JPH06205668A - Bacteria producing acylamino acid racemase - Google Patents

Abstract

PURPOSE:To search microbes capable of advantageously producing acylamino acid racemase. CONSTITUTION:As a result of searching for various kinds of soil microbes capable of advantageously producing acylamino acid racemase, an acylamino acid racemase-productive bacterium, Amycolatopsis sp. TS-1-60 has been found. Using this bacteria, the acylamino acid racemase as a useful enzyme for producing optically active amino acids can be produced in an industrially advantageous way.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel microorganism used for the production of acylamino acid racemase by a fermentation method.

[0002]

Acyl amino acid racemase is a unique enzyme widely distributed in actinomycetes, which converts an optically active N-acyl amino acid into a corresponding optical antipode, which does not act on an optically active amino acid. Detailed properties have already been clarified (Japanese Patent Laid-Open No. 1-137973, Abstracts of the 1990 Conference of the Japanese Society of Agricultural Chemistry, 368 pages, 1
990).

[0003]

Such an acylamino acid racemase is a useful enzyme that can be used for the production of optically active amino acids. However, the development of a method for efficiently and inexpensively producing the enzyme has not been sufficient, and a more advantageous method has been desired.

[0004]

[Means for Solving the Problems] The present inventors have made repeated studies in view of such a current situation, and have conducted research on Amycolatopsis sp. TS-1-60 ( Amycolatopsis sp. TS-1).
-60, IFO 15079, FERM BP-309
2) found that the acylamino acid racemase was produced advantageously, and as a result of further intensive studies, the present invention was completed. That is, the present invention relates to a novel microorganism, Amycolatopsis sp. TS, which has the ability to produce an acylamino acid racemase.
-1-60 and a method for producing an acylamino acid racemase using the microorganism.

[0005] The above IFO number is the Institute for Fermentation (Osaka; I)
FO) and the FERM BP number are the microbial industrial technology research institute (FR) of the Ministry of International Trade and Industry.
The accession numbers in I) are shown, and the same applies hereinafter. Amicolatopsis spices TS-1-60
Is an acylamino acid racemase-producing bacterium isolated from the soil of Shiga Prefecture by the present inventors, and its mycological properties are as follows.

(A) Morphology 1) Liquid culture After inoculation of spores or mycelia of the present bacterium into a nutrient liquid medium such as trypticase soy broth and shaking culture at 28 ° C. for 1 to 2 days, the mycelia are finely broken and Morphology indicates polymorphism. 2) Plate agar culture Basal hyphae grow in a zigzag form in agar medium. Aerial mycelium generally exhibits a straight to slightly bent (RF) shape. In addition, a mycelial mass in which aerial hyphae are entangled with each other is observed (ISP-2 and ISP-7). Spores generally have a cylindrical shape, and their size is (0.3 to 0.5 × 0.6).
.About.2.3 .mu.m) and showed a diversity in length. Its surface is smooth.

(B) Cell composition Diaminopimelic acid as a cell wall component is meso type, and arabinose and galactose are detected as sugars, and the cell wall is type I
Belongs to V and A. Mycolic acid was not detected, menaquinone was 9 (H ₂ , H ₄ ), and phospholipid was type II (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylluinositol, phosphatidylluinositol mannoside, None: phosphatidylcholine).

(C) Growth state in various media The growth states of the TS-1-60 strain in various media are as shown in [Table 1]. The color symbols shown in parentheses are the colors of Container Corporation of America
The one described in Harmony Manual 4th edition was used.

[Table 1] Growth on various media (C) Physiological properties Growth temperature range: 13-3 on yeast / malt agar medium
7 ° C (optimum growth temperature 20-30 ° C) Gelatin liquefaction: Negative Starch hydrolysis: Negative Skim milk coagulation: Negative Skim milk peptone conversion: Negative Melanin-like pigment formation: Negative

