JP3105653B2 - Multifunctional immobilized lipase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lipase immobilized on a water-absorbent resin, a method for producing the same, and a method for optically resolving esters and alcohols using the same.

[0002]

2. Description of the Related Art The use of enzymes as reaction catalysts has been conventionally performed.

[0003] Among them, the reaction using lipase has regioselectivity and stereoselectivity, and is attracting attention as a very effective biocatalyst. Lipases are also relatively stable in organic solvents and are therefore suitable for organic synthesis.

[0004] Lipase is originally a hydrolase,
It is known that the hydrolysis reaction proceeds in an aqueous solution, but the esterification reaction proceeds in an organic solvent system in the presence of a very small amount of water.

[0005] On the other hand, enzymes such as lipase are generally expensive, so that it is required to separate and recover them and reuse them from the viewpoint of economy.

However, in the hydrolysis reaction, since the enzyme is soluble in an aqueous solution, it is difficult to separate and recover only the enzyme after the reaction and reuse the enzyme. Further, in the esterification reaction, a powdery enzyme was used in an organic solvent. However, similarly, after the reaction was completed, it was difficult to separate only the enzyme from the reaction system, and the operability was poor.

[0007] Therefore, when an enzyme is used industrially, it is necessary to immobilize the enzyme in order to facilitate reuse from an economic viewpoint.

[0008] There are various known methods for immobilizing enzymes, such as an entrapment method, a crosslinking method, and a carrier binding method. For example, 1) RBLieberman et al.
Biotechnology and bioengineering
(Biotechnol. Bioeng.), 17 , 1401 (1975), a method in which an enzyme is wrapped in a polyacrylamide gel lattice and immobilized in a state where it cannot be detached, 2) Yuji Inada, "Protein Hybrid" Publishing,
(1987) A method of solubilizing in an organic solvent by covalently bonding an amphiphilic polymer such as polyethylene glycol (PEG) to the surface of an enzyme, and 3) D. Bianchi et al., Journal of Organic. Chemistry (J.Org.Chem.), 53 ,
5531 (1988), for example, a method of adsorbing an enzyme on celite.

However, in these immobilization methods, the immobilization operation is generally complicated and difficult, and it is difficult to use for both the hydrolysis reaction performed in an aqueous solvent and the esterification and transesterification reaction performed in an organic solvent system. there were.
Furthermore, it was impossible to carry out the hydrolysis reaction in an organic solvent.

Therefore, the present invention provides an optical resolution of an alcohol in an organic solvent, an optical resolution of an ester in an aqueous solution, and a progress of optical resolution of an ester in an organic solvent using water retained in a water-absorbing resin. An object of the present invention is to provide a multifunctional immobilized lipase that can be used in a cycle.

[0011]

SUMMARY OF THE INVENTION The present invention relates to a multifunctional immobilized lipase PS and lipase AK and its those of formula, characterized in that it is immobilized on the PQ polymer. Further, the present invention is characterized in that the lipase PS immobilized on the PQ polymer is used as a catalyst, and the optical resolution method of alcohol is used, and the lipase AK immobilized on the PQ polymer is used as a catalyst. The present invention relates to an optical resolution method for esters.

[0012]

Examples The water-absorbing resin used for immobilization according to the present invention may be any water-absorbing resin as long as it has water-retaining property and retains the activity of the enzyme. Is raised.

Examples of the anionic water-absorbing resin include acrylic acid polymer, methacrylic acid polymer, polysaccharide-acrylic acid or methacrylic acid graft copolymer, acrylic acid or methacryl-acrylamide-sulfonated acrylamide terpolymer. Coalescing or alkali metal or alkaline earth metal salts thereof, such as acrylic acid or acrylate polymers, acrylic acid or acrylate-
Methacrylic acid or methacrylate copolymer, starch-acrylic acid or acrylate graft copolymer, polysaccharide-acrylic acid or alkyl methacrylate graft copolymer saponified product, polysaccharide-acrylonitrile graft copolymer Saponified polysaccharide-acrylamide copolymer, alkyl acrylate or alkyl methacrylate-vinyl acetate copolymer, starch-acrylonitrile-acrylamide-2-methylpropanesulfonic acid graft copolymer Unified saponified products, saponified starch-acrylonitrile-vinylsulfonic acid graft copolymer, sodium carboxymethylcellulose, etc., such as acrylic acid, methacrylic acid, and alkali metal salts or alkaline earths thereof. Shokushio or formula instead of these esters, (I):

[0014]

Embedded image

(Wherein the substitution position of the —SO ₃ R ³ group is 2
-, 3-or 4-position, R ³ represents straight-chain or branched alkyl group having 1 to 18 carbon atoms, a hydrogen atom, or an alkali metal or alkaline earth metal, a is 0
Or 1)) or a derivative thereof. These may be used alone or in combination of two or more, and may be used as a crosslinked product.

