JPH1066566A - L-glutamic acid/l-pyroglutamic acid interconversion enzyme produced by streptomyces and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject enzyme useful for improving the taste of a food, isolating D- and L-glutamic acid and D- and L-pyroglutamic acid from a racemic compound, determining L-pyroglutamic acid, etc., capable of being secreted out of the cell of a specific strain belonging to the genus Streptomyces. SOLUTION: The subject enzyme capable of being secreted out of a cell by culturing Streptomyces SY-6 (FERM P-15,429) or Streptomyces SY-164 (FERM P-15,430), having the following properties. (1) Both L-pyroglutamic acid ring opening reaction and L-glutamic acid ring closure reaction have strict substrate specificity. (2) The optimum pH is pH about 7 in the reaction at 30 deg.C for 30 minutes (L-pyroglutamic acid ring opening reaction and L-glutamic acid ring closure reaction). (3) The optimum temperature is about 50 deg.C (L- pyroglutamic acid ring opening reaction and L-glutamic acid ring closure reaction). (4) Molecular weight is about 38,000 (SDS-PAGE).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a reaction for producing L-glutamic acid from L-pyroglutamic acid produced by Streptomyces (hereinafter sometimes referred to as L-pyroglutamic acid ring-opening reaction), And an enzyme that catalyzes the reverse reaction (sometimes referred to as L-glutamic acid ring closure reaction in this specification) and a method for producing the same.

[0002] The enzyme of the present invention can be used to produce L-glutamic acid from L-pyroglutamic acid (for example, to improve the taste of food by converting L-pyroglutamic acid in food into L-glutamic acid), and to produce L-glutamic acid from L-glutamic acid. L-
Production of pyroglutamic acid, isolation of D- and L-glutamic acid from racemic glutamic acid using these reactions, isolation of D- and L-pyroglutamic acid from racemic pyroglutamic acid, L-pyroglutamic acid in sample It is useful in the quantification of etc.

[0003]

2. Description of the Related Art In fermented foods such as miso and soy sauce, L
Glutamic acid and glutamine are very important amino acids in taste. However, it is a well-known fact that these amino acids are non-enzymatically cyclized and converted to tasteless pyroglutamic acid during the aging process of the fermented food. Hitherto, as a means for solving such a problem, the titer of koji mold glutaminase has been improved. However, this method efficiently converts glutamine produced during ripening into glutamic acid and suppresses the production of pyroglutamic acid, and has a drawback that it does not act on pyroglutamic acid once produced.

Further, as a pyroglutamic acid ring-opening enzyme known to date, 5-oxoprolinase (EC 3.
5.2.9, A. Meister et al. Proc. Natl. Acad. Sci. U.
SA, 68 , 2982 (1971)), but these enzymes all depend on ATP, magnesium and potassium ions, and it is difficult to improve the taste of foods due to problems such as ATP regeneration. I can say.

On the other hand, in fields other than food umami,
Pyroglutamic acid has very low toxicity and is used in various fields. For example, it has already been put into practical use as a humectant for cosmetics, and is also used as a raw material for synthesizing various useful substances. It also improves learning in the medical field (F. Drago et al., Acta Ther., 13 , 587 (1987)),
Prevent loss of memory due to alcohol (E. Sinforian
i et al., Minerva Psichiatr., 26 , 339 (1985)), protects the liver from ethanol (H. Geoffroy, Parfumeri
e und Kosmetik, 72 , 94 (1991)) and more than 50 other reports.

[0006] Pyroglutamic acid can also be used as a useful cation carrier (H. Geoffroy, P.
arfumerie und Kosmetik, 72 , 94 (1991), P. Binet et
al., Therapie, 33 , 491 (1978)). Conventionally, a method for producing pyroglutamic acid has been to prepare L-glutamic acid under acidic conditions by one step.
This is performed by heating and dehydrating to 75 ° C. However, this manufacturing method involves a very high temperature of 175 ° C. and a high temperature and a high pressure, and is also dangerous under acidic conditions.
A special reaction tank corresponding to that is required. In addition, since the reaction product is partially racemized, it is difficult to obtain a pure L form.