(D) Carbon source assimilation Positive: glucose, xylose, fructose, mannitol, inositol, arabinose Negative: sucrose, raffinose, rhamnose Based on the above mycological properties, the TS-1-60 strain is amycolatopsis. Since the strain belongs to the genus, the present inventors
1-60 strains Amycolatopsis species TS-1
It was decided to call it -60. Amycolatopsis species TS-1-60 is cultured by a method known per se. When culturing actinomycetes, the medium is 3 as seed medium.
% Glycerol, 0.5% Polypeptone, 0.3%
Meat extract, 0.05% N-acetyl-DL-methionine, pH 7.2, as a production medium, 1.7% pancreatin-treated casein, 0.3% papain-treated defatted soybean flour, 0.5% sodium chloride, 0% .25% dipotassium hydrogen phosphate, 1% glucose, 0.05% N-acetyl-D
L-methionine, pH 7.0 is used. Culture is usually 15
To 40 ° C, preferably 24 to 30 ° C for 10 to 96 hours,
It is preferably carried out for 24 to 72 hours, and aeration and stirring can be added if necessary.

After completion of the culture, the cells and the supernatant are separated by a method known per se. Acyl amino acid racemase remaining in the cells can be isolated by conventional methods in the art, for example, ultrasonic disruption, disruption using French press, mechanical disruption such as grinding, disruption by cytolytic enzyme, etc. Crush and extract. The acylamino acid racemase contained in the thus obtained extract is subjected to usual protein purification methods such as salting out, isoelectric focusing, gel filtration, ion exchange chromatography, hydrophobic chromatography and high performance liquid chromatography. After purification, the target acylamino acid racemase can be obtained.
The acylamino acid racemase activity obtained above is measured by a known method (JP-A-1-137973). That is, measuring method A: a reaction solution containing an appropriate amount of enzyme (25 mM N-
Acetyl-D-methionine, 2 mM cobalt chloride, L
-Aminoacylase 4 U / ml, 5 mM Tris-hydrochloric acid buffer (pH 7.5) (500 µl) at 30 ° C, 5
After reacting for 1 minute, the reaction was stopped by heating at 100 ° C. for 3 minutes. The produced L-methionine was quantified by high performance liquid chromatography. The enzyme unit (U) was defined as 1 unit (U) in which 1 μmol of L-methionine was produced in 1 minute. Assay method B: The reaction solution of Assay method A containing no L-aminoacylase was reacted at 30 ° C. for 5 minutes, and 0.5 ml of hydrochloric acid (2N) was added to stop the reaction. Hydrolysis (120
After 60 ° C. for 60 minutes, the mixture was neutralized with sodium hydroxide (1N), and the L-methionine produced was quantified by high performance liquid chromatography.

[0012]

