JPH02215391A - Method for hydrolyzing amino acid esters - Google Patents

Abstract

PURPOSE:To readily enable obtaining of amino acid esters under mild conditions by carrying out reaction using a lipase as a reaction catalyst. CONSTITUTION:Monoaminomonocarboxylic acids, such as glycine, are reacted with a <=10C alcohol, such as butyl alcohol, to afford amino acid esters, such as glycine butyl ester. The resultant esters in an amount of 1-50wt.% are then dispersed in a buffer solution at a desired pH to provide a raw material dispersion. A lipase, such as derived from a yeast of the genus Candida, in an amount of 1-10000 units (1 unit is the amount of the enzyme liberating fatty acids at 1mumol/min from emulsified olive oil) lipase activity based on 1g raw material is added to the above-mentioned dispersion to provide a reaction solution, which is then reacted at 20-60 deg.C for 3-24hr while being vibrated to hydrolyze the amino acid esters.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel method for hydrolyzing amino acid esters.

(Prior Art) Lipase is called triadylglycerol acylhydrolase, and is known as an enzyme that promotes the stepwise hydrolysis of triadylglycerol into glycerin and fatty acids. Some of the reaction steps are reversible. Even in such a simple reaction, specificity is known depending on the origin of the enzyme. However, its specificity lies in the difference in the decomposition position based on the stereospecificity of triazylglycerol.

Recent advances in biochemistry have led to the discovery of various physiological actions of amino acids. Among them, amino acid esters have attracted attention in the field of neurochemistry as nervous system physiologically active amino acids and their derivatives, and N-acyl-amino acid esters have attracted attention in the field of surface chemistry for their amphoteric surface activity. Its liposome-like structure and antibacterial activity have attracted attention.

Various chemical modifications of amino acids have been investigated to enhance the activity of these compounds. Therefore, there is a need for a mild hydrolysis method that does not affect the steric structure or stability of amino acid esters and N-acylated products that appear during the chemical modification process. The method is unknown.

(Problems to be Solved by the Invention) It is known that esterified products are hydrolyzed under harsh conditions involving high temperature and high pressure using an alkali metal salt, a decomposition catalyst, an autoclave, and the like. Such a method tends to cause side reactions such as migration, isomerization, and cranking in amino acids and acyl groups, and is not preferred for hydrolysis of amino acid esters and N-acyl-amino acid esters.

Therefore, the present inventors investigated a hydrolysis method using an enzyme. Lipase is known as an enzyme that catalyzes hydrolysis using triadylglycerol as a substrate, but the specificity of this enzyme regarding hydrolysis has been studied limited to the stereospecificity of triadylglycerol. Regarding the hydrolysis of other esterified products, there are only the following reports, which cannot be immediately applied to amino acid esters.

Mani et al. investigated the decomposition rate of various fatty acids using diesters of ethylene glycol (V, ν, S, Mani et al.
G. Lakshminayana, Bio
chem, Biophys, octa.

"Kakei, 547.1970), and Mattson et al. investigated the decomposition rates of linear alcohols and fatty acid esters (F.

M, Mattson & R, A, Volpenh
ain, J, Ltpid Res, +10, 271
．． (1969), Plockelhoff p-17 of various fatty acids.
The decomposition rates of 0 lobenzyl, 2-(hexyloxy)ethyl and vinyl esters are being investigated (H, Brock
erhoff, Biochi*, Fliophys
, ^cta.

”■Su, 92. 1970).

As described above, since no studies have been conducted on the hydrolysis of amino acid esters by lipase, the present inventors investigated the use of this enzyme. Therefore, the present inventors synthesized various amino acid esters and N-acyl-amino acid esters that are thought to appear in the amino acid modification process, and proceeded with studies to develop a hydrolysis method using lipase. Ta. As a result, they discovered that by using lipase these amino acid esters can be hydrolyzed without affecting their three-dimensional structure or stability, leading to the completion of the present invention. An object of the present invention is to provide a method for hydrolyzing amino acid esters under mild reaction conditions.

(Means for Solving the Problems) The present invention is a method for hydrolyzing amino acid esters, which is characterized by using lipase as a reaction catalyst.