[0007] Pyroglutamic acid-producing enzymes have been known to date with D-glutamate cyclase (A. Meister et al.
l., Biochem. Z, 338 , 217 (1963)), glutamine synthetase (A. Meister et al., J. Biol. Chem., 237 ,
2932 (1962)), γ-glutamyl cyclotransferase (A. Meister et al., J. Biol. Chem., 253 , 1799).
(1978)), etc., but for industrial production of L-pyroglutamic acid as described above, D-glutamate cyclase has its substrate specificity, glutamine synthetase has its ATP dependence, and γ-glutamyl cyclo Transferase has problems such as an expensive substrate. on the other hand,
D-glutamic acid and D-pyroglutamic acid are important starting materials in the production of drugs, food additives and pesticides. However, since the production process requires organic synthesis and resolution of the racemic form, compared to the L-form and the racemic form,
Expensive.

[0008]

[Related Art] To solve the above-mentioned problems, the present inventors
As a result of intensive search for the L-glutamic acid / L-pyroglutamic acid interconverting enzyme, it was found that bacteria belonging to the genera Alcaligenes and Pseudomonas produce and accumulate the enzyme in the cells (Japanese Patent Application No. 7-155988). However, such strains produce and accumulate enzymes in the cells, and thus have various limitations.

[0009]

SUMMARY OF THE INVENTION Accordingly, the present invention provides a reaction for hydrolyzing L-pyroglutamic acid, which is secreted extracellularly, to open the ring to L-glutamic acid, and for closing the ring by dehydration-condensing L-glutamic acid. To provide a novel enzyme that catalyzes a reaction (reversible reaction) for producing L-pyroglutamic acid by the reaction and a method for producing the same.

[0010]

In order to solve the above-mentioned problems, the present invention provides the following properties: (1) action: reaction for producing L-glutamic acid from L-pyroglutamic acid (L-pyroglutamic acid ring-opening reaction) ),
And catalyzes the reverse reaction (L-glutamic acid ring closure reaction); (2) Substrate specificity: L-pyroglutamic acid ring opening reaction: L
Acting on pyroglutamic acid, D-pyroglutamic acid,
Does not act on proline and hydroxyproline; L-
Glutamic acid ring closure reaction: acts on L-glutamic acid,
Does not act on glutamic acid, L-glutamine, L-aspartic acid, D-aspartic acid, L-asparagine;

(3) Optimum pH and stable pH range: L-pyroglutamic acid ring-opening reaction: The optimum pH is about 7 in a reaction at 30 ° C. for 30 minutes, and the pH stability is 120 at 30 ° C.
Is stable within the range of about 7 to 10 under the processing conditions for one minute;
L-glutamic acid ring closure reaction: The optimum pH is about 7 in a reaction at 30 ° C. for 30 minutes, and the pH stability is 1 at 30 ° C.
It is stable within the range of 7 to 10 under the processing conditions of 20 minutes;

(4) Temperature stability: L-pyroglutamic acid ring-opening reaction: stable up to 50 ° C. under a treatment condition of pH 7 for 30 minutes; L-glutamic acid ring-closing reaction;
(5) Optimal temperature: L-pyroglutamic acid ring-opening reaction: about 5
L-glutamic acid ring closure reaction: having an optimal action temperature of about 50 ° C .; (6) molecular weight: having a molecular weight of about 38,000 as measured by SDS-PAGE. Glutamic acid
An L-pyroglutamic acid interconverting enzyme is provided.

The present invention also relates to the aforementioned L-glutamic acid · L
-In a method for producing pyroglutamic acid interconverting enzyme,
There is provided a method characterized by culturing a microorganism belonging to the genus Streptomyces having the ability to produce the enzyme, and collecting the enzyme from the culture. The present invention further comprises converting the L-glutamic acid to L-pyroglutamic acid by allowing the enzyme to act on a mixture of L-glutamic acid and D-glutamic acid, and converting the L-pyroglutamic acid and the D-glutamic acid. Provided is a method for producing D-glutamic acid or L-pyroglutamic acid, wherein the method is separated from each other.

