JPH05328972A - Novel aminoacylase and its production - Google Patents

Abstract

PURPOSE:To provide the novel aminoacylase excellent in its hydrolytic ability and synthetic ability, and to provide the method for producing the same. CONSTITUTION:Penicillium.funiculosum is cultured, and the new aminoacylase excellent in hydrolytic ability and in synthetic ability is separated and recovered from the culture product. The employment of the new aminoacylase permits the production of N-long chain acyl-amino acids, N-long chain acyl-peptides and N-long chain acyl-proteins at low costs.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel aminoacylase derived from a microorganism and a method for producing the same. More specifically, the present invention relates to Penicillium (Penicinium).
The present invention relates to a novel aminoacylase obtained by culturing a microorganism belonging to the genus llium) and a method for producing the same.

[0002]

2. Description of the Related Art Generally, aminoacylase is an enzyme that hydrolyzes N-acyl amino acids into organic acids and amino acids, and is widely present in animal tissues, plants and microorganisms. Further, since aminoacylase has a high optical specificity and acts only on L-amino acids, conventionally, N-acetyl-amino acids and other short-chain N-amino acids have been used for the purpose of optical resolution of amino acids.
Many reports have been made on aminoacylases that optically hydrolyze -acyl-amino acids.

In recent years, the safety and excellent biochemical degradability of N-long chain acyl-amino acids have been confirmed.
Since long-chain acyl-amino acids have a surface active action and a bactericidal action, they have been widely used in soaps, shampoos, detergents, emulsifiers, antibacterial agents, and various applications have been attracting attention. Accordingly, hydrolysis of N-long-chain acyl-amino acids using aminoacylase and its reverse reaction are expected to be applied to the synthesis of N-long-chain acyl-amino acids using aminoacylase.

However, Journal of Biochemistry, Vol. 91, 1731.
~ 1738 (1982) on aminoacylases that hydrolyze N-long chain acyl-amino acids, and JP-A-57-129,696 discloses N-long chain acyl-amino acids.
There are reports on aminoacylase, which is an amino acid synthase, but the number is very small, and N-long-chain acyl-
There is no known aminoacylase capable of efficiently hydrolyzing and synthesizing amino acids.

[0005]

Heretofore, there have been few reports on aminoacylases capable of synthesizing N-long chain acyl-amino acids, and JP-A-57-129,696 reports synthesis of glutamic acid and various fatty acids. But 3
The synthesis rate in the reaction for a day was as low as 5% or less, and no case was reported when other amino acids were used.

Therefore, an object of the present invention is to provide a novel aminoacylase excellent in hydrolyzing ability and synthesizing ability and a method for producing the same.

[0007]

Means for Solving the Problems The present inventors have eagerly studied for a microorganism which produces an aminoacylase excellent in hydrolyzing ability and synthetic ability. As a result, they have found that a microorganism belonging to the genus Penicillium produces an aminoacylase that specifically acts on a long-chain acyl group, which has not been found in filamentous fungi until now, and completed the present invention.

The aminoacylase of the present invention has the following physicochemical properties.

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

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 buffers (pH 5.5-6.
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 to 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 ions 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.

The present invention also relates to Penicillium (Penicilliu).
m) A method for producing aminoacylase, which comprises culturing an aminoacylase-producing bacterium belonging to the genus and having the above-mentioned properties, and separating and recovering the aminoacylase from the culture.

Further, the present invention provides a Penicillium (Penicinium)
Lactobacillus sp., which has the ability to produce the above aminoacylase, is a penicillium funicolosam.
niculosum) aminoacylase.

[0019]

The novel aminoacylase used in the present invention is produced by using a microorganism, belongs to the genus Penicillium as a producing bacterium, and has the ability to produce an enzyme having the above properties. Good.

This strain is called Micro Engineering Kenjoyori No. 3176 (FER
It has been deposited as MBP-3176) and its mycological properties are as follows.

(A) Growth state in various media (a) Tuapeck's medium Rapidly grows and has a diameter of 30 to 40 mm at 25 ° C. for 7 days.
Reach A colony consists 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 hyphae Methre: 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 elliptical 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.