Amino acid esters to be hydrolyzed in the present invention include amino acid esters and N-acyl-amino acid esters, and the amino acids include the following substances. Examples include aliphatic and aromatic amino acids, such as monoaminomonocarboxylic acids, oxyamino acids, sulfur-containing amino acids, and diaminomonocarboxylic acids. Particularly preferred are monoaminomonocarboxylic acids of aliphatic amino acids. Specific amino acids of monoamino monocarboxylic acids include glycine, alanine, T-aminobutyric acid, δ-aminovaleric acid, and ε-aminocaproic acid.

The carbon chain length of the alcohol bonded to the carboxyl group of the above amino acid is preferably 10 or less from the viewpoint of decomposition rate. That is, specific esters include methyl, ethyl, propyl,
Examples include butyl, hexyl, octyl and decyl, and corresponding alcohols are used. An ester reaction between an amino acid and an alcohol proceeds under dehydration conditions and in the presence of an esterification catalyst, and an amino acid ester is synthesized.

Therefore, the amino acid esters targeted by the present invention include all compounds constituted by various combinations depending on the specific types of amino acids and alcohols, and the following are exemplified as representative compounds. That is, glycine butyl ester, β-alanine butyl ester,
These include T-aminobutyric acid octyl ester, ε-aminocaproic acid methyl ester, glycine decyl ester, α-alanine propyl ester, γ-aminobutyric acid pentyl ester, and ε-aminocaproic acid octyl ester.

Furthermore, the present invention also targets N-acyl-amino acid esters in which an acyl group is bonded to the free amino group of the above-mentioned amino acid ester. In this case, since the acyl group used means a fatty acid, the bond type is an aminocarbonyl bond, that is, a peptide bond. The fatty acid bonded to the amino group of the above amino acid ester has a carbon chain length of 8 to 24
Those having 0 to 6 unsaturated bonds are advantageous from the viewpoint of ease of handling the reaction product.

As raw fatty acids, saturated acids can be obtained at industrially high purity using molecular distillation, etc., and monoenoic acids and dienoic acids can be obtained at industrially high purity using a combination of crystal fractionation. Further, highly unsaturated acids that can be used include docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid, lyllunic acid, and T-lyllunic acid, which have been highly purified by a combination of molecular distillation, urea addition, and column chromatography.

These fatty acids and the above-mentioned amino acid esters are induced into N-acyl-amino acid esters using a basic catalyst typified by a nitrogen-containing heterocyclic compound. At this time, the catalysts used include imidazole, carbonyldiimidazole, N-
Among these, the derivative of the model substance of the present invention can be prepared by adding 1.1 to 1.3 moles of carbonyldiimidazole.

Specific examples include N-docosahexanoyl-T-aminobutyric acid octyl ester, N-eicosapentanoyl-grincybutyl ester, N-lauroyl-β-alanine-butyl ester, N-α-lylnoyl-β-alanine-octyl. ester, N-arachitonyl-γ-aminobutyric acid pentyl ester, N-valmitoyl-ε-aminocaproic acid propyl ester, and the like.

There are no particular limitations on the lipase used in the hydrolysis method of the present invention. Although it is well known that lipase performs position-specific decomposition of triglycerides depending on the origin of the enzyme, its decomposition properties regarding amino acid esters and N-acyl-amino acid esters have been completely unknown. Therefore, the present inventors synthesized a group of model compounds of these amino acid derivatives and found that this group of compounds undergoes hydrolysis by lipase to become amino acids and N-acyl-amino acids.

The lipase used in the present invention includes pancreatic juice, gastric juice, serum, urine,
There are those of animal origin present in milk, etc., those of plant origin present in castor seeds, rapeseed seeds, rice bran seeds, etc., and those of microbial origin present in molds, yeasts, bacteria, etc.

Because of its easy availability, ease of handling, and low cost,
In the present invention, preferred are those originating from yeasts of the genus Candida, molds of the genus Aspergillus, Rhizobus, Geotrichum, and Mucor, and freeze-dried products of extracts of porcine pancreas.

The aforementioned amino acid esters and N-acyl-amino acid esters were used as raw materials for inducing amino acids and N-acyl-amino acids by the action of such enzymes. The hydrolysis reaction is carried out by dispersing the amino acid ester or N-acyl-amino acid ester in water or a buffer solution with a pH appropriate for the enzyme to a concentration of 1 to 50% by weight, preferably 2 to 10% by weight, and dispersing this dispersion. per 1g of raw material, lipase activity is 1~
Toooo.

unit (1 unit is 1 μmol/mol from emulsified olive oil)
Add lipase in an amount equivalent to the amount of enzyme that liberates 1000 fatty acids, preferably 100 to 5000 units, and heat the reaction mixture at 20 to 60°C, more preferably 25 to 40°C, while shaking the reaction solution. It is best to react for 24 hours.