The present invention further comprises converting the L-pyroglutamic acid into L-glutamic acid by reacting the enzyme with a mixture of L-pyroglutamic acid and D-pyroglutamic acid. Disclosed is a method for producing D-pyroglutamic acid, which comprises separating residual D-pyroglutamic acid from each other. The present invention also provides a method for producing L-pyroglutamic acid, which comprises allowing the enzyme to act on L-glutamic acid.

The enzyme of the present invention is an L-glutamic acid / L-pyroglutamic acid interconverting enzyme having the following properties. (1) action: acts on L-pyroglutamic acid to generate L-glutamic acid (L-pyroglutamic acid ring-opening reaction) and catalyzes the reverse reaction (L-glutamic acid ring-closing reaction); (2) substrate specificity : L-pyroglutamic acid ring-opening reaction: L
Acting on pyroglutamic acid, D-pyroglutamic acid,
Does not act on proline and hydroxyproline; L-
Glutamic acid ring closure reaction: acts on L-glutamic acid,
Does not act on glutamic acid, L-glutamine, L-aspartic acid, D-aspartic acid, L-asparagine;

(3) Optimum pH and stable pH range: L-pyroglutamic acid ring-opening reaction: The optimum pH is about 7 in the reaction at 30 ° C. for 30 minutes, and the treatment at 30 ° C. for 120 minutes. L-glutamic acid ring-closure reaction: the optimum pH is about 7 in a reaction at 30 ° C. for 30 minutes, and 7-about under a treatment condition of 30 ° C. for 120 minutes. (4) Temperature stability: L-pyroglutamic acid ring-opening reaction: pH
L-glutamic acid ring closure reaction at 30 ° C. for 30 minutes; stable to 50 ° C. under pH 7 for 30 minutes;

(5) Optimum temperature: L-pyroglutamic acid ring-opening reaction: having an optimum working temperature of about 50 ° C .; L-glutamic acid ring-closing reaction: having an optimum working temperature of about 50 ° C .; (6) molecular weight : Having a molecular weight of about 38,000 as measured by SDS-PAGE; (7) isoelectric point: 7 to 8; (8) independent of ATP, Mg ²⁺ , K ⁺ ; (9) ) It is an extracellular secretory enzyme.

The enzyme of the present invention belongs to the genus Streptomyces, and any enzyme capable of producing the aforementioned enzyme can be used. Examples of preferred strains used in the present invention include Streptomyces SY-6 and SY-164 newly isolated from soil by the present inventors. This microorganism was supplied to FERM P-15429 and FERM P-154 by the Institute of Biotechnology and Industrial Technology, respectively.
Deposit 30. Streptomyces SY-6
And Streptomyces SY-164 have the following properties.

[0019] Spores micro morphological SY-6 produces a branched whorled branched short straight chain on aerial mycelium (up to 10 spores per chain). The spores have a smooth surface. No SY-164 spores are formed. Cell wall hydrolyzates of cell wall compositions SY-6 and SY-164 cultures both contain LL-diaminopimelic acid (DAP). Both menaquinone components SY-6 and SY-164 have MK-9 (H6) and MK-9 (H8) as menaquinone components.

[0020] The amount SY-6 in good growth of growth, glucose asparagine agar, glycerin-asparagine agar, starch agar, tyrosine agar, nutrient agar, yeast-malt agar, and on oatmeal agar And moderate growth is observed on sucrose / nitrate agar. In SY-164, good growth was observed on glucose-asparagine agar, starch agar, tyrosine agar, yeast malt agar, and oatmeal agar, and moderate growth was observed on glycerin-asparagine agar. Insufficient growth is observed on sucrose-nitrate agar and nutrient agar.

The vegetative mycelium SY-6 has a vegetative mycelium prominent on a medium in which good growth occurs, and has a yellowish-gray color tone. In addition, SY-164 has a vegetative hypha on a medium where good growth occurs, and has a brown color tone. In the color of the aerial mycelium and the spores, SY-6, the color of the aerial mycelium and / or the sporule is white to light gray, and the aerial mycelium of SY-164 is white.

