms in the column. The unreacted indoline-2-carboxylic acid glycerol ester Id that had been adsorbed was hydrolyzed to II , desorbed and eluted. 300 ml of the alkaline buffer fraction containing II
Was processed in the same manner as in Reference Example 1, and the specific rotation ▲ [α] ²⁵ _D ▼ -2
9.2 DEG (c = 1.0, dimethylformamide) white powder with a (R) - indoline-2-carboxylic acid (R) - II
0.94 g was obtained.

Examples 2 to 3 Cultures, immobilization, asymmetric hydrolysis and separation were performed by the same operations as in Comparative Example 1 while changing the strains, and the results shown in Table 3 were obtained. The substrate was loaded with 4 g of (R, S) -Ia .

Claims (5)

[Claims] 1. A general formula I (In the formula, R is an alkyl group or alkenyl group having 2 to 10 carbon atoms, an alkyl group or alkenyl group having 2 to 10 carbon atoms, which is independently or simultaneously substituted with a hydroxyl group and a halogen atom, an unsubstituted or substituted lipid group. Cyclic hydrocarbon group,
It represents an unsubstituted or substituted phenyl group or a benzyl group. (R, S) -indoline-2-carboxylic acid ester represented by the formula (I) and an (R) -stereoselective esterase activity-immobilized Arthrobacter (Arthrobacter) immobilized on a hydrophobic carrier.
Obacter) genus Brevibacterium (Brevibacterium) belong to contacting the immobilized microorganism or immobilized enzyme using an enzyme derived from a microorganism belonging, reacted with structural formula (R) - II And an optically active (R) -indoline-2-carboxylic acid represented by the general formula (S) -I (In the formula, R is the same as above) and is hydrolyzed to an optically active (S) -indoline-2-carboxylic acid ester, which is a hydrophilic indoline-
2-Carboxylic acid (R) -II is eluted with water or a buffer solution, collected, and then adsorbed on and immobilized on a carrier for immobilization.
2-carboxylic acid ester (S) - I and alkaline hydrolysis, and the resulting ahead (R) - II and the optically active indoline-2-carboxylic acid with opposite optical rotation (S) - II the elution is collected A method for producing an optically active indoline-2-carboxylic acid, which comprises: 2. The enzyme is Arthrobacter paraffineus, Arthrobacter nicotianae, Brevibacterium protophormiae.
The method according to claim 1, wherein the enzyme is derived from a microorganism. 3. A carrier for immobilizing a hydrophobic enzyme or microorganism is a synthetic adsorbent, a resin for hydrophobic chromatography,
The production method according to claim 1, which is a hydrophobic photocrosslinkable resin or a polymer substance introduced by chemically bonding a hydrophobic group. 4. The production method according to claim 1 or 2, wherein the asymmetric hydrolysis is carried out with an immobilized enzyme or immobilized microorganism packed in a column. 5. The production method according to claim 1 or 2, wherein the asymmetric hydrolysis is carried out batchwise.