JPH0622776A - Production of n-long-chain acylamino acid by fermentation method - Google Patents

Abstract

PURPOSE:To efficiently produce various N-long-chain acylamino acids by using an aminoacylase. CONSTITUTION:The method for producing N-long-chain acylamino acids is to make an aminoacylase produced by a microorganism belonging to the genus Penicillium, or a microorganism belonging to the genus Penicillium capable of producing the aminoacylase, react with a mixture of an amino acid with a long-chain fatty acid. The N-long-chain amino acids, especially an N-long-chain acylglycine, an N-long-chain acylserine, etc., can simply be synthesized in a high yield.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing an N-long chain acylamino acid by an enzymatic method. More specifically, the present invention relates to a method for producing an N-long chain acylamino acid from an amino acid and a long chain fatty acid by using an enzymatic method using aminoacylase or a microorganism capable of producing an acylamylase.

[0002]

2. Description of the Related Art N-long-chain acylamino acids are amphipathic substances composed of natural components, have been confirmed to be safe and have excellent biochemical degradability, and also have a surface activity and a bactericidal action. is doing. Therefore, the N-long chain acylamino acid is
It is a substance that has been attracting attention as a surfactant that is safe and has excellent biochemical degradability, and that it can be an antioxidant by combining an amino acid and a fatty acid. And
At present, N-long-chain acylamino acids are being studied for use in various soaps, shampoos, detergents, emulsifiers, antibacterial agents, etc., and some of them have been put into practical use.

Conventionally, N-long chain acylamino acids have been chemically synthesized from amino acids and fatty acids (M. Tahara).
kehara) et al., Journal of The American O
il Chemist's Society.), Volume 49, pages 157-16
1 (1972), Y. Lapidot et al.
Journal of Lipid Research
id Research), Volume 8, Pages 142-145 (1967)
Year), Japanese Patent Publication No. 31-9,568, Japanese Patent Publication No. 46-8,6
No. 85). However, these chemical synthesis methods are carried out under severe conditions such as temperature, pressure and pH, and have a drawback that many by-products are produced. In addition, these chemical synthesis methods have various drawbacks such as the problem of their persistence because dangerous reagents are used in the synthesis.

[0004] In order to solve such a problem, N-type amino acids and fatty acids were used instead of amino acids and fatty acids using Pseudomonas diminuta, Xanthomonas albinianus, and Gluconobacter liquifaciens cells. A method for synthesizing a long-chain acylamino acid (JP-A-57-129,696) has been proposed. However, according to the above-mentioned method, only about 1 to 2% of N-long chain acylglutamic acid is produced in the molar ratio of the fatty acid used. Further, the above-mentioned method has many problems such as reaction efficiency when industrialization is considered such that the amino acid is limited to glutamic acid.

[0005]

As described above, a method for easily and efficiently synthesizing an N-long chain acylamino acid using a microorganism has not been found yet.

Therefore, an object of the present invention is to provide a method for easily and efficiently producing various N-long chain acylamino acids using aminoacylase.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that penicillium (P
penicillium funiculosum (p. enicillium)
The present inventors have found that an aminoacylase (Japanese Patent Application No. 3-2,096) produced by enicillium funiculosum efficiently synthesizes various N-long chain acylamino acids from amino acids and long chain fatty acids, and completes the present invention. Came to.

That is, the above-mentioned object is caused by allowing a mixture of amino acids and long-chain fatty acids to act on an aminoacylase produced by a microorganism belonging to the genus Penicillium or a microorganism belonging to the genus Penicillium capable of producing aminoacylase. This is achieved by the method for producing an N-long chain acylamino acid.

The present invention also shows a method for producing an N-long chain acylamino acid in which aminoacylase has the following physicochemical properties.

1) Action It acts on N-long chain acyl amino acids to release L-amino acids.