【Example】

Example 1 Purification of Acyl Amino Acid Racemase from Amycolatopsis sp. TS-1-60 Strain Amycolatopsis sp. TS-1-60 was formed by culturing yeast extract-malt extract agar slope medium (ISP2 medium) at 28 ° C for 7 days. One platinum loop of the spores was dispensed into a 200 ml Erlenmeyer flask in an amount of 20 ml. Sterilized seed medium (3% glycerol, 0.5% polypeptone, 0.3% meat extract, 0.3%).
05% N-acetyl-DL-methionine, pH 7.2)
On a rotary shaker (200 rpm) at 28 ° C. for 48 hours.
Incubated for hours. 8 ml of this culture broth was dispensed into a sterilized 15 ml vial and frozen and stored at -80 ° C to obtain a frozen-preserved bacterial suspension. The first seed was prepared by inoculating 1 ml of the cryopreserved bacterial solution into the above seed medium and culturing under the same conditions. The second seed was prepared by inoculating 20 ml of the first seed culture liquid into a 500 ml seed medium sterilized by dispensing into a 2 liter Sakaguchi flask and culturing on a reciprocal shaker (78 spm) at 28 ° C. for 72 hours. Enzymes are produced by using a production medium (1.7% pancreatin-treated casein, 0.3% papain-treated defatted soybean flour,
0.5% sodium chloride, 0.25% dipotassium hydrogen phosphate, 1
% Glucose, 0.05% N-acetyl-DL-methionine, pH 7.0) 100 liters were sterilized by pipetting into a 200 liter fermenter, and 4 liters of the second seed culture was transplanted into the broth and aerated (80 vvm). The cells were cultured at 28 ° C for 42 hours with stirring (160 rpm). 98 liters of the culture broth was collected using a continuous centrifuge (13000 × g, Sharpless) to obtain 1,880 g of wet microbial cells. This was suspended in 5 liters of 50 mM Tris-HCl buffer (pH 7.5),
Egg white lysozyme was added to a final concentration of 1 mg / ml. The suspension was kept at 30 ° C., lysed with gentle stirring for 2 to 3 hours, and a small amount of DNase I was added to decompose DNA leaking from the cells. After lysis, increase the liquid temperature to 5
The temperature was raised to 5 ° C. and heat treatment was performed for 30 minutes while stirring.
When this solution is heat-treated, about 60 to 70% of L and D-aminoacylase activity is inactivated, and the total protein amount is about 70%.
% Protein was insolubilized. After ammonium sulfate was added to the heat-treated solution so that the degree of saturation was 20%, the precipitate was removed using a centrifuge (18100 × g, 30 minutes) to obtain 4 liters of supernatant. The supernatant was preliminarily equilibrated with 50 mM Tris-hydrochloric acid buffer (pH 7.5) containing 20% saturated ammonium sulfate, and a butyl-Toyopearl 650M column (60 × 42) was used.
0 mm 1 liter) at a flow rate of 400 ml / h,
It was washed with 3 liters of the same buffer. Subsequently, the same buffer solution in which the degree of saturation of ammonium sulfate was decreased stepwise by 5% was flown in 1 liter increments for elution. When the acylamino acid racemase was changed from ammonium sulfate 15% saturation to 10%, D-aminoacylase was eluted with a slight delay. Acyl amino acid racemase fractions were collected and desalted, and then 50 mM Tris-HCl buffer (pH 7.
After being equilibrated in 5), it was adsorbed on a DEAE-Toyopearl 650M column (30 × 470 mm, 500 ml). After washing with 2 liters of the same buffer, the concentration of sodium chloride contained in the same buffer was linearly changed (0 to 0.5 M) for elution. This removed a small amount of mixed D- and L-aminoacylase. The active fractions are collected, and the ultrafiltration membrane (P
After desalting and concentrating using M10, Grace Japan, molecular sieve column (G3000SW, 21.5 x 600 mm)
Fully automatic preparative HPLC (HLC-8) equipped with Tosoh
37, Tosoh), and 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride was run at a rate of 4 ml / min for molecular sieve chromatography. The obtained active fraction was electrophoretically uniform, the degree of purification of the protein was about 340 times that of the lysozyme-treated solution, and the yield was about 28%. The above results are shown in [Table 2].

[0013]

[Table 2] ─────────────────────────────── Step Total protein Total activity Specific activity Purity Yield (mg) (units) ) (units / mg) (fold) (%) ─────────────────────────────── Lysozyme treatment 134000 1950 0.015 1.0 100 Heat treatment 39900 1550 0.039 2.6 80 BUTYL-Toyopearl 537 1140 2.12 141 59 DEAE-Toyopearl 194 781 4.03 268 40 G3000SW 107 542 5.07 338 28 ────────────────────── ──────────

Example 2 Properties of Acyl Amino Acid Racemase Derived from Amycolatopsis sp. TS-1-60 Strain Properties of the enzyme prepared by the method shown in Example 1 were investigated.
The enzyme activity was measured by either of the above measuring methods A and B. A) Substrate specificity It acted on the optically active N-acyl amino acid, but did not act on the corresponding optically active amino acid. Among the acyl groups, it showed the highest activity for a propionyl group having 3 carbon atoms. The activity was measured according to the measuring method B, and the activity for each substrate was 10 for N-acetyl-D-methionine.
It is shown in [Table 3] with a relative value of 0.

[Table 3] Ac: N-acetyl, CA: N-chloroacetyl, Fr: N-formyl, Pr: N-propionyl, Bu: N-butyryl

