JP3928046B2 - New lactonase - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel lactonase. More specifically, lactonase that stereoselectively hydrolyzes (S) -5-oxo-2-tetrahydrofurancarboxylic acid (L-α-hydroxyglutaric acid γ-lactone), a method for producing the same, and the ability to produce the enzyme It relates to a new strain belonging to the genus Erunia.
[0002]
[Prior art]
Generally, lactonase is a general term for enzymes that hydrolyze lactones into hydroxycarboxylic acids, and is expected to be applied to the production of optically active hydroxycarboxylic acids from lactones. The lactonase reported so far includes gluconolactonase derived from mammalian liver that acts on glucono-δ-lactone (Non-patent Document 1), 4-pyridoxolactone that acts on 4-pyridoxolactone. Lactonase derived from lactonase (Non-patent document 2), 5-pyridoxolactonase (Non-patent document 2) acting on 5-pyridoxolactone, Fusarium oxosporum acting widely on aldonic acid lactones and aromatic lactones (Non-patent document 3), lactonase derived from Agrobacterium tumefaciens (Non-patent document 4) that hydrolyzes L-pantoyl lactone to L-pantothenic acid is known.
[0003]
[Non-Patent Document 1]
J. Biol. Chem., 212, 677-685 (1955)
[Non-Patent Document 2]
J. Biol. Chem., 261, 15112-15114 (1986)
[Non-Patent Document 3]
Eur. J. Biochem., 209, 383-390 (1992)
[Non-Patent Document 4]
Biosci. Biotechnol. Biochem., 64, 1255-1262 (2000)
[0004]
[Problems to be solved by the invention]
However, these conventionally reported lactonases do not act on (S) -5-oxo-2-tetrahydrofurancarboxylic acid, and L-α-hydroxyglutaryl is converted from (S) -5-oxo-2-tetrahydrofurancarboxylic acid. In the case of producing an acid, side reactions are likely to occur, and an acid that requires post-treatment or an alkaline hydrolysis must be relied upon.
[0005]
[Means for Solving the Problems]
In order to solve the above-mentioned problems of the prior art, the present inventors have newly developed a lactonase (hereinafter referred to as L-) that hydrolyzes (S) -5-oxo-2-tetrahydrofurancarboxylic acid to L-α-hydroxyglutaric acid. α-hydroxyglutarate γ-lactonase) was obtained from microorganisms, and as a result of extensive screening, a new strain belonging to Erwinia newly isolated from soil by the present inventors was found to be a novel L-α- The inventors have found that hydroxyglutarate γ-lactonase can be produced and completed the present invention.
That is, the present invention relates to the following (1) to (5).
(1) L-α-hydroxyglutarate γ-lactonase having the following characteristics (a) to (f):
(A) Action: Hydrolysis of (S) -5-oxo-2-tetrahydrofurancarboxylic acid to L-α-hydroxyglutaric acid.
(B) Substrate specificity: Acts on (S) -5-oxo-2-tetrahydrofurancarboxylic acid and weakly acts on 2-oxotetrahydrofuran-4,5-dicarboxylic acid.
(C) Optimal pH: pH 6.5-7.5
(D) Optimal temperature: 25-45 ° C
(E) pH stability: pH 6.5-8.0
(F) Temperature stability: ~ 50 ° C
(G) Molecular weight: Beckman Ultra spheroplasts gel SEC3000 (7.5mm × 30 cm) to about by gel permeation chromatography using a column 79,000, 41,000 at SDS-PAGE
(2) The L-α-hydroxyglutarate γ-lactonase according to claim 1, wherein the N-terminus has the following amino acid sequence.
Gln-Glu-Thr-Pro-Ser-Tyr-Val-Tyr-Val-Ser-Asn-Gly-Asp-Ser-Gly-Thr-Val-Thr-Ala-Tyr-Gln-Leu-Asp-
(3) A strain belonging to Erwinia and having the ability to produce L-α-hydroxyglutarate γ-lactonase according to claim 1 or 2 is cultured in a nutrient medium, and the L-α-hydroxyglutarate is obtained. The method for producing L-α-hydroxyglutarate lactonase according to claim 1 or 2, wherein γ-lactonase is produced and accumulated and collected.
(4) The method for producing L-α-hydroxyglutarate lactonase according to claim 3, wherein the nutrient medium contains citric acid.