The soluble dye SY-6 produces a pale yellow pigment on glucose-asparagine agar, starch agar and oatmeal agar, and a pale orange pigment on glycerin-asparagine agar. SY-164 produces a brown pigment on glycerin / asparagine agar, tyrosine agar and yeast / malt agar, a pale green pigment on star agar, and a greenish brown pigment on oatmeal agar.

Physiological reaction growth temperature range: SY-6 grows between 10 ° C and 40 ° C and shows good growth between 18 ° C and 33 ° C. ; S
Y-164 grows between 10 ° C and 45 ° C,
Shows good growth between 30 and 34 ° C; SY-6 and SY
No liquefaction of gelatin for both SY-164: Strong hydrolysis of starch in SY-6, weak hydrolysis in SY-164: Peptone yeast iron agar medium (I
SP medium No. 6) and tyrosine agar medium (ISP medium N
o.7) On the above, there is melanin-like pigment production and SY-16
In No. 4, peptone yeast / iron agar medium (ISP medium N
o.6) and tyrosine agar medium (ISP medium No. 7) produce melanin-like pigment; assimilation of each carbon source: SY-
6, good utilization of glucose; moderate utilization of inositol; insufficient utilization of D-fructose; no utilization of L-arabinose, D-xylose, sucrose, L-rhamnose, raffinose, D-mannitol . S
For Y-164, moderate utilization of glucose; no utilization of L-arabinose, D-xylose, D-fructose, sucrose, inositol, L-rhamnose, raffinose, D-mannitol. Culture properties in various media

[0024]

[Table 1]

[0025]

[Table 2]

The taxonomic properties of SY-6 and SY-164 “BERGEY'S MANUAL OF Systematic Bacteriology Vol. 4
(1989) "and" Experimental method for identification of actinomycetes ", edited by the Japanese Society of Actinomycetes (1988)," Key for Classificati "by H. Nonomura
on and Identification of 458 Species of the Strept
omycetes Included in ISP ”, J.Ferment.Technol., Vo
l.52, No.2, p.78-92,1974, SY-6 and SY
The observation of the culture, physiology and morphological characteristics of -164 identified the following. In SY-6, a ring-like spore chain was observed, and it was classified into the genus Streptoverticillium based on the stereoisomeric form of DAP and the molecular species of menaquinone. However, the International Journal of S
In ystematic Bacteriology, since Streptoverticillium genus integrated into the genus Streptomyces, Streptom the SY-6 strain
yces . No sporulation was observed with SY-164, but the stereoisomeric form of DAP and the molecular species of menaquinone identified SY-164 as a genus of Streptomyces . SY
-6 and SY-164 strains have the characteristics shown above,
Identified as Streptomyces.

When the microorganism of the present invention is cultured, the medium may be a carbon source such as sugars such as sucrose, glucose, fructose, inositol and soluble starch, acetic acid,
Organic acids such as citric acid and malic acid, and hydrocarbons can be used. As the nitrogen source, organic and inorganic nitrogen sources such as meat extract, peptone, pyroglutamic acid and ammonium sulfate, and ammonia are used. Furthermore, phosphates such as potassium monophosphate and potassium phosphate, magnesium salts such as magnesium sulfate and magnesium chloride, metal ions such as iron, manganese, zinc and calcium, and trace amounts required for the growth of each strain A substance to which an appropriate amount of a substance or the like is added is used.

Cultivation is carried out under conditions such as shaking culture or aeration and stirring culture. The culture temperature is 20 to 40 ° C, preferably 25 to 37 ° C, pH 5.0 to 8.0, and preferably pH 7.0.
Perform at 0-8.0. The culture time is usually 13 to 72 hours. L-glutamic acid / L-pyroglutamic acid interconverting enzyme is produced in the culture supernatant and cells cultured by the above method. The enzyme is purified by ammonium sulfate salting-out, ion exchange chromatography, hydrophobic chromatography, gel filtration, or a combination thereof for separation and collection.