As a result of identification of a bacterium having the above-mentioned mycological properties, this strain was identified as a strain belonging to the genus Penicillium. In addition, comparing this strain with strains of the same genus, Penicillium funiculosum
llium funiculosum).

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.

The novel aminoacylase-producing bacterium of the present invention can be cultured according to the information known in the field of fermentation. 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 hydrolyzate, sugars such as molasses, molasses, natural carbohydrates such as wheat and rice, alcohols such as glycerol, methanol and ethanol,
Considering the assimilability of microorganisms to be used from common carbon sources such as fatty acids such as acetic acid, gluconic acid, pyruvic acid and citric acid, hydrocarbons such as normal paraffin, amino acids such as glycine, glutamine and asparagine. Then, one kind or two or more kinds may be selected and used. As a nitrogen source,
Organic nitrogen compounds such as meat extract, peptone, yeast extract, soy hydrolyzate, milk casein, casamino acid, various amino acids, corn steep liquor, other animal, plant, microbial hydrolysates, ammonia, ammonium nitrate, ammonium sulfate, chloride One or more of ammonium salts such as ammonium, nitrates such as sodium nitrate, and inorganic nitrogen compounds such as urea are selected in consideration of the assimilation of the microorganism used.

In order to efficiently produce the present aminoacylase, N-long chain acyl-amino acids, Amisoft CS-11, LS-11 and GS-, are used as inducers.
11, HS-11 (Ajinomoto Co., Inc.) and similar substances can be used alone or in combination of two or more.

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 produced and accumulated in the culture as described above can be collected and fractionated by the following method.

Since the aminoacylase of the present invention is accumulated in the cells, the cells are collected by a method such as filtration or centrifugation after the culture is completed, and the cells are washed with a buffer solution or the like, for example, freeze-thaw treatment or ultrasonic wave treatment. Physical treatment such as treatment, pressure treatment, osmotic pressure difference treatment, grinding treatment, etc., or biochemical treatment such as cell wall lysing enzyme treatment such as lysozyme or chemical treatment such as contact treatment with a surfactant alone or By carrying out in combination, the cells can be crushed and aminoacylase can be extracted. The crude aminoacylase thus obtained is subjected to salting out, fractional precipitation with an organic solvent, ion exchange chromatography,
Purification using gel filtration chromatography, hydrophobic chromatography, dye chromatography, hydroxyapatite chromatography, affinity chromatography, etc., and high performance liquid chromatography (HPLC), etc., alone or in combination. You can

An example thereof is as follows. That is, the bacterial cells collected by filtration are bead beater (Bead-Bea
ter) (Biospec product (BIOSPEC PRODUCT
S)) to obtain an aminoacylase extract, and then salting out with ammonium sulfate, DEAE-Toyopearl 650M ion exchange chromatography, Toyopearl HW55F gel filtration chromatography, butyl (BUTHYL). ) -Toyopearl 650M hydrophobic chromatography and Toyopearl HW55F gel filtration chromatography can be used to perform a single purification by polyacrylamide gel electrophoresis.

The aminoacylase activity was measured by adding 200 μl of distilled water to 250 μl of 100 mM phosphate buffer (pH 7) containing 10 mM N-oleoyl-L-glutamic acid and incubating 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.

[0036]

EXAMPLES Next, the present invention will be explained with reference to examples.
It goes without saying that the scope of the present invention is not limited by these. In addition, the percentage in the following examples means "wt%" unless otherwise specified.

Example 1 Culture composition Peptone 2.0%, glucose 0.5%, N
H ₄ NO ₃ 0.3%, KH ₂ PO ₄ 0.4%, K ₂
HPO ₄ 0.1%, NaCl 0.1%, MgSO ₄
500 ml of 60 ml of 0.02% pH 6 medium
Place in a 1-volume Sakaguchi flask and inoculate with one platinum loop of penicillium funiculosum.
It was cultured at 30 ° C. for 48 hours to obtain a seed culture solution. 0.5% Amisoft GS-11 (Ajinomoto Co., Inc.) was added to the above culture composition to prepare 5 liters of a medium adjusted to pH 6.
Place in a liter Erlenmeyer flask and add seed culture 6
0 ml was added and the cells were cultured for about 120 hours. After a total of 9 liters of main culture was completed, about 570 g (wet weight) of the bacterial cells obtained by suction filtration were washed with 0.85% NaCl aqueous solution, and then 5 times the wet weight of the bacterial cells was added. The cells were suspended in 50 mM Tris-hydrochloric acid buffer (pH 8), and the cells were disrupted by a grinding treatment for 4 minutes (Bead-Beater, manufactured by Biopec Products). The obtained bacterial cell treatment liquid was centrifuged (20000).
(0 × g, 30 minutes) to obtain an aminoacylase extract. As a result, the total activity of the aminoacylase was 20,
The specific activity was 817 U and the specific activity was 1.5 U / mg. The protein was quantified according to the dye binding method.