(5) Erwinia cypripedii 314B (FERM P-19195) strain.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The L-α-hydroxyglutaric acid γ-lactonase of the present invention has an action of hydrolyzing (S) -5-oxo-2-tetrahydrofurancarboxylic acid to L-α-hydroxyglutaric acid. It is a novel enzyme with no knowledge. The L-α-hydroxyglutaric acid γ-lactonase of the present invention is obtained by culturing a strain belonging to the genus Erunia and having the ability to produce the enzyme in a nutrient medium. This is based on the fact that the present inventors found that a strain belonging to the genus Erunia newly isolated from soil has the L-α-hydroxyglutarate γ-lactonase.
[0007]
The mycological properties of this strain are as follows.
1. Form: Gram negative, Neisseria gonorrhoeae (0.7-0.9 × 1.5-2.5 μm)
2. Sensitivity to 3% KOH: Lysis
3. Aminopeptidase: positive
4. Oxidase: negative
5. Catalase: positive
6. Acid production: Acid is produced from glucose, fructose, mannose, maltose, xylose, sucrose, trehalose, arabinose, rhamnose, galactose, inositol, and acid is produced from adonitol, arabitol, dulcitol, erythritol, α-methylglucoside. do not do.
7. ONPG test: positive
8. ADH test: negative
9. LDC test: negative
10. ODC test: negative
11 Urease test: negative
12. Simmons citrate test: positive
13. Phenylalanine deaminase test: positive
14. Hydrogen sulfide production: negative
15. Gas production from glucose: positive
16. Degradation of gelatin: negative
17. DNA degradation: negative
18. Tween 80 decomposition: negative
19. Indole production: negative
20. Use of malonic acid: positive
21. Esculin hydrolysis: positive
22. MR test: positive
23. VP reaction: negative
24. Reduction of nitrate: positive
25. Denitrification: Positive
The fatty acid composition of this bacterium is in good agreement with that of the family Enterobacteriaceae, and the above bacteriological properties are in good agreement with the characteristics of the known Erunia cypripedi. However, the base sequence of 16S rDNA of this bacterium showed 98.5% homology with that of Erunia cypripedi. That is, this strain is a new strain belonging to Erunia cypripedi, named Elunia cypripedi 314B, and deposited with the National Institute of Advanced Industrial Science and Technology (BIRM P-19195).
[0009]
The culture method for producing and accumulating the L-α-hydroxyglutarate γ-lactonase of the present invention using this strain may be either a liquid culture method or a solid culture method, but preferably a liquid culture method is performed. . Specifically, in the case of a liquid culture medium, the culture method contains a carbon source, a nitrogen source, an inorganic salt, a necessary nutrient source, etc. necessary for the microorganism to grow well and produce an enzyme smoothly. A synthetic medium or a natural medium.
For example, as the carbon source, carbohydrates such as starch or a fraction thereof, roasted dextrin, modified starch, starch derivative, physically treated starch and α-starch can be used. Specific examples include soluble starch, corn starch, potato starch, sweet potato starch, dextrin, amylopectin, and amylose.
[0010]
Furthermore, in the present invention, a significant amount of L-α-hydroxyglutarate γ-lactonase can be produced by adding citric acid as a carbon source for the medium.
Examples of nitrogen sources include polypeptone, casein, meat extract, yeast extract, corn steep liquor, organic nitrogen source materials such as extracts of soybeans and soybean meal, inorganic nitrogen compounds such as ammonium sulfate and ammonium phosphate, and amino acids such as glutamic acid Is mentioned.
Examples of inorganic salts include ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, and other iron ion-containing compounds, phosphates such as potassium phosphate and sodium phosphate, magnesium sulfate Magnesium salts such as calcium chloride, calcium salts such as calcium chloride, and sodium salts such as sodium chloride.
[0011]
The culture is performed under aerobic conditions such as shaking culture or aeration and agitation culture, and the medium is adjusted to pH 2.0 to 7.0, preferably pH 5.0, and the temperature is in the range of 10 to 40 ° C., preferably 25 to Although it is desirable to carry out at 30 ° C. and culture for 1 to 15 days in general, there is no particular limitation as long as the microorganism grows and the target enzyme is produced even under these conditions.
Next, the bacterial cells are collected from the culture solution and crushed to obtain a crude enzyme solution. Further, the enzyme is recovered by ammonium sulfate salting out or organic solvent precipitation. Thereafter, a high purity L-α-hydroxyglutarate γ-lactonase preparation can be obtained by combining various chromatographies.