The cationic water-absorbing resin is represented by the following general formula (II):

[0017]

Embedded image

(Wherein R ⁴ is hydrogen or methyl, R ⁵
Represents a linear or branched alkyl group having 1 to 18 carbon atoms or a hydrogen atom, and J represents 1, 2, 3, 4 or 5
Wherein Y represents an oxygen atom or a nitrogen atom, and Z represents a sulfonate ion or a halogen ion), or a copolymer with N-alkyl-acrylamide or N-alkyl-methacrylamide. And so on.

The shape of such a water-absorbent resin includes powder,
There are various things such as sphere, bead, block, etc.
One that maintains a shape such as a sphere independently without gelling even after water absorption, especially when the enzyme disperses in water-absorbent resin, is easy to handle, and reacts with the substrate when the reaction using the enzyme is performed. It is preferable because reactivity becomes high.

The particle size of these particles is 5 μm to 1 cm,
Preferably, those having a thickness of 10 μm to 2 mm are used.

Commercial products of such water-absorbing resins include:
Aqualic (trade name, manufactured by Nippon Shokubai Co., Ltd.), Poise and Wonder Gel (trade name, manufactured by Kao Corporation), Aqua Keep (trade name, manufactured by Sumitomo Seika Co., Ltd.), Arasorb (Arakawa Chemical Co., Ltd.) ), Diamond Wet (Mitsubishi Yuka Co., Ltd., trade name), Sumikagel (Sumitomo Chemical Co., Ltd.)
(Product name), Sunwet (product name, manufactured by Sanyo Kasei Co., Ltd.), PQ polymer (product name, manufactured by Osaka Organic Chemical Industry Co., Ltd.), Xslana (product name, manufactured by Nippon Xlan Industrial Co., Ltd.), Evergreen (Kuraray Co., Ltd., trade name), Aqua Reserve (Nihon Gosei Co., Ltd., trade name), KI gel (Kuraray Isoprene Co., Ltd., trade name), etc.
PQ polymers (grade: BL-
100) (manufactured by Osaka Organic Chemical Industry Co., Ltd.)
56512).

The lipase which can be used in the present invention includes genus Candida, genus Pseudomonas, and Penicillium.
Genus, Aspergillus, Chromobacterium, Mucor, Rhizopus, Bacillus, Alcaligenes, Micrococcu
s) genus, Geotrichum, Humicola
genus, genus Enterobacter, genus Arthrobactor, Brevibacterium (Bre
vibacterium), Microbacterium
Genus, Corynebacterium, Lactobacillus, Trichoderma
ma), Saccharomyces, Rhodotorula, Cryptococcus
Genus, Tolulopusis, Aureobasidium, Actinomuc
or), Nocardia, Streptomyces
A lipase produced by a microorganism belonging to the genus (Streptomyces), Hansenura, or Achromobactor, or a lipase produced in the pancreas of an animal. Any organic solvent can be used as long as it is capable of stereoselectively esterifying an alcohol.A preferred example of a lipase having excellent stereoselectivity is Pseudomonas sp. Pseudomonas sp.), Candida rugosa, Candida cylindracea (C
andida cylindracea), Aspergillus niger
gillus niger), Chromobacterium biscosum (Chr
omobacterium viscosum), Rhizops sp.
pus sp.). Commercially available products of these enzymes include, for example, Pseudomonas sp. Lipase “Amano” PS (manufactured by Amano Pharmaceutical Co., Ltd.
Brand name), Pseudomonas lipase "Amano"
AK (manufactured by Amano Pharmaceutical Co., Ltd.), lipase type VII derived from Candida cylindracea (trade name, manufactured by Sigma), lipase “Amano” A derived from Aspergillus niger A (manufactured by Amano Pharmaceutical Co., Ltd., trade name) ), Lipase derived from Chromobacterium biscosum (trade name, manufactured by Toyo Brewing Co., Ltd.), lipase "Amano" D derived from Rhizos sp. (Trade name, manufactured by Amano Pharmaceutical Co., Ltd.), lipase derived from Pseudomonas sp. "Amano" CES
(Trade name, manufactured by Amano Pharmaceutical Co., Ltd.), and the like,
Above all, "Amano" AK, "Amano" PS, "Amano"
CES and the like are preferred.