In addition, regarding the hydrolysis reaction of lipase, anionic and nonionic surfactants act in an inhibitory manner,
Cationic activators promote at low concentrations and inhibit at high concentrations;
Amino acid derivatives belonging to amphoteric surfactants are not affected by such ionizing properties.

(Effects of the Invention) According to the present invention, amino acid esters and N-acyl-amino acid esters that are thought to appear during the modification process of physiologically active amino acids and their derivatives are produced using commercially available lipases of various origins. can be modified by hydrolysis.

A feature of the hydrolysis method of the present invention is that all reaction steps can be carried out near room temperature, thereby preventing changes in amino acids and production of by-products. It is particularly suitable for decomposing N-acyl-amino acid esters in which heat- and enzyme-labile unsaturated acids are introduced as acyl groups, so it is effective as a means for modifying physiologically active amino acids used in the field of neurochemistry, for example. .

(Example) Hereinafter, the present invention will be specifically explained based on Examples.

Example 1 1 equipped with calcium chloride pipe, cooler, and moisture metering receiver
n-butyl alcohol 3-O in the eggplant flask of 001d.
g (40mM) and glycine 2.25g (30mM)
7.4g (3.9mM) of p-)luenesulfonic acid and about 50mf of xylene were weighed out, heated to 100°C, and reacted for 7 hours.

After the reaction, the mixture was cooled to room temperature, benzene was added, and xylene was azeotropically distilled off. Ether was added to the azeotropic residue, the mixture was transferred to a separatory funnel, and water was further added to perform extraction. Powdered sodium carbonate was added to the lower aqueous layer from which the ether layer was removed, and the pH was adjusted to 1.
1-12 and an oil separated on top of the aqueous layer. Dissolve this oil in ether, add water and wash with water for 5 minutes.
Repeated times.

The ether layer was distilled off to obtain 4.4 g of an oily substance.

The entire amount of the oil was subjected to silica gel column chromatography, and hexane/ether/acetic acid (50150/1 v/
Elution was performed using a mixed eluent (v/v). Fractionate 10 parts of the eluate and perform thin layer chromatography on each fraction [Developing solvent: hexane/ether/acetic acid (70/30/1 v/v/v
)) and checked for eluates. As a result, a small amount of n-butyl alcohol was observed at the Rf value of 0.2, and a spot of glycine butyl ester, the main component, was observed at Rf(i[0,1), based on the polarity of the compound.

The amount of the target product recovered from both parts was 3.3 g, with a yield of 84%.

The analytical values of the recovered material were as follows.

Appearance: Slight yellow oily liquid Dissolution state: Water, hot ethanol soluble Thin layer chromatography ■Hexane/ether/acetic acid (50150/1 v/v
/v) Rf value: 0.4, dichlorofluorefcene reagent color development: orange ■Chloroform/methanol/water (65/25/4)
v/v/v) Rf value: 0.7, ninhydrin reagent color development: reddish-purple FAB-MS F (M+H)'' 132 Based on the above results, the recovered material was confirmed to be glycine butyl ester.

Next, 1.0 g of the obtained glycine butyl ester and 0.0 g of glycine butyl ester were added.
2M boric acid buffer (pH 7°0) 25- and lipase OF (origin: yeast Candtda Cylindrac
eas Meito Sangyo ■) 3,000 units were placed in a 50-mm brown eggplant flask and incubated at 37 to 40°C for 6 hours. After the reaction, ethanol was added to the reaction solution and water was azeotropically distilled off. Next, 30% aqueous ethanol was added to the azeotropic residue, transferred to a centrifuge tube, and centrifuged at 3500 rpm+s for 15 minutes.

Concentrate the supernatant liquid to an appropriate amount and pass it through a microfilter equipped with a glass filter to completely remove lipase O.
F was removed. After concentrating the filtrate, this concentrated solution was used as an eluent [chloroform/methanol/water (65/25/4 v/ν
/ν)] and subjected to silica gel column chromatography (manufactured by Merck & Co., Kieselgel 60, particle size 70-230).
The mixture was applied to a mesh column (2φ×25c column) and eluted with this eluent. A 10M portion of the eluate was fractionated, and both fractions were developed using thin layer chromatography [developing solvent: chloroform/methanol/water (65/25/4V/C/V)], and then a ninhydrin reagent was sprayed and heated.