Example 2 The crude aminoacylase extract obtained according to Example 1 was saturated with 30% ammonium sulfate and the precipitate was removed using Celite. The obtained supernatant was saturated with ammonium sulfate 80%, and the precipitate was recovered by centrifugation (12,000 × g, 30 minutes). The precipitate was dissolved in 20 mM Tris-HCl buffer (pH 8) and dialyzed against the same buffer. As a result, the total activity of the aminoacylase was 21,285 U and the specific activity was 10.
It was 7 U / mg.

This aminoacylase solution was passed through DEAE-Toyopearl 650M equilibrated with the same buffer solution to adsorb aminoacylase, and then thoroughly washed with the same buffer solution to remove non-adsorbed substances, and then 0 to 0. Aminoacylase was eluted by a linear gradient elution method with 5 M NaCl.
After concentrating this aminoacylase active fraction with an ultrafiltration membrane,
20 mM Tris-HCl buffer (pH 8) + 0.1 M Na
Gel filtration was performed using Toyopearl HW55F equilibrated with Cl. The obtained aminoacylase active fraction was saturated with 25% ammonium sulfate and equilibrated with 20 mM tris-hydrochloric acid buffer solution (pH 8) saturated with 25% ammonium sulfate (BUTHY).
L) -After passing through Toyopearl 650M to adsorb aminoacylase, it was thoroughly washed with the same buffer solution to remove non-adsorbed substances, and aminoacylase was eluted by a linear concentration gradient elution method of 25 to 0% ammonium sulfate saturation. did. The obtained aminoacylase active fraction was concentrated with an ultrafiltration membrane, and then gel filtration was performed by the same operation using the above Toyopearl HW55F. When the obtained aminoacylase active fractions were collected, the total activity was 3,497 U and the specific activity was 624.5 U / mg. Further, when this active fraction was subjected to polyacrylamide gel electrophoresis, the aminoacylase showed a single band, indicating that it was purified to a single polyacrylamide gel electrophoresis. The results of purification are shown in Table 1.

[0040]

[Table 1]

Example 3 Using the purified aminoacylase obtained according to Example 2, a 100 mM boric acid-hydrochloric acid buffer solution (pH 8.00) was used in place of the 100 mM phosphate buffer solution (pH 7) described above. 5) was used) and the decomposition activity was compared.

Tables 2 and 3 show examples of the results. Table 2,
The enzyme activity in Table 3 was expressed as a relative activity (%) with the decomposition activity for N-oleoyl-L-glutamic acid as 100.

[0043]

[Table 2]

[0044]

[Table 3]

Example 4 Using the purified aminoacylase obtained according to Example 2, a 100 mM tricine-hydrochloric acid buffer solution (pH 8.5) was used in place of the 100 mM phosphate buffer solution (pH 7) described above for the activity measurement method. ) Was used, and the reaction time was set to 30 minutes), and the decomposition activity was compared in the presence of various metal ions and an inhibitor.

Table 4 shows an example of the result. The enzyme activity in Table 4 was expressed as a relative activity (%) with the degradation activity without addition as 100.