2) Substrate specificity It acts on the L-form of N-acylamino acid having an acyl group having 4 or more carbon atoms, and does not act on the D-form. It has a wide range of specificity for acyl groups and acts on N-acyl compounds composed of saturated fatty acids, unsaturated fatty acids, and aromatic carboxylic acids.

3) Optimum pH and pH stability When N-oleoyl-L-glutamic acid is used as a substrate, the optimum pH is 30 ° C and various buffers (pH 5.5 to 6.
0: Acetate buffer, pH 6.0-8.0: Phosphate buffer,
pH 8.0-9.5: boric acid-hydrochloric acid buffer, pH 9.5
.About.11.0 in carbonate buffer). It is stable up to pH 6-9 in the same buffer solution at 30 ° C. for 5 hours.

4) Optimum temperature and thermal stability The optimum temperature when N-oleoyl-L-glutamic acid was used as a substrate was 25-35 ° C. when measured using a phosphate buffer (pH 7). is there. It is not inactivated at all in 20 mM Tris-hydrochloric acid buffer (pH 8) at 40 ° C. for 15 minutes, and the residual activity after treatment at 45 ° C. for 15 minutes is about 80%.

5) Effect of inhibitors Parachloromercury benzoate or dithiothreitol inhibits the reaction.

6) Effect of metal ion Hg activated by Ni ²⁺ , Cu ²⁺ , Mg ^2+, etc.
^Inhibited by ²⁺ .

7) Molecular weight of subunit Molecular weight of about 4 by SDS-polyacrylamide gel electrophoresis
8,000.

8) Molecular weight: Approximately 192,000 by gel filtration method.

Further, the present invention provides a Penicillium (Penicinium)
llium) is a microorganism belonging to the genus Penicillium funiculosum
(Penicillium funiculosum) N-long chain acylamino acid production method.

[0019]

The present invention is to synthesize various N-long chain acylamino acids easily and in high yield by adding fatty acid and aminoacylase to an amino acid aqueous solution.

The aminoacylase used in the present invention is produced by a microorganism belonging to the genus Penicillium, preferably penicillium funiculosum.

The above Penicillium funiculosum
llium funiculosum) is a micromachine research article No. 3176 (FER
It has been deposited as MBP-3176) and its mycological properties are as follows.

(A) Growth state in various culture media (a) Tuapeck culture medium Rapidly grows and has a diameter of 30 to 40 mm at 25 ° C. for 7 days.
Reach The colony is composed of a dense mycelium layer in which yellow vegetative mycelium and gray to yellow-green conidiophore are mixed. The back side of the colony is pink to crimson. The morphological properties are as follows.

Conidia peduncle: 25-40 × 2.0-3.0 μm branched from aerial mycelium Metre: 5-8 turns into a ring-shaped 10-13 × 2.0-3.0 μm Phialide: 3 to 6 are ring-shaped. Tip-shaped 10 to 12 × 1.5 to 2.5 μm Conidia: Subspherical to elliptic and small Surface is smooth to finely rough 2.5 to 3 0.5 × 2-2.5 μm (b) Malt extract agar medium The growth state and morphological properties are almost the same as those of Tuapeck agar medium. However, the silk formation is superior to that of Tuapec agar medium.

(B) Physiological and ecological properties pH: Approximately 6 shows the best growth Temperature: Almost no growth is observed at 5 ° C. Optimal growth is observed at approximately 25 to 37 ° C. The microorganism used in the present invention is not limited to the strain of the present invention and its variants and mutants as long as it has an enzyme having the above-mentioned properties.