These enzymes can be used alone or in combination as needed. These enzymes are obtained by culturing the microorganisms that produce them.

The multifunctional immobilized lipase of the present invention can be produced by dissolving or suspending lipase in water or a buffer solution and allowing the lipase to penetrate into a water-absorbing resin. Specifically, for example, a method in which an enzyme is well suspended in distilled water using a mechanical stirrer or a magnetic stirrer, and then a water-absorbent resin is charged and stirred, and the water-absorbent resin and the enzyme are mechanically or magnetically stirred. It can be prepared by a very simple method such as a method of adding distilled water while stirring well using a stirrer or the like and stirring.

The preparation of such an immobilized enzyme is carried out at 0 ° to 50 °.
The reaction can be carried out under stirring at a temperature of 20 DEG C., preferably 20 DEG to 40 DEG C., for 0.1 to 5 hours, preferably 0.5 to 1 hour.

In this case, the amount of the enzyme, the amount of the water-absorbing resin and the amount of water are not particularly limited, but the amount of the enzyme is 1 to 200% by weight, preferably 10 to 50% by weight based on the water-absorbing resin. If the amount of the enzyme is larger than this range, the enzyme will not elute or a granular immobilized enzyme will not be obtained. If the amount is smaller than this range, the reaction time with the substrate will be longer if the reaction is carried out using the enzyme.

The amount of water is from 1 to 200% by weight, preferably from 10 to 30% by weight, based on the water-absorbing resin. If the amount of water exceeds this range, the reactivity becomes poor, and if it is less than this range, no immobilized enzyme is formed.

The type of water is not particularly limited as long as it does not hinder the reaction, but purified water and distilled water are preferably used.

The immobilized enzyme thus obtained is a spherical one in which the enzyme is incorporated into a water-absorbent resin.

The immobilized enzyme may be used as it is,
20 ~ 100 ℃ (0.5mmHg ~ 760mmH if necessary)
g) at 20 ° C to 100 ° C using an appropriate organic solvent such as normal hexane or cyclohexane.
C. (0.5 mmHg to 760 mmHg) may be used after azeotropic dehydration to remove water.

For example, when the reaction is not completed in the presence of a large amount of water as in the case of the esterification reaction, water is distilled off to the extent that the esterification reaction of each substrate is completed. When water is required for the reaction, it may be used as it is.

The immobilized enzyme of the present invention is, for example, an enantiomer mixture of a secondary or tertiary alcohol such as ethyl 3-hydroxybutyrate, 2-octanol, 1-indanol, 1-phenyl-1-propanol, α-hydroxyethylbenzene and the like. And an optical resolution of alcohol by using an acylating agent such as vinyl acetate, acetic anhydride and acetic acid.

The optical resolution of alcohol using the immobilized enzyme of the present invention can be carried out by stirring a secondary or tertiary alcohol compound as a substrate and an acylating agent together with the immobilized enzyme in an organic solvent. it can.

The organic solvent that can be used in the optical resolution of the present alcohol is not particularly limited as long as esterification proceeds with a secondary or tertiary alcohol and an acylating agent. Examples thereof include normal hexane, cyclohexane, and toluene. Benzene, xylene, octane, heptane, petroleum ether, methyl isobutyl ketone and the like.

The reaction temperature is usually from 0 to 50 ° C., and the reaction time is generally from 1 to 48 hours, but is not limited thereto.

Although this reaction is carried out in an organic solvent system, it is necessary that a very small amount of water is present.
Depending on the substrate and the enzyme, water of several ppm to several tens% can be retained in the immobilized enzyme by adjusting the amount of dehydration of the hydrous immobilized enzyme.

After completion of the reaction, the immobilized enzyme is separated by filtration or the like, and the solution is removed from the filtrate by distillation under reduced pressure to obtain a mixture of an optically active alcohol compound and an ester. These can be isolated and purified by a usual method.