The method for measuring the activity of L-glutamic acid ring closure reaction and the method for indicating the activity are as follows. That is, 0.5 ml of 0.12 ML-glutamic acid, 0.45 ml of 0.4 M Tris-HCl buffer (pH 7.0), 50 μl of enzyme solution
And left at 30 ° C. for 30 minutes. Then 100
At 10 ° C. for 10 minutes to deactivate the enzyme.
H using m-pack SCR-101H (manufactured by Shimadzu)
The amount of pyroglutamic acid generated by PLC is determined.
The unit of the enzyme activity is 1 μmo per minute under the conditions described above.
l of the enzyme required to produce pyroglutamic acid
Display as a unit. The method for measuring the activity of the pyroglutamic acid ring-opening reaction and the method for indicating the activity are as follows.

That is, the enzyme solution 1 was placed on a titer plate.
0 µl, 0.1 µM Tris-HCl buffer (pH 7.0) 40 µ
1 and 50 μl of 1% pyroglutamic acid (pH 7.0) are mixed and left at 30 ° C. for 30 minutes. 7.5 mM N in the reaction solution
AD, 1.2 mM INT, 1.5 U diaphorase,
100 μl of 0.2 M triethanolamine buffer (pH 8.6) containing 1% Triton X-100 and 6 U of glutamate dehydrogenase are added, and the mixture is left at room temperature for 15 minutes. afterwards,
Glutamic acid produced is quantified by measuring the absorbance at 492 nm. The unit of enzyme activity is expressed as the amount of enzyme required to produce 1 μmol of glutamic acid per minute under the above conditions.

The enzyme of the present invention is obtained by converting L-glutamic acid into L-glutamic acid.
-Can be used to produce pyroglutamic acid. Accordingly, the present invention provides a method for producing or producing L-pyroglutamic acid, which comprises causing the enzyme of the present invention to act on L-glutamic acid.

The enzyme of the present invention is a mixture of L-pyroglutamic acid and D-pyroglutamic acid (for example, a racemic form).
Can be used to resolve D-pyroglutamic acid. Therefore, the present invention provides a method for producing or producing D-pyroglutamic acid, which comprises allowing the enzyme of the present invention to act on a mixture of L-pyroglutamic acid and D-pyroglutamic acid (for example, racemic form). For example, L-pyroglutamic acid in a mixture (for example, racemic) of L-pyroglutamic acid and D-pyroglutamic acid is converted to L-glutamic acid, and the resulting D-pyroglutamic acid and L-glutamic acid are separated from each other according to a conventional method. , D-pyroglutamic acid can be obtained.

The enzyme of the present invention can also be used for resolving a mixture of L-glutamic acid and D-glutamic acid (for example, a racemate). For example, by allowing the enzyme of the present invention to act on a mixture of L-glutamic acid and D-glutamic acid, only L-glutamic acid is specifically converted to L-pyroglutamic acid, and D-glutamic acid and L-glutamic acid are converted.
-D-glutamic acid can be easily separated from pyroglutamic acid. A mixture (for example, racemic) of L-glutamic acid and D-glutamic acid as raw materials can be produced by various methods such as a synthesis method and an enzymatic method, and these are not limited.

[0034]

Next, the present invention will be described more specifically with reference to examples. Embodiment 1 FIG . Streptomyces SY-6 was used as the inoculum. Meat extract 10g / l, polypeptone 10g / l,
8 ml of a medium containing 10 g / l of glucose, 3 g / l of NaCl, and 0.15 g / l of adecanol (pH 7.0) was placed in a test tube having an inner diameter of 22 mm, and sterilized at 120 ° C. for 15 minutes. One platinum loop of the strain was inoculated into the above medium,
The cells were cultured with shaking for 8 hours. After completion of the culture, 0.5 ml of the culture solution was inoculated into three 500 ml Sakaguchi flasks containing 100 ml of the above medium, and cultured with shaking at 30 ° C. for 48 hours. After cultivation,
All of the cultures were inoculated into 30 l of the main culture in a 50 l jar fermenter. Main culture is 30 ℃, 300rpm
Per minute, aeration rate 1 liter / medium 1 liter for 1 minute for 30 hours.