Penicillium in the present invention
Microorganisms belonging to the genus m) can be cultured according to information known in the field of fermentology. As a medium to be used, either a synthetic medium or a natural medium can be used as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance and other nutrients, and can be cultured using a liquid medium or a solid medium. . Specifically, as a carbon source, glucose, fructose, maltose, galactose, starch,
Starch hydrolysates, sugars such as molasses and molasses, natural carbohydrates such as wheat and rice, alcohols such as glycerol, methanol and ethanol, fatty acids such as acetic acid, gluconic acid, pyruvic acid and citric acid, and normal paraffin One or two or more species may be selected from the general carbon sources such as hydrocarbons, amino acids such as glycine, glutamine, and asparagine in consideration of the assimilation ability of the microorganism used.
As the nitrogen source, meat extract, peptone, yeast extract, soybean hydrolyzate, milk casein, casamino acid, various amino acids, corn steep liquor, other animals, plants,
Organic nitrogen compounds such as microbial hydrolysates, ammonia,
Considering the assimilability of the microorganism used, one or more of ammonium salts such as ammonium nitrate, ammonium sulfate and ammonium chloride, nitrates such as sodium nitrate and inorganic nitrogen compounds such as urea are selected and used.

Further, in order to efficiently produce the aminoacylase used in the present invention, as an inducer, N-long chain acylamino acid Amisoft CS-1 is used.
One or two or more of 1, LS-11, GS-11, HS-11 (Ajinomoto Co., Inc.) and similar substances can be selected and used.

Further, as inorganic salts, trace amounts of magnesium, manganese, calcium, sodium, potassium,
One or more selected from phosphates such as copper and zinc, hydrochlorides, sulfates and acetates can be used.
Moreover, you may add a defoaming agent, such as vegetable oil and surfactant, as needed.

Culturing can be carried out in a liquid medium containing the above-mentioned medium components by using a usual culturing method such as shaking culture, aeration stirring culture, continuous culture and the like.

The culture conditions may be appropriately selected depending on the type of medium and the culture method, and there is no particular problem as long as the strain can grow and produce aminoacylase. Usually, it is desirable to adjust the pH at the start of culture to about 6 and culture under the temperature condition of 25 to 35 ° C. When culturing is carried out using a 5 liter Erlenmeyer flask, 4 to 6 days are suitable for culturing.

The aminoacylase used in the present invention accumulates in the microbial cells. Therefore, after culturing under the above-mentioned culturing conditions, the microbial cells themselves may be used as an enzyme agent or at any stage after extraction from the microbial cells. Enzymes may be used. Specific examples of the method for extracting aminoacylase from bacterial cells include the following. The bacterial cells are collected by a method such as filtration and centrifugation, and the bacterial cells are washed with a buffer solution or the like, and then, for example, physical means such as freeze-thawing treatment, ultrasonic treatment, pressure treatment, osmotic pressure difference treatment, and grinding treatment, Alternatively, a biochemical treatment such as cell wall lysing enzyme treatment with lysozyme or a chemical treatment such as contact treatment with a surfactant may be performed alone or in combination to crush the cells and extract aminoacylase. . The crude aminoacylase thus obtained is subjected to chromatography such as salting out, fractional precipitation with an organic solvent, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, dye chromatography, hydroxyapatite chromatography and affinity chromatography. Purification can be performed using means such as liquid chromatography (HPLC) and electrophoresis alone or in combination.