B) Optimum pH The optimum pH was 7.4 to 7.8. In measurement method A, instead of Tris-HCl buffer (pH 7.5), Bis-Tris-HCl buffer (pH 6.0, 6.4, 6.6, 6.
8, 7.0, 7.2), Tris-HCl buffer (7.0,
7.4, 7.6, 7.8, 8.0, 8.4, 9.0, 9.5
10.0) was used. The relative activity was determined from the amount ratio of L-methionine produced, and the result is shown in FIG. 1 C) Optimum temperature The optimum reaction temperature was 40 ° C to 50 ° C. After the reaction was carried out using the reaction solution from which L-aminoacylase was removed in the measuring method A, 1 U of L-aminoacylase was added and reacted at 30 ° C for 30 minutes. The relative activity was determined from the amount ratio of L-methionine produced, and the results are shown in FIG. D) Thermostability This enzyme showed 73% residual activity after treatment at 60 ° C for 30 minutes. The heat treatment was performed at each temperature for 30 minutes, and the activity was measured according to the measuring method A. The results are shown in [Fig. 3]. E) Molecular weight The molecular weight is measured by gel filtration chromatography and S
DS-polyacrylamide electrophoresis (SDS-PAG
E) method. Gel filtration chromatography is T
SK-gel G3000SW (7.5 x 600 mm,
Tosoh Co., Ltd. column, 0.5 ml of 50 mM phosphate buffer containing 0.2 M sodium chloride as a mobile phase
The flow rate was / min. The molecular weight was estimated to be about 300,000. SDS-PAGE is SDS-PAGE plate 10 /
20 (Daiichi Pure Chemicals Co., Ltd.) was used at a constant current of 60 mA. The molecular weight of the subunit was estimated to be about 40,000.

F) Effect of metal salt The enzyme activity is 1 mM of cobalt chloride or manganese sulfate.
When it was added, it was promoted 4 to 5 times as compared to when it was not added. When ferrous sulfate was added at 10 mM, the activity of this enzyme was promoted about 4-fold. The activity was measured according to the measuring method B, and the relative activity was determined from the amount ratio of L-methionine produced. [Table 4] shows the results of examining the effects of various metal salts on the enzyme activity.

[Table 4] Effect of metal salt on enzyme activity ────────────────────────── Metal salt concentration (mM) Relative activity (%) ─ ───────────────────────── None 100 MgSO ₄・ 7H ₂ O 1 139 MnSO ₄・ 4-6H ₂ O 1 386 FeSO ₄・ 7H ₂ O 1 104 10 382 CoCl ₂ · 6H ₂ O 1 496 NiCl ₂ · 6H ₂ O 1 157 CuSO ₄ · 5H ₂ O 1 150 10 0 ZnSO ₄ · 7H ₂ O 1 154 AlCl ₃ · 6H ₂ O 1 71 1100 PdCl ₂ 17 SnCl ₂・ 2H ₂ O 1 71 BaCl ₂・ 2H ₂ O 1 75 CaCl ₂・ 2H ₂ O 1 25 NaCl 1 104 KCl 1 82 82 ────────────────── ─────────

G) Effect of Inhibitor The SH inhibitor p-chloromercuribenzoic acid (PCMB) at 1 mM inhibited this enzyme activity by 94%. The activity was measured according to the measuring method B, and the relative activity was determined from the amount ratio of L-methionine produced. The results of examining the effects of various inhibitors on the enzyme activity are shown in [Table 5].

[Table 5] Effect of inhibitor on enzyme activity ────────────────────────────────── Inhibitor concentration ( mM) Relative activity (%) ────────────────────────────────── None 100 Hydrazine sulfate 172 Diphenylcarbazide 169 Hydroxylamine hydrochloride 186 N-ethylmaleimide 189 mercuric P-chlorobenzoate (PCMB) 16 phenylmethanesulfonyl fluoride (PMSF) 182 Nα-P-tosyl-L-lysine chloromethylketone hydrochloride 185 Ethylenediaminetetraacetic acid (EDTA) 1 90 5 3 ───────────────────────────────────

[0018]

INDUSTRIAL APPLICABILITY According to the present invention, an acylamino acid racemase, which is a useful enzyme that can be used for producing an optically active amino acid, can be industrially produced in good yield.

[Brief description of drawings]

FIG. 1 Relationship between enzyme activity and pH obtained in Example 2B

FIG. 2 Relationship between enzyme activity and reaction temperature obtained in Example 2C

FIG. 3 Relationship between enzyme stability and temperature obtained in Example 2D

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1:01)

Claims (2)

[Claims] 1. Amycolatopsis spices TS-1-60 having an ability to produce an acylamino acid racemase. 2. Amycolatopsis species TS-
A method for producing an acylamino acid racemase, which comprises culturing 1-60, accumulating the acylamino acid racemase in the culture solution in the culture, and collecting the same.