JPH05993B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a novel hydantoinase.
More specifically, the present invention relates to a novel hydantoinase having the activity of cleaving and hydrolyzing a 5-substituted hydantoin to produce an N-carbamoyl-L-amino acid. [Prior art] Hydantoinase that acts on 5-substituted hydantoins is present in livers and kidneys of various animals, leguminous plants, and microorganisms, and cleaves and hydrolyzes dihydrouracil or dihydrothymine to produce N-carbamoyl, respectively. - Converts to β-alanine or N-carbamoyl-β-amino-isobutyric acid, and also acts on 5-substituted hydantoins, stereospecifically cleaves and hydrolyzes them, resulting in optically active N-carbamoyl-D-amino acids. Dihydropyrimidinase (EC3.5.2.2) which also has the action of converting to On the other hand, L-
Carboxymethylhydantoinase (EC3.5.2.4) that generates N-carbamoyl-L-aspartic acid from 5-carboxymethylhydantoin is known (Enzyme Handbook, Asakura Shoten, 1982, p. 597). As a method for producing L-amino acids from 5-substituted hydantoin, Japanese Patent Publication No. 42-13850 describes a method for producing L-amino acids using bacterial cells, culture fluids, and disrupted fluids of various microorganisms. −
No. 8633 describes a method for producing L-lysine, and Japanese Patent Publication No. 54-2274 and Japanese Patent Publication No. 54-8749 disclose a method for producing L-lysine from 5-substituted hydantoin by the action of Flavobacterium aminogenes. A method for producing alanine and L-tryptophan is disclosed. The present inventors have also proposed a method for producing L-amino acids by reacting Arylobacter strain DK-200 with a 5-substituted hydantoin (Japanese Patent Application No. 70906/1982). These methods are extremely effective in producing L-amino acids useful as medicines, raw materials for the chemical industry, and food additives, and are expected to have practical effects. [Problems to be Solved by the Invention] However, in these L-amino acid production methods, the enzyme that cleaves and hydrolyzes the 5-substituted hydantoin has not been clarified. If such an enzyme is available, useful L-amino acids can be produced economically and have great industrial value. Therefore, the present inventors developed a reaction to produce the corresponding L-amino acid from a 5-substituted hydantoin. As a result of research on microbial enzymes involved in This discovery led to the completion of the present invention. [Means for Solving the Problems] That is, the present invention has the following physical and chemical properties, and cleaves and hydrolyzes 5-substituted hydantoin to produce N-
It is a novel hydantoinase with the activity of producing carbamoyl-L-amino acids. Substrate specificity: Does not act on 5-carboxymethylhydantoin. Optimum pH: Hydrolysis reaction from 5-substituted hydantoin PH8.0-9.0 Reverse reaction from N-carbamoyl-amino acid
PH5.0 to 7.0 Stable PH range; PH5.0 to 10.0 Suitable temperature range for action; 30 to 40℃ Temperature stability range; Up to 35℃, influence of stability inhibitors and metal ions; p-chloromercury benzoate ( PCMB), which is inhibited by ethylenediaminotetraacetic acid. The activity is promoted by cobalt ions, manganese ions, and zinc ions, and cobalt ions improve thermal stability. The present invention will be explained in more detail below. First, the physicochemical properties of this enzyme will be described below. (1) Action and substrate specificity This enzyme is a 5-substituted hydantoin in L form and D form.
The amino acids undergo cleavage hydrolysis and are converted into the corresponding N-carbamoyl-L-amino acids and N-carbamoyl-D-amino acids, respectively. Among 5-substituted hydantoins, it acts particularly well on 5-benzylhydantoin, 5-indolylmethylhydantoin, etc., which have an aromatic ring as a substituent. However, 5-carboxymethylhydantoin (DL-aspartate hydantoin) and DL
- Does not act on glutamate hydantoin and hydantoin. Therefore, it is a novel hydantoinase different from known carboxymethyl hydantoinases. Also, dihydrouracil and dihydrothymine,
It has little effect and is different from dihydropyrimidinase. (2) Optimal PH The optimal PH is determined by using ammonia buffer (0.05M aqueous ammonia-ammonium chloride buffer, PH8.0-10.0) as a buffer solution, using 5-benzylhydantoin as a substrate, and at a temperature of 35°C for 30 minutes. When the N-carbamoyl-DL-phenylalanine produced by the reaction is measured, the pH is 8.0-9.0. In addition, acetic acid buffer (0.05M
N
-carbamoyl-L-phenylalanine or N
The optimum pH is 5.0 to 7.0 when 5-benzylhydantoin production is measured using -carbamoyl-D-phenylalanine as a substrate. This enzyme is
It has a reaction to produce 5-substituted hydantoin from N-carbamoyl-L-amino acid and N-carbamoyl-D-amino acid, that is, it has a reverse reaction action. (3) Measurement method of potency 5-benzylhydantoin
To 0.8 ml of 0.02M Tris-HCl buffer containing 2.5 mg and adjusted to pH 8.0, 0.1 ml of 10 mM cobalt chloride and 0.1 ml of enzyme solution are added, and the reaction is carried out at 35°C for 30 minutes. After 30 minutes, 1 ml of 0.1N hydrochloric acid is added to stop the reaction. This reaction was spotted on a thin layer plate, and n-butanol:acetic acid:water=4:
Develop with a 1:1 developing solvent. After drying, 10% p
- Color is developed with an acetone solution of dimethylaminobenzaldehyde (containing 1.5N hydrochloric acid), and the amount of N-carbamoylphenylalanine produced is determined by densitometry. 1.0 μmol N per minute
- The amount of hydantoinase that produces carbamoylphenylalanine is taken as one unit, and the hydantoinase titer of the sample is calculated. (4) Stable PH range As buffer solutions, acetate buffer (0.05M acetic acid-sodium acetate PH4.0-5.5), phosphate buffer (0.05M phosphate-potassium-sodium phosphate buffer PH5.5-5.5) 8.0), ammonia buffer (0.05M aqueous ammonia-ammonium chloride buffer PH8.0-10.0), borate buffer (0.05M
The stable PH range was determined by measuring the residual titer after incubating the enzyme solution at 35℃ for 30 minutes at each pH using boric acid-sodium borate (PH9.5-11.5). It is 10.0. (5) Range of suitable temperature for action When allowed to work for 10 to 30 minutes at pH 8.0, the optimum temperature range is 30 to 40°C. (6) Conditions for inactivation due to PH, temperature, etc. Incubation at 35°C for 30 minutes at PH4.0 or below and PH11 or above completely inactivates. 20 at 60℃ at PH8.0
It is completely inactivated by heat treatment for a minute. (7) Inhibition, activation and stabilization ○a Inhibition The enzyme activity value without the addition of inhibitor is 100, and the enzyme activity value is 100, respectively.