The aminoacylase used in the present invention is a penicillium funicu.
The following is a specific example of the method obtained from (losum). 60 ml of medium (peptone 2.0%, glucose 0.5%, ammonium nitrate 0.3%, potassium dihydrogen phosphate)
0.4%, dipotassium hydrogen phosphate 0.1%, sodium chloride 0.1%, magnesium sulfate 0.02%, p
H6.0) in a 500 ml Sakaguchi flask and put into a penicillium funiculosum
1 platinum loop is inoculated and cultured at 30 ° C. for 48 hours to obtain a seed culture solution. Amisoft GS-11 was added to the medium of the above culture composition.
(Ajinomoto Co., Inc.) 0.5% was added, and 1 liter of the medium whose pH was adjusted to 6.0 was placed in a 5 liter Erlenmeyer flask.
Incubate for 0 hours. In this way, a total of 9 liters of main culture was performed, and the cultured cells were collected by suction filtration,
About 570 g (wet weight) of bacterial cells are collected. Next, the cells were washed with a 0.85% sodium chloride aqueous solution, suspended in 50 mM Tris-hydrochloric acid buffer solution (pH 8.0) in an amount 5 times the wet weight of the cells, and the cells were suspended. Minute grinding process (Bead-Beater), biospec
Crush cells with the product (manufactured by BIOSPEC PRODUCTS). The treatment liquid thus obtained is centrifuged (2
30,000 × g for 30 minutes) to obtain an aminoacylase extract. At this time, the total activity of aminoacylase was 20,817 U and the specific activity was 1.5 U / mg. The protein was quantified according to the dye binding method. This crude aminoacylase extract was saturated with 30% ammonium sulfate, the precipitate was removed with Celite, and the resulting supernatant was saturated with 80% ammonium sulfate and centrifuged (12,000).
Xg, 30 minutes) to recover the precipitate. Next, this precipitate was dissolved in 20 mM Tris-hydrochloric acid buffer (pH 8.0) and dialyzed against the same buffer. This aminoacylase solution was equilibrated with the same buffer solution DEAE-Toyopearl 6
After passing through 50M to adsorb aminoacylase,
Non-adsorbed substances were removed by thorough washing with the same buffer solution, and aminoacylase was eluted by a linear concentration gradient elution method using a 0 to 0.5 M sodium chloride aqueous solution. Furthermore, this aminoacylase active fraction was concentrated,
Dialyze against water and freeze-dry. The aminoacylase preparation thus obtained had a total activity of 1400.
0 U, specific activity 79 U / mg.

The method for measuring the activity of the aminoacylase of the present invention is to add 250 μl of distilled water to 250 μl of 100 mM phosphate buffer (pH 7) containing 10 mM N-oleoyl-L-glutamic acid and incubate at 30 ° C. for 5 minutes. Then, 50 μl of enzyme solution was added and reacted for 15 minutes, and the produced L-glutamic acid was treated with the ninhydrin-hydrindantine method (Journal of Biological Chemistry, No. 2).
Vol. 11, p. 907 (1957)). Note that 1 U was the amount of enzyme that catalyzes the production of 1 μmol of L-glutamic acid in 1 minute.

The amino acids that can be used in the present invention are L-form, D-form, DL-form amino acids and salts or esters thereof. Specifically, alanine, β-alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine,
Glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine,
Examples include arginine, aspartic acid, glutamic acid, hydroxylysine, doper, ornithine and salts and esters thereof.

The fatty acid is a saturated or unsaturated fatty acid having 6 to 24 carbon atoms, preferably 6 to 20 carbon atoms and salts thereof, and examples thereof include caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, Examples include stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid and salts thereof.

In the present invention, the method for synthesizing an N-long chain acylamino acid from an amino acid and a fatty acid is carried out by adding a fatty acid to an aqueous amino acid solution, adding aminoacylase or a microorganism capable of producing an aminoacylase, and shaking or stirring at a predetermined temperature. Can be achieved by doing. The pH of the amino acid aqueous solution at this time is 6 to 9, preferably 7 to 8.5.
Is. The amino acid concentration is preferably close to the saturation concentration of the amino acid used. The amount of fatty acid is preferably 50 to 200 mg per 1 ml of the amino acid aqueous solution.
Furthermore, the amount of aminoacylase added is preferably 50 U or more per 1 ml of the amino acid aqueous solution. The reaction temperature is 20 to 50 ° C, preferably 30 to 40 ° C.
Further, it is preferable to disperse the fatty acid during the reaction, and specific examples thereof include a shaking method and a stirring method. When the reaction time satisfies the above-mentioned method, the equilibrium time of the reaction is almost reached in 30 hours or more. When a fatty acid having a high melting point such as lauric acid, myristic acid, palmitic acid, stearic acid, or arachidic acid is used, an inert organic solvent such as hexane may be added to the reaction solution to such an extent that the used fatty acid is uniformly dispersed. Good.