[0047]

[Table 4]

Example 5 When subjected to SDS-polyacrylamide electrophoresis,
It showed a single band with a molecular weight of about 48,000. According to the gel filtration method using TSK gel G3000SW (moving layer: 0.2 M phosphate buffer (pH 7)), the molecular weight was 192,000. However, phosphorylase b (molecular weight 97,400), serum albumin (65,4)
00), ovalbumin (42,700), carbonic anhydrase (29,000), tryptophan inhibitor (2)
0,100), α-lactalbumin (14,400)
Was used as a standard substance for determining the molecular weight of aminoacylase by SDS-polyacrylamide gel electrophoresis, and glutamate dehydrogenase (290,000), lactate dehydrogenase (142,000), and enolase (67,00).
0), adenylate kinase (32,000), and cytochrome C (12,400) were used as standard substances for determining the molecular weight of aminoacylase by gel filtration.

Example 6 The purified aminoacylase obtained according to Example 2 was used to measure the optimum pH and pH stable range of the enzyme when N-oleoyl-L-glutamic acid was used as a substrate.

The results are shown in FIGS. 1 and 2. In FIG. 1, activity is measured at 30 ° C. in various buffer solutions (pH 5.5-5.5).
6.0: Acetate buffer, pH 6.0 to 8.0: Phosphate buffer, pH 8.0 to 9.5: Boric acid-hydrochloric acid buffer, pH
9.5 to 11.0: carbonate buffer). The activity was expressed as a relative activity (%) when the activity value at the maximum value was 100. In Figure 2, the enzyme solution is pH
Incubate in various buffers 3-11 and
The residual activity was measured after a lapse of time. The activity is the residual activity rate (%) when the activity value of the enzyme before incubation is 100.
Expressed as

Using the purified aminoacylase, the optimum temperature and thermostability of the enzyme when N-oleoyl-L-glutamic acid was used as a substrate were measured.

The results are shown in FIGS. 3 and 4. In FIG. 3, the activity measurement was performed at each temperature. The activity was expressed as a relative activity (%) when the activity value at the maximum value was 100. In FIG. 4, the enzyme solution (20 mM Tris-hydrochloric acid buffer solution (pH 8)) was treated for 15 minutes at each temperature shown on the horizontal axis, followed by cooling with ice and measuring the residual activity. The activity was represented by the residual activity rate (%) when the activity value of the untreated enzyme was 100.

Example 7 An example of the synthetic ability of the present aminoacylase will be shown. 0.1M Tris-HCl buffer (pH
8.5M) 3M glycine solution: 1 ml, oleic acid: 100 mg, enzyme solution: 20 μl (20 U) were added, and the mixture was placed under conditions of 40 ° C. and 120 rpm (reciprocal shaking).
Reacted for hours. After completion of the reaction, the reaction was stopped with hydrochloric acid, and N-oleoyl-glycine was extracted by the Horch method to obtain HPL.
C _{(Column: shim-pack FLC-NH 2} , mobile phase: hexane / 2-propanol / 10% phosphoric acid, 9
5: 5: 0.1 v / v / v, detection: 208 nm), the synthesis of about 30 mg of N-oleoyl-glycine was confirmed.

[0054]

As described above, the present invention is a novel aminoacylase having the physicochemical properties as described above and a method for producing aminoacylase using a microorganism of the genus Penicillium. Therefore, by using the aminoacylase, It enables low-cost production of N-long-chain acyl-amino acids, N-long-chain acyl-peptides, and N-long-chain acyl-proteins.

[Brief description of drawings]

FIG. 1 shows the optimum pH of aminoacylase used in the present invention.

FIG. 2 shows pH stability of aminoacylase used in the present invention.

FIG. 3 shows the optimum temperature of aminoacylase used in the present invention.

FIG. 4 shows the thermostability of aminoacylase used in the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Junji Terao 2-810-103 Azuma Tsukuba, Ibaraki

Claims (3)

[Claims] 1. An aminoacylase having the following physicochemical properties: 1) Action It acts on an N-long chain acyl-amino acid to liberate an 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 subunits Molecular weight of about 4 by SDS-polyacrylamide gel electrophoresis.
8,000. 8) Molecular weight: about 192,000 by gel filtration method. 2. A method for producing aminoacylase, which comprises culturing the aminoacylase-producing bacterium according to claim 1 belonging to the genus Penicillium, and separating and recovering the aminoacylase from the culture. 3. The strain having aminoacylase-producing ability according to claim 1, which belongs to the genus Penicillium, is a penicillium funiculo.
Sum), The method for producing an aminoacylase according to claim 2.