In order to collect the enzyme from 30 l of the culture termination liquid, the liquid was centrifuged (15,000 rpm, Sharpless A).
S-16 (Tomo Kogyo) at 20 ° C.) to obtain 38 l of a culture supernatant. Add ammonium sulfate to this culture supernatant and add 80%
A saturated ammonium sulfate precipitation fraction was obtained. This precipitate was dissolved in 3 l of a 0.1 M Tris-HCl buffer (pH 7.0). Ammonium sulfate was added to the resulting solution to 25% saturation, and 2
Butyl Toyopearl 6 equilibrated with 0.05 M Tris-HCl buffer (pH 7.0) containing 5% saturated ammonium sulfate
Adsorb to 50C, wash with the same buffer, and 0.05M Tris-HCl buffer containing 20% saturated ammonium sulfate (pH 7.0).
Eluted at 0). The active fraction was dialyzed against 5 mM Tris-HCl buffer (pH 8.0). 0.02M of dialysis solution
Adsorbed on DEAE Toyopearl 650M equilibrated with Tris-HCl buffer (pH 8.0), washed with the same buffer, and 0 →
Elution was with a 0.15 M NaCl gradient. The active fraction was dialyzed against 5 mM Tris-HCl buffer (pH 7.0).

Ammonium sulfate was added to the inner solution of the dialysis so as to be 30% saturated, and adsorbed on 650 M of butyl toyopearl equilibrated with a 0.05 M Tris-HCl buffer (pH 7.0) containing 30% saturated ammonium sulfate. Wash with the same buffer, and then add 0.0
Elution was performed with 5M Tris-HCl buffer (pH 7.0). After concentration of the active fraction, gel filtration was performed with Toyopearl HW-50F. By the above operation, a single enzyme preparation (16 mg) was obtained by SDS polyacrylamide gel electrophoresis (gel concentration: 12%, pH: 8.8). The activity yield is 44.9
%, And the specific activity of the L-pyroglutamic acid ring-opening reaction was 2,240 U / mg. Further, the specific activity of the L-glutamic acid ring closure reaction was 42,600 U / mg.

Embodiment 2 FIG. Streptomyces S as inoculum
Y-164 was used. Meat extract 10g / l, polypeptone 10g / l, glucose 10g / l, NaCl 3g
Of a medium containing 0.15 g / l and 0.15 g / l of decanol (pH 7.0) was placed in a test tube having an inner diameter of 22 mm, and the temperature was adjusted to 120 ° C.
For 15 minutes. One platinum loop of the strain was inoculated into the above medium, and cultured with shaking at 30 ° C. for 48 hours. After completion of the culture, 0.5 ml of the culture solution was inoculated into three 500 ml Sakaguchi flasks containing 100 ml of the above medium, and cultured with shaking at 30 ° C. for 48 hours. After completion of the culture, the whole culture was inoculated into 30 l of the main culture in a 50 l jar fermenter. Main culture is 3
The operation was carried out at 0 ° C., 300 rpm / min, aeration rate 1 l / culture medium 1 l / min for 30 hours.

In order to collect the enzyme from 30 l of the culture termination liquid, the liquid was centrifuged (15,000 rpm, Sharpless A).
S-16 (Tomo Kogyo) at 20 ° C.) to obtain 38 l of a culture supernatant. Add ammonium sulfate to this culture supernatant and add 80%
A saturated ammonium sulfate precipitation fraction was obtained. This precipitate was dissolved in 3 l of a 0.1 M Tris-HCl buffer (pH 7.0). Ammonium sulfate was added to the resulting solution to 25% saturation, and 2
Butyl Toyopearl 6 equilibrated with 0.05 M Tris-HCl buffer (pH 7.0) containing 5% saturated ammonium sulfate
Adsorb to 50C, wash with the same buffer, and 0.05M Tris-HCl buffer containing 20% saturated ammonium sulfate (pH 7.0).
Eluted at 0).