The N-long chain acylamino acid produced in the reaction solution can be obtained by using various extraction methods and crystallization methods.
It can be easily purified.

[0037]

EXAMPLES Next, the present invention will be described with reference to Examples.
It goes without saying that the scope of the present invention is not limited by these.

Example 1 60 ml of medium (peptone 2.0%, glucose
0.5%, ammonium nitrate 0.3%, potassium dihydrogen phosphate 0.4%, dipotassium hydrogen phosphate 0.1
%, Sodium chloride 0.1%, magnesium sulfate
0.02%, pH 6.0) was put into a 500 ml Sakaguchi flask and the penicillium funiculosum was added.
m. funiculosum) was inoculated and cultured at 30 ° C. for 48 hours to obtain a seed culture solution. Amisoft GS-11 (Ajinomoto Co., Inc.) 0.5% was added to the medium having the above culture composition to adjust the pH.
1 liter of the medium adjusted to 6.0 was placed in a 5 liter Erlenmeyer flask, and 60 ml of the seed culture solution was added to it.
It was cultured at 30 ° C. for about 120 hours.

A total of 9 liters of main culture was carried out in this way, and the cultured cells were collected by suction filtration to give about 57
0 g (wet weight) of bacterial cells was collected.

Next, the bacterial cells 57 thus collected
0 g (wet weight) was washed with 0.85% sodium chloride aqueous solution, and the amount of 50 mM Tris- was 5 times the wet weight of the cells.
The bacterial cells were suspended in a hydrochloric acid buffer solution (pH 8.0), and the bacterial cells were disrupted by a grinding treatment (Bead-Beater, manufactured by Biospec Products, Inc.) for 4 minutes. The treatment solution thus obtained was centrifuged (20,000 × g, 30 minutes) to obtain an aminoacylase extract. At this time, the total activity of aminoacylase was 20,817 U and the specific activity was 1.5 U / mg.
Met. The protein was quantified according to the dye binding method.

This crude aminoacylase extract was diluted with ammonium sulfate 30
% And saturated with celite to remove the precipitate, the resulting supernatant is saturated with ammonium sulfate 80% and centrifuged (12,
The precipitate was recovered by (000 × g, 30 minutes). next,
This precipitate was added to 20 mM Tris-HCl buffer (pH 8.0).
, And dialyzed against the same buffer. This aminoacylase solution was passed through DEAE-Toyopearl 650M equilibrated with the same buffer solution to adsorb aminoacylase, and then sufficiently washed with the same buffer solution to remove non-adsorbed substances, and further 0 to 0. Aminoacylase was eluted by a linear concentration gradient elution method using a 5M aqueous sodium chloride solution. Further, this aminoacylase active fraction was concentrated, dialyzed against water, and freeze-dried. In this way, the total activity is 14,000 U and the specific activity is 79 U / m.
An enzyme preparation of aminoacylase of g was obtained.

Examples 2 to 5 To 1 ml of the amino acid solution shown in Table 1 and 100 mg of oleic acid as a fatty acid, 80 U of the aminoacylase obtained in Example 1 was added. 40 this mixed solution
750r using a magnetic stirrer at ℃
Stirred at pm.

After stirring for 20 hours, the remaining fatty acid was completely extracted with a mixed solution of n-hexane: isopropanol (3: 2 (v / v)), and the amount of the fatty acid decreased by the titration method using sodium hydroxide. I checked. The results are shown in Table 1.

The pH of the amino acid solution shown in Table 1
Is 100 mM Tris-HCl buffer (pH 8.5) at pH 8.5 and 100 mM phosphate buffer (pH 7.0) at pH 7.0.
Each prescribed pH using sodium hydroxide aqueous solution
Adjusted to.