The optically active esters obtained by this reaction are ethyl (R) -3-acetoxybutyrate, (S) -2
-Acetoxyoctane, (R) -1-acetoxyindane, (R) -1-acetoxy-1-phenylpropane,
(R) -α-acetoxyethylbenzene and the like.

On the other hand, the immobilized enzymes of the present invention include, for example, butyl 2,3-epoxybutyrate, butyl glycidate,
It can be used for optical resolution of enantiomeric mixtures of esters such as methyl chloropropionate and ethyl 2-chloropropionate.

In the asymmetric hydrolysis reaction carried out using the immobilized enzyme of the present invention, an enantiomer mixture of an ester serving as a substrate and the immobilized enzyme are mixed with water or a buffer or one of them and an organic solvent. The reaction is carried out by stirring in a mixed solution of the above, but the use of a buffer allows the reaction to proceed while maintaining the enzyme activity within the optimum pH of the enzyme, so that the amount of the enzyme used can be reduced. . Examples of the buffer used include a commonly used buffer of an inorganic acid salt such as sodium phosphate and potassium phosphate, a buffer of an organic acid salt such as sodium acetate and sodium citrate, and the like. PH of liquid
Is adjusted to the optimum pH of the lipase, that is, pH 2 to 12, preferably pH 4 to 11. On the other hand, by using an anion exchange resin and an organic amine in combination, the reaction can proceed without using a buffer solution.
Even when an aqueous solution of sodium hydroxide, potassium hydroxide or the like is used and the pH of the reaction solution is controlled using a pH stat, the same effect as when a buffer solution is used can be obtained. The organic solvent used is not particularly limited as long as it satisfies the requirement of dissolving the substrate and not inhibiting the enzyme activity. Specific examples of such an organic solvent include acetonitrile, 1,4-dioxane, tetrahydrofuran and the like.

The characteristic feature is that the optical resolution of the ester using the immobilized enzyme can be carried out in an organic solvent.

In this case, the reaction can be carried out in an organic solvent using water retained in the immobilized enzyme as water required for the hydrolysis reaction. In this case, the amount of water is not less than the amount required for hydrolysis and may be any amount that does not elute the retained water into the organic solvent.

In this reaction, the above-mentioned immobilized enzyme may be used as it is in an organic solvent, but the generated carboxylic acid may be trapped to promote the reaction. Examples of the trapping agent include, but are not limited to, anion exchange resins (Amberlite (trade name, manufactured by Rhom & Hass Co.), Dowex (Dow Chemical C.)).
o.), product name), Diaion (Mitsubishi Chemical Co., Ltd.)
Examples thereof include amines such as triethylamine, and pyridine derivatives such as pyridine. Preferred examples include Amberlite IR-45 and triethylamine.

The reaction solvent is not particularly limited as long as it dissolves the substrate and does not inhibit the enzyme activity. The reaction solvent is not particularly limited as long as the reaction proceeds. Preferred examples thereof include normal hexane, cyclohexane, acetonitrile, 1,4 -Dioxane and tetrahydrofuran.

Since the hydrolysis reaction in an organic solvent is different from the reaction in an aqueous solution, water is retained in the immobilized enzyme.
It is characterized in that the extraction operation is unnecessary.

When any solvent is used, the reaction temperature is usually 0 to 50 ° C., and the reaction time is generally 1 to 48 hours, but is not limited thereto.

The time and end point of the reaction can be confirmed by HPLC or GC. After completion of the reaction, the immobilized enzyme is filtered. When the reaction is carried out with an aqueous solution, an appropriate organic solvent such as dichloromethane or ethyl acetate is added to the filtrate for extraction, and when the organic solvent is used, the filtrate is used. By applying a conventional method such as distillation or column chromatography, only the optically active ester which has not been hydrolyzed can be purified and obtained. Such a reaction has a shorter reaction time than using an enzyme that is not immobilized.

In any of the above-mentioned esterification reaction and hydrolysis reaction, the immobilized enzyme recovered by ordinary methods such as filtration, centrifugation and the like after liquid separation has a lipase activity, It is possible to continuously use the reaction as it is by a column method or the like.

In the batch system, the recovered immobilized enzyme is reused, while in the column system, the reaction can be continuously performed using the immobilized enzyme as a filler.