Ammonium sulfate was added to the active fraction to a concentration of 30%, adsorbed again to butyl Toyopearl 650C, washed with the same buffer, and 0.05M Tris-HCl buffer (pH 7) containing 30 → 15% saturated ammonium sulfate. .0). The active fraction was treated with 5 mM Tris-HCl buffer (pH 9.
Dialyzed against 0). Q-Sepharo equilibrated in dialysis solution with 0.05 M Tris-HCl buffer (pH 9.0)
The cells were adsorbed on se Fast Flow, washed with the same buffer, and eluted with a 0 → 0.5M NaCl gradient.

After concentrating the active fraction, Toyopearl HW-50
Gel filtration was performed by F. After concentrating the active fraction,
Gel filtration was performed using TSKgel G2000SW. By the above operation, 5.2 mg of a single enzyme preparation was obtained by SDS polyacrylamide gel electrophoresis (gel concentration: 12%, pH: 8.8). The activity yield was 19.9%, and the specific activity of the L-pyroglutamic acid ring-opening reaction was 2,2.
It was 10 U / mg. The specific activity of the L-glutamic acid ring closure reaction was 42,000 U / mg.

Embodiment 3 FIG. Streptomyces SY-6 is 0.3% meat extract, 1% peptone and 0.1% sodium chloride.
Inoculate 8 ml of medium (pH 7.0) containing 5%
After culturing with shaking for 0 hour, the culture solution was centrifuged after completion of the culture,
The cells were separated into 7 ml of culture supernatant and cells. The obtained cells were sonicated to obtain 5 ml of a cell-free extract. The titer of the L-pyroglutamic acid ring-opening reaction of the culture supernatant was 3.3 U / ml, and the L-glutamic acid ring-closing reaction was 62.7 U / ml. The titer of the L-pyroglutamic acid ring-opening reaction of the cell-free extract was 0.0
At 54 U / ml, L-glutamic acid ring-closure reaction is 1.03 U
/ Ml.

Embodiment 4 FIG. Streptomyces SY-164
Was inoculated into 8 ml of a medium (pH 7.0) containing 0.3% of meat extract, 1% of peptone and 0.5% of sodium chloride.
After culturing with shaking for 60 hours, the culture solution was centrifuged after completion of the culture to separate 7 ml of the culture supernatant and cells. The obtained cells were sonicated to obtain 5 ml of a cell-free extract. L- of culture supernatant
The titer of the pyroglutamic acid ring-opening reaction is 0.43 U / ml,
The L-glutamic acid ring closure reaction was 8.17 U / ml.
The titer of L-pyroglutamic acid ring-opening reaction of the cell-free extract is 0.037 U / ml, and the titer of L-glutamic acid ring-closing reaction is 0.17 U / ml.
It was 703 U / ml.

Embodiment 5 FIG. 305 nmol of L-glutamic acid,
10 ml of 20 mM Tris-HCl buffer (pH 7.0) containing 63 ng of the enzyme obtained in Example 1 was mixed and left at 30 ° C. to produce pyroglutamic acid. As a result, 295 nmol of pyroglutamic acid (96.7% yield) was produced.

Embodiment 6 FIG. After dissolving 5.0 g of D, L-pyroglutamic acid (Tokyo Kasei) in 1N NaOH, the solution was dissolved in 0.1N NaOH.
Add 1N NaOH to 5N and add 10N with distilled water.
Make up to 0 ml. The solution was autoclaved at 121 ° C. for 20 minutes to perform hydrolysis. After completion of the hydrolysis, 3N HCl
The pH was adjusted to 7.1 with and adjusted to 200 ml with distilled water. The solution contained 2.6 g of D-glutamic acid and 2.3 g of L-glutamic acid. 73 μg (161 U) of the enzyme obtained in Example 2 was added to the solution, and the mixture was allowed to act at 40 ° C. for 6 hours. After the completion of the reaction, heat treatment was performed at 100 ° C. for 5 minutes to inactivate the enzyme.