[0012]
The enzymatic chemistry of the L-α-hydroxyglutarate γ-lactonase thus obtained was as follows.
(A) Optimal pH: pH 6.5-7.5
(B) Optimal temperature: 25-45 ° C
(C) pH stability: pH 6.5-8.0 (95% or more remaining: 4 ° C., 24 hours)
(D) Temperature stability: -50 ° C (95% or more activity remains even at 10 minutes at a temperature up to 50 ° C (pH 7.0))
(E) Substrate specificity: Acts on 2-oxotetrahydrofuran-4,5-dicarboxylic acid in addition to (S) -5-oxo-2-tetrahydrofurancarboxylic acid. However, relative activity of 2-oxotetrahydrofuran-4,5-dicarboxylic acid is 8%. (R) -5-oxo-2-tetrahydrofurancarboxylic acid, D-glucono-δ-lactone, D- and L-galactono 1,4-lactone, L-mannono-1,4-lactone, D- and L-pant Does not act on lactone, γ-butyrolactone, γ-valerolactone, or γ-caprolactone.
(F) Molecular weight: about 79,000 by gel permeation chromatography using Beckman Ultraspherogel SEC3000 (7.5 mm × 30 cm) column, 41,000 by SDS-PAGE.
(G) Inhibitors / activators: PMSF (phenylmethylsulfonyl fluoride), hydroxyamine, O-phenanthroline, 2,2'-bipyridyl, EDTA, N-ethylmaleimide, PCMB, silver nitrate, iodoacetamide, Mn It is not substantially inhibited or activated by ²⁺ , Co ²⁺ , Fe ³⁺ , Fe ²⁺ , Ca ²⁺ , or Mg ²⁺ . Inhibited by N-bromosuccinimide, Cu ²⁺ .
[0013]
Further, the amino acid sequence at the N-terminal of L-α-hydroxyglutarate γ-lactonase of the present invention was as follows.
N-terminal amino acid sequence: Gln-Glu-Thr-Pro-Ser-Tyr-Val-Tyr-Val-Ser-Asn-Gly-Asp-Ser-Gly-Thr-Val-Thr-Ala-Tyr-Gln-Leu- Asp-
[0014]
The L-α-hydroxyglutarate γ-lactonase obtained by culturing the Erunia cypripedi 314B strain has been described in detail above. In the present invention, a part of L-α-hydroxyglutarate γ-lactonase or Even a lactonase in which one or more amino acids are deleted or added from the lactonase and / or one or more amino acids in the lactonase are replaced with other amino acids, Any material having the biological properties of L-α-hydroxyglutarate γ-lactonase is included in the present invention.
Further, the novel L-α-hydroxyglutarate γ-lactonase of the present invention can be obtained, for example, by culturing a strain derived from Ernia cypripedi as described above, and the amino acid sequence of the lactonase is analyzed, It can also be obtained by genetic engineering by preparing DNA encoding it.
[0015]
Hereinafter, the present invention will be described in detail by way of examples. The present invention is not limited to these.
【Example】
[Example 1]
After inoculating Erunia cypripedi 314B in a liquid medium having the following composition, it was cultured at 30 ° C. for 1 day.
Medium: Citric acid 20.0, KH ₂ PO ₄ 3.0, (NH ₄ ) ₂ SO ₄ 2.0, yeast extract 1.0, MgSO ₄・ 7H ₂ O 0.3, CaCl ₂・ 2H ₂ O 0.1, NaCl 0.1 g / l, pH 5.0
The cells were collected by centrifugation, washed twice with 50 mM Tris buffer (pH 7.0), crushed with an MSK cell homogenizer manufactured by Braun, and the supernatant after centrifugation was used as a crude enzyme solution. Ammonium sulfate was added to the crude enzyme solution so as to be 60% saturation to precipitate the enzyme. The precipitated enzyme was collected by centrifugation, redissolved in 50 mM Tris buffer (pH 7.0) containing 4 M NaCl, and applied to a butyl Toyopearl column equilibrated with the same buffer. The adsorbed active fraction was eluted by linearly decreasing the NaCl concentration in the mobile phase. The active fraction obtained was dialyzed against 10 mM Mes buffer (pH 7.0), adsorbed on a fructogel EMD COO-column equilibrated with the same buffer, and then NaCl was added linearly into the mobile phase. Elute. The obtained active fraction is dialyzed with 10 mM phosphate buffer, adsorbed on a hydroxyapatite column equilibrated with the same buffer, and the target enzyme is obtained by linearly increasing the phosphate concentration in the mobile phase. Elute. The enzyme preparation thus obtained gave a single band by SDS-PAGE. Using this purified enzyme, the enzyme chemistry as described above was clarified.