In the batch method, after completion of the reaction, the reaction solution and the immobilized enzyme are separated by the above-mentioned method, concentrated, and the optically active compound is purified by applying a conventional method such as distillation or column chromatography. , Can be obtained.

In the case of the column method, the solvent in which the substrate is dissolved is injected into a column filled with the immobilized enzyme alone or together with another packing material, and the reaction solution, which has completed the reaction flowing out of the column, is concentrated and distilled. Alternatively, an optically active compound can be purified and obtained by applying a conventional method such as column chromatography. In order for the reaction to proceed efficiently and for the reaction solution to flow out, it is desirable to use the immobilized enzyme in a mixture with other fillers, as long as the other fillers used do not inhibit the enzyme activity. It is not particularly limited. Examples of such packing materials include silica gel for column separation, acidic, basic and neutral alumina, cation and anion exchange resins, celite, quartz sand and sea sand, and these may be used alone or as a mixture. Suitable examples include silica gel (200 to 300 mesh) (manufactured by Wako Pure Chemical Industries, Ltd., Celite (521) (manufactured by Aldrich, USA)).

Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.

Example 1 (Esterification) (Preparation of immobilized enzyme) 1 g of enzyme (Lipase PS) was placed in 300 ml of distilled water and stirred well, and a water-absorbing resin (PQ polymer: BL-100) was added to the suspension. , Diamond wet,
10 g of Poise SA or Aqua Reserve) was added, and the mixture was stirred with a mechanical stirrer. Swollen immobilized enzyme at 40 ° C
The water in the polymer is removed by dehydration under reduced pressure (1 mmHg or less) (may be azeotropically dehydrated with an organic solvent such as normal hexane), and the water in the polymer varies depending on the type of the substrate and the enzyme. The amount of dehydration was adjusted so as to retain 100 to 1000 ppm of water to obtain an immobilized lipase. The amount of the enzyme was 10% by weight based on the water-absorbing resin.

(Reaction) 1 g (7.6 mmol) of ethyl D, L-3-hydroxybutyrate, 1 g (11.6 mmol) of vinyl acetate
l) 100 ml of normal hexane was weighed into a 300 ml eggplant-shaped flask and thoroughly stirred. 1 g of the immobilized enzyme prepared above was added thereto, and the mixture was stirred at room temperature for 24 hours. Thereafter, the immobilized enzyme was separated by filtration, and the filtrate was distilled off under reduced pressure at 40 ° C. or lower (20 mmHg) to obtain a mixture of ethyl 3-hydroxybutyrate and ethyl 3-acetoxybutyrate.

The obtained mixture was subjected to high performance liquid chromatography (LC-6A, manufactured by Shimadzu Corporation) using an optically active column under the following conditions to determine the optical purity of the product and to calculate E according to the following equation.

(Analysis conditions) LC column: CHIRALCEL OD (manufactured by Daicel Chemical Industries, Ltd.) Developing solvent: hexane: isopropyl alcohol = 95: 5 Flow rate: 0.5 ml / min Detector UV: 220 nm (as a measure of selectivity) E value)

[0057]

(Equation 1)

The higher the value of E, the better the selectivity.

Table 1 shows the results.

[0060]

[Table 1]

Example 2 (Esterification with Various Substrates) (Reaction) Secondary alcohol (10 mmol), vinyl acetate 1.
The same operation as in Example 1 was performed using 29 g (15 mmol), 130 ml of normal hexane and 1.29 g of the immobilized enzyme (water-absorbent resin used: PQ polymer: BL-100) prepared in the same manner as in Example 1. An optically active secondary alcohol and an acetyl form were obtained.

The conditions for analysis of the obtained mixture were as follows, and E was calculated from the optical purity.

(Analytical conditions) 1-indanol, 1-phenyl-1-propanol and α-hydroxyethylbenzene were the same as those in Example 1 for ethyl D, L-3-hydroxybutyrate.

2-octanol was analyzed by gas chromatography using an optically active column (GC-8A, Shimadzu Corporation).
The optical purity was determined under the following conditions.

Column: S-2614 Chiraldex G-
TA (manufactured by ASTEC), L: 20 m × 0.25 mm, film thickness: 0.125 μm,
Column temperature: 60 ° C, injection temperature: 200 ° C, carrier gas:
He, pressure: 1.0 kg The results are shown in Table 2.