In the glycine elution fraction of the decomposition product, a reddish-purple color was confirmed at the origin of thin layer chromatography. The amount of glycine recovered was 0.4 g, and the yield was 70%.

From this result, it was found that the lipase according to the present invention acts on amino acid esters and efficiently performs the hydrolysis reaction.

Example 2 1 equipped with calcium chloride pipe, cooler, and moisture metering receiver
5゜n-octyl alcohol in a 00- eggplant flask
g (40111M) and ε-aminocaproic acid 3.93
g (30mM) and p-toluenesulfonic acid 7.4g
(39mM) and about 50cm of xylene, and heated to 100°C.
The temperature was raised to 100%, and the reaction was allowed to proceed for 6 hours. After the reaction, the mixture was cooled to room temperature, benzene was added, and xylene was azeotropically distilled off. Ether was added to the azeotropic residue, the mixture was transferred to a separatory funnel, and water was further added for extraction. Powdered sodium carbonate was added to the lower aqueous layer from which the ether layer was removed to adjust the pH to 11-12, and an oil was separated on top of the aqueous layer. This oil was dissolved in ether, water was added, and water washing was repeated. The ether layer was distilled off to obtain 7.5 g of an oil.

The entire amount of the oil was fractionated by silica gel chromatography in the same manner as in Example 1, and a check of thin layer chromatography revealed that a small amount of n-octyl alcohol was present at an Rf value of 0.2, and mainly a small amount of n-octyl alcohol was present at an Rf value of 0.1. A spot of the component 6-aminocaproic acid octyl ester was confirmed. The amount of the target product recovered from both parts was 5.6 g, and the yield was 76%.

The analytical values of the recovered material were as follows.

Appearance: Yellow oily liquid soluble &'Water, hot ethanol soluble Thin layer chromatography ■Hexane/ether/acetic acid (50150/1ν/v/
v) Rf value 1.4, dichlorofluoreracene reagent coloring, two orange colors ■Chloroform/methanol/water (65/25/l)
v/v/v) Rf value: 0.1, ninhydrin reagent color development: reddish-purple FAB-MS: (M+H)'' 244 From the above results, the recovered material was confirmed to be ε-aminocaproic acid octyl ester.

Next obtained ε-aminocaproic acid octyl ester 1
．． 0g and 0.1M boric acid buffer (pH 8,0) 2
ON+1 and pancreatic lipase
as: Funakoshi Pharmaceutical ■) 1500 units were placed in a 50-mm brown eggplant flask and incubated at 37°C for 10 hours. After the reaction, 0.36 g of ε-aminocaproic acid was recovered by the same procedure as in Example 1, and the yield was 68
%Met.

These results revealed that the lipase according to the present invention acts on amino acid esters and efficiently performs the hydrolysis reaction.

Example 3 1 equipped with calcium chloride pipe, cooler, and moisture metering receiver
3.0g of n-butyl alcohol in a 00d eggplant flask
(40mM), 2.67g (30mM) of β-alanine, 7.4g (39mM) of p-toluenesulfonic acid, and about 50% of xylene were heated to 100°C and reacted for 7 hours. Hereinafter, the same procedure as in Example 1 was carried out to obtain the oily substance 4.
7g was obtained. Next, the entire amount of the oil was fractionated by silica gel column chromatography in the same manner as in Example 1, and checked by thin layer chromatography to find that the Rf value was 0.
．． A spot of β-alanine butyl ester, the main component, was confirmed in No. 1. The amount recovered from the target object and both parts was 3.4
The yield was 78%.

The analytical values of the recovered material were as follows.

Appearance: Yellow oily liquid Dissolution state: Water, hot ethanol soluble Thin layer chromatography ■Hexane/ether/acetic acid (50150/1 v/v
/v) Rf value: 0.4, dichlorofluorefcene reagent color development: orange ■Chloroform/methanol/water (65/25/4)
v/v/v) Rf value: 0.1, ninhydrin reagent color development: reddish-purple FAB-MS: (M+H)'' 146 From the above results, the recovered material was confirmed to be β-alanine butyl ester.