[0045]

[Table 1]

Examples 6 to 7 Instead of 1 ml of the amino acid solution shown in Table 1 and 100 mg of oleic acid as the fatty acid, 1 ml of the amino acid solution shown in Table 2 and 100 m of capric acid as the fatty acid.
An experiment was conducted in the same manner as in Examples 2 to 5 except that g was used.

The results are shown in Table 2.

Examples 8 to 9 Instead of 1 ml of the amino acid solution shown in Table 1 and 100 mg of oleic acid as the fatty acid, 1 ml of the amino acid solution shown in Table 2 and 100 of myristic acid as the fatty acid were used.
The experiment was conducted in the same manner as in Examples 2 to 5 except that mg was used.

The results are shown in Table 2.

[0050]

[Table 2]

Example 10 5 ml of a 3M glycine solution adjusted with 100 mM Tris-hydrochloric acid buffer (pH 8.5) (final pH was adjusted to pH 8.5 with 4N aqueous sodium hydroxide solution) was added to olein. Add 500 mg of acid and 400 U of aminoacylase obtained in Example 1, and add at 40 ° C.
The reaction was carried out for 60 hours while stirring the reaction solution at 750 rpm using a magnetic stirrer. After completion of the reaction, the remaining oleic acid is removed by hexane extraction,
The reaction solution was acidified with hydrochloric acid, N-oleoylglycine was extracted with ethyl acetate, and then crystallized in ethyl acetate to give 170
Obtained mg of N-oleoylglycine.

Example 11 5 ml of a 3M glycine solution adjusted with 100 mM Tris-hydrochloric acid buffer (pH 8.5) (final pH was adjusted to pH 8.5 with 4N aqueous sodium hydroxide solution) was added to caprin. Add 500 mg of acid and 400 U of aminoacylase obtained in Example 1, and add at 40 ° C.
The reaction was carried out for 60 hours while stirring the reaction solution at 750 rpm using a magnetic stirrer. After the reaction is completed, the remaining capric acid is removed by hexane extraction,
The reaction solution was acidified with hydrochloric acid, N-caprinoylglycine was extracted with ethyl acetate, and crystallized in ethyl acetate.
Obtained 3.5 mg of N-caprinoylglycine.

Example 12 5 ml of a 2.9 M serine solution adjusted with 100 mM Tris-hydrochloric acid buffer (pH 8.5) (final pH was 4N).
The pH was adjusted to 8.5 using an aqueous solution of sodium hydroxide), 500 mg of oleic acid and 400 U of the aminoacylase obtained in Example 1 were added, and at 40 ° C.,
The reaction was carried out for 60 hours while stirring the reaction solution at 750 rpm using a magnetic stirrer. After completion of the reaction, the remaining oleic acid is removed by hexane extraction,
The reaction solution was acidified with hydrochloric acid, N-oleoylserine was extracted with ethyl acetate, and crystallized in ethyl acetate to give 196 m.
g of N-oleoyl serine was obtained.

Example 13 5 ml of a 2.9 M serine solution adjusted with 100 mM Tris-HCl buffer (pH 8.5) (final pH was 4N)
The pH was adjusted to 8.5 with an aqueous sodium hydroxide solution), 500 mg of capric acid and 400 U of the aminoacylase obtained in Example 1 were added, and at 40 ° C.,
The reaction was carried out for 60 hours while stirring the reaction solution at 750 rpm using a magnetic stirrer. After the reaction is completed, the remaining capric acid is removed by hexane extraction,
The reaction solution was acidified with hydrochloric acid, N-caprinoylserine was extracted with ethyl acetate, and crystallized in ethyl acetate.
0.5 mg of N-caprinoylserine was obtained.