[0066]

[Table 2]

From this example, it can be seen that the use of the immobilized lipase has superior effects such as a shorter reaction time and a larger E than the use of the non-immobilized lipase. .

Example 3 (Cycle test of esterification) (Reaction) 4.4 g of ethyl D, L-3-hydroxybutyrate (33
mmol), 4.31 g (50 mmol) of vinyl acetate and 440 ml of normal hexane were weighed into a 1-liter eggplant-shaped flask and thoroughly stirred. An immobilized enzyme prepared by the same method as in Example 1 (water-absorbing resin used: PQ
(Polymer: BL-100) (4.31 g) was added and the mixture was stirred at room temperature for 24 hours. Thereafter, the immobilized enzyme was separated by filtration, and the filtrate was distilled off under reduced pressure at 40 ° C. or lower (20 mmHg).
A mixture of optically active ethyl 3-hydroxybutyrate and ethyl 3-acetoxybutyrate was obtained. Subsequently, the same operation was performed using the separated immobilized enzyme, and 10 cycles of the reaction were performed.

The mixture obtained in each cycle was measured under the same analytical conditions as in Example 1, and E was calculated.

Table 3 shows the results.

[0071]

[Table 3]

Example 4 (Hydrolysis) (Preparation of immobilized enzyme) 5 g of an enzyme (lipase AK) was placed in 400 ml of distilled water and stirred well, and a water-absorbent resin (PQ polymer: BL-100) was added to the suspension. , Sunwet, Poise, KI gel or Aqua Keep) and stirred with a mechanical stirrer to obtain immobilized lipase.
The amount of the enzyme was 16.7% by weight based on the water-absorbing resin.

(Reaction) 1 g of butyl epoxybutyrate (6.3 m
mol) and immobilized lipase 4.35g phosphate buffer _{pH7.2 (2M-KH 2 PO 4} , 2N-NaOH) was added into the eggplant-shaped flask 100ml containing the 9 g, optical purity 96% at room temperature It stirred until it became ee or more. After the reaction,
The immobilized lipase was separated by filtration, and the filtrate was extracted three times with 5 ml of normal hexane. The separated immobilized lipase was washed with 5 ml of normal hexane, and the combined extract was dried using anhydrous sodium sulfate.
Thereafter, anhydrous sodium sulfate was separated by filtration, and the filtrate was distilled off under reduced pressure at 40 ° C. or lower (20 mmHg) (2R, 3S)-(−).
-Obtained butyl epoxybutyrate.

The optical purity of the obtained compound was determined by gas chromatography (GC-8A, Shimadzu Corporation) under the following conditions, and E was calculated.

(Analytical conditions) Column: S-2614 Chiraldex G-TA (Astec, manufactured by ASTEC), L: 20 m × 0.25 mm, film thickness: 0.125 μm, column temperature: 100 ° C., injection temperature: 200
° C, carrier gas: He, pressure: 1.0 kg The results are shown in Table 4.

[0076]

[Table 4]

Example 5 (Hydrolysis when Using Various Substrates) (Reaction) 1.8 g of ester, immobilized lipase prepared in the same manner as in Example 4 (water-absorbing resin used: PQ polymer: BL- 100) 8.7g, pH 7.2 phosphate buffer solution 15
Using g, the same operation as in Example 4 was carried out to obtain an optically active ester.

The analysis conditions of the obtained mixture were as follows, and E was calculated from the optical purity.

(Analysis Conditions) Butyl glycidate was prepared in Example 4.
Same conditions as for butyl epoxybutyrate in

The methyl α-chloropropionate was the same as above except for the column temperature (70 ° C.).

Ethyl α-chloropropionate is the same as above except for the column temperature (60 ° C.).

Table 5 shows the results.

[0083]

[Table 5]

According to this example, the use of the immobilized lipase was more effective than the use of the non-immobilized lipase.
It can be seen that excellent effects such as a shortened reaction time are obtained.

Example 6 (Hydrolysis in Organic Solvent) (Reaction) 1 g (6.3 mmol) of butyl epoxybutyrate, 4.35 g of immobilized lipase prepared in the same manner as in Example 4,
100 ml of normal hexane and a trapping agent (0.15 g in the case of ion-exchange resin, equimolar to the substrate in the case of triethylamine or pyridine) were placed in a 300 ml eggplant-shaped flask and stirred at room temperature for 24 hours. Thereafter, the immobilized lipase was separated by filtration, and the separated immobilized lipase was washed with 10 ml of normal hexane. The washed liquid and the filtrate were combined, dried over anhydrous sodium sulfate, and then separated by filtration. The filtrate was distilled off under reduced pressure at 40 ° C. or lower (20 mmHg) to obtain (2R, 3S)-(−)-butyl butylbutyrate.