Next, 2.0 g (lomM) of lauric acid, 60 d of dehydrated tetrahydrofuran, and 1.78 g (11 mM) of N,N''-carbonyldiimidazole were weighed into a 200 m eggplant flask equipped with a calcium chloride tube, and reacted for 3 hours at room temperature. 1.45g (10mM) of β-alanine butyl ester obtained above was added to this reaction solution, and the reaction was further allowed to proceed overnight at room temperature.Tetrahydrofuran in the reaction solution was distilled off, and 4.1g of distillation residue was obtained. chloroform/methanol (2/1 v/v) was added to this distillation residue to make a homogeneous solution. This solution was transferred to a separating funnel and
Extraction was performed by adding hydrochloric acid, and the upper water-methanol layer was removed. Water was added to the lower chloroform layer and water washing was repeated. Separate the chloroform layer and remove the solvent, 3.1
g of the target product was obtained.

The entire amount recovered was added to the eluent [chloroform/methanol (1
0/1 v/v)) and subjected to silica gel column chromatography (manufactured by Merck & Co., Kieselgel 60, particle size 7).
0 to 230 mesh, 2φ x 30cm column) and eluted with this eluent. Fractionate 10 d of eluate,
After developing each portion by thin layer chromatography [developing solvent: chloroform/methanol (10/1 v/v)], a dichlorofluoreracene reagent was sprayed, and spots were observed by UV irradiation. From the polarity of the compound, a trace amount of β-alanine butyl ester was observed at an Rf value of 0.4, and a spot of the main component, N-lauroyl-β-alanine butyl ester, was observed at an Rf value of 0.7. The amount of the target product recovered from both parts was 2.5 g, and the yield was 76%.

The analytical values of the recovered material were as follows.

Appearance: Slight yellow oily liquid Dissolution state: Chloroform, tetrahydrofuran, benzene soluble Thin layer chromatography ■Chloroform/methanol/water (10/1 v/v)
Rf4fi: 0.7, dichlorofluorescene reagent color development: orange FAB-MS: (M+H) 3328 From the above results, the recovered material was confirmed to be N-lauroyl-β-alanine butyl ester.

Next, 1.0 g of the obtained N-lauroyl-β-alanine butyl ester was added with 0.1 M phosphate buffer (pH 7.0).
4,000 units of 30- and Lipase F (origin: Resobs javanicus, Amano Pharmaceutical) were placed in a 50-brown eggplant flask and incubated at 37 to 40°C for 6 hours.

Add chloroform/methanol (2/1 v/v) to the reaction solution.
Add 30 - of ) and shake vigorously, transfer to a centrifuge tube and
Centrifugation was performed for 15 minutes at 0 rpH1. The chloroform layer was concentrated to obtain 0.7 g of the desired product. The entire amount of concentrate,
Eluent [chloroform/methanol (10/1 v/v
)) and silica gel column chromatography (
The sample was applied to a column (manufactured by Merck & Co., Ltd., Kieselgel 60, particle size 70-230 mesh, 1.2φ x 25cm column) and eluted with this eluent. The eluate ioMi was fractionated, and both fractions were developed by thin layer chromatography (developing solvent: same as eluent), then dichlorofluoreracene reagent was sprayed, UV irradiated, and the C spot was observed. Orange color development was confirmed at the origin of thin layer chromatography in the eluted fraction of the decomposition product.

The recovered decomposition product was 0.6 g, and the yield was 72%.

The analytical values of the recovered material were as follows.

Appearance: White powder Dissolution state: Equal amounts of chloroform/methanol mixed? Can it be used as a container? Thin layer chromatography developing solvent, chromatography/methanol (10/1
v/v) Rf value: Origin dichlorofluoreracene reagent Color: Orange FAB-MS; (M+H)” 2
From the results of 72 or above, the recovered material was confirmed to be N-lauroyl-β-alanine. This revealed that the lipase according to the present invention acts on N-acyl-amino acid esters and efficiently performs the hydrolysis reaction.

Example 4 1 equipped with calcium chloride pipe, cooler, and moisture metering receiver
5.2 n-octyl alcohol in a 00- eggplant flask
g (40mM) and γ-aminobutyric acid 3.09g (30++
+M) and p-) luenesulfonic acid? -4g (39mM)
About 50 ml of xylene was weighed out, heated to 100°C, and reacted for 7 hours. Thereafter, the same procedure as in Example 1 was carried out to obtain 7.1 g of an oily substance. Next, the entire amount of the oil was subjected to silica gel column chromatography and fractionated in the same manner as in Example 1, and by checking by thin layer chromatography, a spot of γ-aminobutyric acid octyl ester, the main component, was found at an Rf value of 0.1. was confirmed. The amount of the target product recovered from both parts was 6.1 g, with a yield of 74%.