[0016]
[Example 2]
For 100 mM Hepes buffer (pH 7.0) containing 25 mM (S) -5-oxo-2-tetrahydrofurancarboxylic acid, the amount of L-α-hydroxyglutarate γ-lactonase obtained in Example 1 was 0.9 U / Added to ml and kept at 37 ° C. FIG. I shows changes with time of the concentrations of (S) -5-oxo-2-tetrahydrofurancarboxylic acid and α-hydroxyglutaric acid in the reaction solution.
After 6 hours, all (S) -5-oxo-2-tetrahydrofurancarboxylic acid was converted to α-hydroxyglutaric acid. The generated α-hydroxyglutaric acid was analyzed by high performance liquid chromatography using CHIRALPAK MA (+) (manufactured by Daicel), and all were confirmed to be L-type.
The activity of L-α-hydroxyglutarate γ-lactonase was measured by the following method.
[Activity Measurement Method] 125 mM Hepes buffer solution (pH 7.0) containing 125 mM (S) -5-oxo-2-tetrahydrofurancarboxylic acid is used as a substrate solution. After warming 0.4 ml of substrate solution at 37 ° C for 3 minutes, add 0.1 ml of enzyme solution to start the reaction. After reacting at 37 ° C for 30 minutes, put on a boiling water bath for 1 minute to stop the reaction.
After stopping the reaction, the mixture was centrifuged, and the supernatant fraction was quantified by high-performance liquid chromatography using GL Sciences Inertosyl ODS-3 column (4.6 x 250 mm) to determine the amount of L-α-hydroxyglutaric acid produced by the enzymatic reaction. To do. Enzyme 1 U was defined as the amount of enzyme that liberates 1 μmol of L-α-hydroxyglutaric acid per minute under the above conditions.
[0017]
【The invention's effect】
The present invention provides a novel lactonase that hydrolyzes (S) -5-oxo-2-tetrahydrofurancarboxylic acid to L-α-hydroxyglutaric acid. The enzyme can be obtained from microorganisms, and the enzyme can be usefully used in various fields such as the food field and the pharmaceutical field.
[Brief description of the drawings]
FIG. 1 shows (S) -5-oxo-2-tetrahydrofurancarboxylic acid when L-α-hydroxyglutaric acid γ-lactonase of the present invention is allowed to act on (S) -5-oxo-2-tetrahydrofurancarboxylic acid. It is a graph which shows the result of having measured the time-dependent change of acid concentration and L-α-hydroxyglutaric acid concentration.

Claims (5)

L-α-hydroxyglutarate γ-lactonase having the following characteristics (a) to (f):
(A) Action: Hydrolysis of (S) -5-oxo-2-tetrahydrofurancarboxylic acid to L-α-hydroxyglutaric acid.
(B) Substrate specificity: Acts on (S) -5-oxo-2-tetrahydrofurancarboxylic acid and weakly acts on 2-oxotetrahydrofuran-4,5-dicarboxylic acid.
(C) Optimal pH: pH 6.5-7.5
(D) Optimal temperature: 25-45 ° C
(E) pH stability: pH 6.5-8.0
(F) Temperature stability: ~ 50 ° C
(G) Molecular weight: Beckman Ultra spheroplasts gel SEC3000 (7.5mm × 30 cm) to about by gel permeation chromatography using a column 79,000, 41,000 at SDS-PAGE The L-α-hydroxyglutarate γ-lactonase according to claim 1, wherein the N-terminus has the following amino acid sequence.
Gln-Glu-Thr-Pro-Ser-Tyr-Val-Tyr-Val-Ser-Asn-Gly-Asp-Ser-Gly-Thr-Val-Thr-Ala-Tyr-Gln-Leu-Asp-   A strain belonging to Erwinia and having the ability to produce L-α-hydroxyglutarate γ-lactonase according to claim 1 or 2 is cultured in a nutrient medium, and the L-α-hydroxyglutarate γ- The method for producing L-α-hydroxyglutarate lactonase according to claim 1 or 2, wherein lactonase is produced and accumulated and collected.   The method for producing L-α-hydroxyglutarate lactonase according to claim 3, wherein the nutrient medium contains citric acid.   Erwinia cypripedii 314B (FERM P-19195) strain.

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