Example 14 Myristin was added to 5 ml of a 3M glycine solution (final pH was adjusted to pH 8.5 using a 4N sodium hydroxide aqueous solution) adjusted with 100 mM Tris-hydrochloric acid buffer (pH 8.5). 500 mg of acid and 400 U of aminoacylase obtained in Example 1 were added, and the reaction was carried out at 40 ° C. for 60 hours while stirring the reaction solution at 750 rpm using a magnetic stirrer. After completion of the reaction, residual myristic acid was removed by extraction with hexane, the reaction solution was acidified with hydrochloric acid, N-myristoylglycine was extracted with ethyl acetate, and crystallized in ethyl acetate,
143.7 mg of N-myristoylglycine was obtained.

Example 15 5 ml of a 2.9 M serine solution adjusted with 100 mM Tris-HCl buffer (pH 8.5) (final pH was 4N)
The pH was adjusted to 8.5 with an aqueous sodium hydroxide solution), myristic acid (500 mg) and the aminoacylase (400 U) obtained in Example 1 were added, and the reaction solution was stirred at 40 ° C. with a magnetic stirrer at 750 rpm. While carrying out the reaction, the reaction was carried out for 60 hours. After completion of the reaction, residual myristic acid was removed by extraction with hexane, the reaction solution was acidified with hydrochloric acid, N-myristoylserine was extracted with ethyl acetate, and crystallized in ethyl acetate.
Obtained 65.8 mg of N-myristoylserine.

[0057]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, by using an aminoacylase produced by penicillium funiculosum, N-long chain acylamino acids, particularly N-long chain acylglycine, N-
It has become possible to synthesize long-chain acylserine etc. easily and in high yield. Furthermore, the present invention makes it possible to provide an N-long chain acylamino acid at a lower cost. From these, it can be expected that the application development of N-long chain acylamino acids will become more active.

Claims (3)

[Claims] 1. An N-long chain obtained by reacting a mixture of amino acids and long chain fatty acids with an aminoacylase produced by a microorganism belonging to the genus Penicillium or a microorganism belonging to the genus Penicillium capable of producing aminoacylase. A method for producing an acylamino acid. 2. The N- according to claim 1, wherein the aminoacylase is an aminoacylase having the following physicochemical properties.
Method for producing long-chain acylamino acid. 1) Action Acts on N-long chain acylamino acid to release L-amino acid. 2) Substrate specificity It acts on the L-form of N-acyl amino acid having an acyl group having 4 or more carbon atoms, and does not act on the D-form. It has a wide range of specificity for acyl groups and acts on N-acyl compounds composed of saturated fatty acids, unsaturated fatty acids, and aromatic carboxylic acids. 3) Optimum pH and pH stability When N-oleoyl-L-glutamic acid is used as a substrate, the optimum pH is 30 ° C and various buffer solutions (pH 5.5 to 6.
0: Acetate buffer, pH 6.0-8.0: Phosphate buffer,
pH 8.0-9.5: boric acid-hydrochloric acid buffer, pH 9.5
.About.11.0 in carbonate buffer). It is stable up to pH 6-9 in the same buffer solution at 30 ° C. for 5 hours. 4) Optimum temperature and thermal stability The optimum temperature when N-oleoyl-L-glutamic acid is used as a substrate is 25 to 35 ° C. when measured using a phosphate buffer (pH 7). It is not inactivated at all in 20 mM Tris-hydrochloric acid buffer (pH 8) at 40 ° C. for 15 minutes, and the residual activity after treatment at 45 ° C. for 15 minutes is about 80%. 5) Effects of inhibitors Parachloromercury benzoate or dithiothreitol inhibits the reaction. 6) Effect of metal ions Hg activated by Ni ²⁺ , Cu ²⁺ , Mg ^2+, etc.
^Inhibited by ²⁺ . 7) Molecular weight of subunit Molecular weight is about 4 by SDS-polyacrylamide gel electrophoresis.
8,000. 8) Molecular weight: about 192,000 by gel filtration method. 3. The microorganism belonging to the genus Penicillium is penicillium funiculosum.
funiculosum). The method for producing an N-long chain acylamino acid according to claim 1.