The analytical values of the recovered material were as follows.

Appearance: Yellow oily liquid Dissolution state: Water, hot ethanol soluble Thin layer chromatography developing solvent ■Hexane/ether/acetic acid (50150/1 v/v
/v) Rf value: 0.5, dichlorofluoreracene reagent coloring dichroic ■Chloroform/methanol/water (65/25/4)
v/v/v) Rf value: 0.7, ninhydrin reagent color development: reddish-purple FAB-MS N (M+H)'' 215 From the above results, the recovered material was confirmed to be γ-aminoacetic acid octyl ester.

Next, 3.2 g (10 mM) of docosahexaenoic acid, 1.78 g (11 mM) of dehydrated tetrahydrofuran 60- and N,N''-carbonyldiimidazole were weighed into a 200 d eggplant flask equipped with a calcium chloride tube, and the mixture was heated at room temperature for 3 hours. γ- obtained above was added to this reaction solution.
2.15 g (10 mM) of aminobutyric acid octyl ester was added, and the mixture was further reacted day and night at room temperature. Tetrahydrofuran in the reaction solution was distilled off to obtain 4.4 g of distillation residue.

This distillation residue was mixed with chloroform/methanol (2/I V
/V) was added to form a homogeneous solution. Transfer this solution to a separatory funnel and perform extraction by adding IN hydrochloric acid.
The methanol layer was removed. The lower chloroform layer was separated and the solvent was removed to obtain 3.8 g of the desired product. Example 3
It was fractionated by column chromatography in the same manner as above and checked by thin layer chromatography. As a result, based on the polarity of the compound, a trace amount of T-aminobutyric acid octyl ester was found at an Rf value of 0.45, and a spot of the main component, N-docosahexaenoyl-γ-aminobutyric acid octyl ester, was found at an Rf value of 0.8. It was done. The amount recovered from both sides of the target object was 3.8
g, and the yield was 72%.

The analytical values of the recovered material were as follows.

Appearance: Slightly yellow oily liquid Dissolution state: Soluble in chloroform, tetrahydrofuran, benzene Thin layer chromatography developing solvent, chloroform/methanol (10/1V/ν
) Rf value: 0.8 Dichlorofluoreracene reagent Coloration: Mi color FAB-M
S: (M+H)" 517 From the above results, the recovered material was confirmed to be N-docosahexaenoyl-T-aminobutyric acid octyl ester. Next, the obtained N-docosahexaenoyl-T-aminobutyric acid Octyl ester 1.
Og, 0.1M phosphate buffer (pH 7,0) 251
5000 units of R1 and lipase A (origin: Aspergillus niger, Amano Pharmaceutical) were placed in a 50-brown flask and incubated at 37-40°C for 6 hours. The reaction solution was operated in the same manner as in Example 3, fractionated by column chromatography, and checked by thin layer chromatography.

As a result, it was confirmed that the eluted fraction of the decomposition product developed an orange color at the origin. The recovered decomposition product was 0.4 g, and the yield was 7.
It was 2%.

The analytical values of the recovered material were as follows.

Appearance: Yellowish brown oily liquid Dissolved state B: Soluble in equal amounts of chloroform/methanol mixed solvent Thin layer chromatography developing solvent, chloroform/methanol (10/IV/V
i Rf value: Origin dichlorofluoreracene reagent Color: Orange FAB-MS
: (M+H)” 405GC: 2%0V-10,
6m column, column temperature 130-220℃, 7.5℃/
A 7% methanol trifluoride solution was added to the ether solution of the recovered fraction, heated for 30 minutes, extracted with hexane, and used as a GC sample. Besides the small peak of methyl docosahexaenoate, there was only a single main peak of N-docosahexaenoyl-T-aminobutyric acid methyl ester.

From the above results, the recovered material is N-docosahexaenoyl-
It was confirmed that it is γ-aminobutyric acid and that its acyl group is also stable.

This revealed that the lipase according to the present invention acts on N-acyl-amino acid esters and efficiently performs the ester hydrolysis reaction.

Claims (1)

[Claims] A method for hydrolyzing amino acid esters, characterized by using lipase as a reaction catalyst.