The reaction solution contained 2.6 g of D-glutamic acid and 88 mg of L-glutamic acid (see FIG. 1). The reaction solution was concentrated under reduced pressure to 40 ml with an evaporator, the pH was adjusted to 3.2 with 3N HCl, and the mixture was allowed to stand at 5 ° C. overnight. The generated white crystals are collected by filter paper with No.2,
After washing with cold ethanol, it was dried. White crystals are 1.
41 g were obtained. The melting point (decomposition) of the obtained crystals of D-glutamic acid is 214-216 ° C., [α] ²² _D = −2.
4.4 °, containing 0.6% L-glutamic acid.

[Brief description of the drawings]

FIG. 1 is a graph showing the time-dependent changes in the total amount of D, L-glutamic acid and the amount of L-glutamic acid when the enzyme of the present invention is allowed to act on a racemate of glutamic acid.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication (C12N 9/14 C12R 1: 465) (C12P 13/14 C12R 1: 465) (C12P 41/00 (C12N 1:20) (C12N 1/20 C12R 1: 465) (72) Inventor Sawao Murao 2-8-12 Horigami Midoricho, Sakai-shi, Osaka

Claims (5)

[Claims] 1. The following properties: (1) Action: reaction for producing L-glutamic acid from L-pyroglutamic acid (L-pyroglutamic acid ring-opening reaction);
And catalyzes the reverse reaction (L-glutamic acid ring closure reaction); (2) Substrate specificity: L-pyroglutamic acid ring opening reaction: L
Acting on pyroglutamic acid, D-pyroglutamic acid,
Does not act on proline and hydroxyproline; L-
Glutamic acid ring closure reaction: acts on L-glutamic acid,
-Does not act on glutamic acid, L-glutamine, L-aspartic acid, D-aspartic acid, L-asparagine; (3) Optimal pH and stable pH range: L-pyroglutamic acid ring-opening reaction: optimal pH is 30 ° C Is about 7 in the reaction for 30 minutes at 30 ° C., and the pH stability is stable within the range of about 7 to 10 under the treatment conditions at 30 ° C. for 120 minutes; L-glutamic acid ring closure reaction: the optimal pH is 30. About 7 in a reaction at 30 ° C. for 30 minutes, and the pH stability is stable within a range of 7 to 10 under a treatment condition at 30 ° C. for 120 minutes; (4) Temperature stability: L-pyroglutamic acid cleavage Ring reaction: pH
L-glutamic acid ring-closure reaction at 30 ° C. for 30 minutes; L-glutamic acid ring-closing reaction; stable to 50 ° C. at 30 minutes at pH 7; (5) Optimal temperature: L-pyro Glutamic acid ring opening reaction: about 5
L-glutamic acid ring closure reaction: having an optimal action temperature of about 50 ° C .; (6) molecular weight: having a molecular weight of about 38,000 as measured by SDS-PAGE. Glutamic acid
L-pyroglutamic acid interconverting enzyme. 2. L-glutamic acid • L according to claim 1,
-In a method for producing pyroglutamic acid interconverting enzyme,
A method comprising culturing a microorganism belonging to the genus Streptomyces having the ability to produce the enzyme, and collecting the enzyme from the culture. 3. The method according to claim 1, wherein the enzyme is reacted with a mixture of L-glutamic acid and D-glutamic acid to convert the L-glutamic acid into L-pyroglutamic acid, and the L-glutamic acid and the D-glutamic acid are converted to L-pyroglutamic acid. -A method for producing D-glutamic acid or L-pyroglutamic acid, comprising separating glutamic acid and glutamic acid from each other. 4. The method according to claim 1, wherein the enzyme is reacted with a mixture of L-pyroglutamic acid and D-pyroglutamic acid to convert the L-pyroglutamic acid to L-glutamic acid, and the resulting L-glutamic acid is produced. And separating residual D-pyroglutamic acid from each other. 5. A method for producing L-pyroglutamic acid, wherein the enzyme according to claim 1 is allowed to act on L-glutamic acid.