For the obtained compound, the optical purity was determined under the same conditions as in Example 4, and E was calculated.

Table 6 shows the results.

[0088]

[Table 6]

Example 7 (Hydrolysis cycle test 1)
(Batch method)) (Preparation of immobilized enzyme) 0.15 g of enzyme (lipase AK) was put in 12 ml of distilled water and stirred well, and 0.9 g of a water-absorbent resin (PQ polymer: BL-100) was added to the suspension. in addition,
The mixture was stirred with a mechanical stirrer to obtain immobilized lipase.

(Reaction) 3 g of butyl epoxybutyrate (19 mm
ol) and phosphate buffer immobilized lipase 13.05g and _{pH7.2 (2M · KH 2 PO 4} · 2N-NaOH) 27g 3
Put into a 00 ml eggplant-shaped flask, and the optical purity is 96% at room temperature.
It stirred until it became ee or more. After completion of the reaction, the immobilized lipase was separated by filtration, and the immobilized lipase was washed once with 15 ml of normal hexane.

Further, the filtration was carried out three times with 15 ml of normal hexane, combined with the washing solution and dried with anhydrous sodium sulfate. Thereafter, anhydrous sodium sulfate was separated by filtration, and the filtrate was distilled off under reduced pressure at 40 ° C. or lower (20 mmHg) to give (2R, 3S)-
(-)-Butyl epoxybutyrate was obtained. Subsequently, the same operation was performed using the separated immobilized lipase, and the reaction was performed for 10 cycles.

The compound obtained in each cycle was measured under the same analytical conditions as in Example 4, and E was calculated.

Table 7 shows the results.

[0094]

[Table 7]

Example 8 (Hydrolysis cycle test 2)
(Column packing method)) (Preparation of immobilized enzyme packed column) Enzyme (Lipase AK)
1 g, water-absorbent resin (RQ polymer: BL-100) 6 g
Immobilized lipase was prepared in the same manner as above using 80 ml of distilled water and packed in an open column.

(Reaction) A mixture of 10 g (63.2 mmol) of butyl epoxybutyrate and 90 g of a phosphate buffer having a pH of 7.2 was dropped into the open column, and the optical purity of the effluent from the column became 96% ee or more. The effluent was repeatedly dropped into the open column until the flow. After the reaction was completed, the effluent was extracted with 15 ml of normal hexane. In addition, the column was developed with 15 ml of normal hexane, and the effluent was combined with the previous extract,
It was dried over anhydrous sodium sulfate and the sodium sulfate was separated by filtration. Normal hexane was distilled off under reduced pressure at 40 ° C or less (20 mmHg).
(2R, 3S)-(-)-Butyl epoxybutyrate was obtained.

Further, the same operation was repeated for the open column, and 10 cycles of the reaction were performed.

The compound obtained in each cycle was measured under the same analytical conditions as in Example 4, and E was calculated.

Table 8 shows the results.

[0100]

[Table 8]

[0101]

According to the present invention, it can be used not only for optical resolution of alcohol but also for optical resolution of ester.
The present invention provides a multifunctional immobilized lipase that can be used in a cycle by a batch method, a column packing method, or the like.

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁷ , DB name) C12N 11/00-11/14

Claims (6)

(57) [Claims] 1. A multifunctional immobilized lipase PS, which is immobilized on a PQ polymer. 2. A multifunctional immobilized lipase AK, which is immobilized on a PQ polymer. 3. A process for producing immobilized lipase PS of claim 1 Symbol mounting, characterized in that the lipase PS is dissolved or suspended in water or buffer is infiltrated into the PQ polymer. 4. Dissolve lipase AK in water or a buffer.
Or suspended and penetrated into the PQ polymer.
The method for producing the immobilized lipase AK according to claim 2. 5. An optical resolution method for alcohol, wherein lipase PS immobilized on a PQ polymer is used as a catalyst. 6. An optical resolution method for esters, wherein lipase AK immobilized on a PQ polymer is used as a catalyst.