Add PCMB, ethylenediaminetetraacetic acid, sodium nitride, hydroxylamine hydrochloric acid, and PH
8.0, the residual activity after reaction at 30°C for 30 minutes is shown in the table. Note that cobalt ions were not added in this activity measurement.

[Table] As is clear from the table, this enzyme is significantly inhibited by ethylenediaminetetraacetic acid in addition to PCMB, and is considered to be a metalloenzyme. ○B Activation When this enzyme is reacted in the presence of 0.1 to 10mM of metal ions such as cobalt ions, manganese ions, zinc ions, magnesium ions, and iron ions, the activity is promoted compared to when no additives are used. Ru. ○C Stabilization The thermostability of this enzyme improves when 0.1-10mM cobalt ions coexist,
When incubated with 1mM cobalt ion, the residual activity of the enzyme without additives is 30-40%, but no decrease in activity is observed in the presence of 1mM cobalt ion. (8) Purification method Centrifuging the culture to collect wet bacterial cells, suspending the bacterial cells in 0.02M Tris-HCl buffer (PH8.0, containing 1mM manganese chloride), and applying sonication to Crush the bacterial cells, remove solids by centrifugation,
Obtain crude enzyme solution. Next, 3% protamine sulfate aqueous solution (PH7.0) was added to the crude enzyme solution, the resulting precipitate was removed by centrifugation, and ammonium sulfate was added to the supernatant.
Add 0.30 to saturation, and remove the precipitate by centrifugation. Ammonium sulfate was further added to the obtained supernatant until the saturation reached 0.60, and the resulting precipitate was separated and dissolved in 0.02M Tris-HCl buffer (PH8.0, containing 1mM manganese chloride), and this solution was added to the same solution. Dialyze for 24 hours against buffer. The ammonium sulfate fraction obtained as described above is subjected to liquid chromatography using an anion exchange resin column for protein purification (MONO Q manufactured by Pharmacia) equilibrated with the above buffer solution, and the enzyme is adsorbed onto the column.
Then, the above-mentioned buffer solution with increasing sodium chloride concentration linearly from 0 to 1M is passed through the tube, and the effluent is fractionated with a fraction collector to collect the active fraction. The active fraction was then transferred to a gel filtration column (G3000SW) equilibrated with 0.05M Tris-HCl buffer (containing 0.2M sodium chloride, 1mM manganese chloride).
×2 manufactured by Toyo Soda Kogyo Co., Ltd.), elute with a buffer solution of the same composition, and collect the active fraction. This active fraction is applied to a gel filtration column again, chromatography is performed, and the active fraction is used as a purified sample. (9) Molecular weight The molecular weight of this enzyme was measured by gel filtration using high-performance liquid chromatography (G3000SW x 2 manufactured by Toyo Soda Kogyo Co., Ltd.) and was approximately 200,000, and when measured after treatment with ethylenediaminetetraacetic acid, it was approximately 200,000. It is 100,000. These enzymes with different molecular weights are similar in their physicochemical properties except for their different molecular weights. (10) Isoelectric point The result was 4 to 4.5 as measured by the chromatofocusing method using MONOP (manufactured by Famacia). This enzyme is recognized as a novel hydantoinase based on the above properties, and by utilizing the properties of this enzyme, it becomes possible to produce extremely useful N-carbamoyl-L-amino acids. Next, specific means for producing the present enzyme will be described below. Any method may be used to produce the present enzyme, including the following methods. First, the bacteria used to produce the present enzyme may be any bacteria that belongs to the genus Arylobacter and has the ability to produce the above-mentioned hydantoinase, or may be a variant or mutant strain of these bacteria. A specific example of a bacterium belonging to the genus Arimalobacter and having the ability to produce a novel hydantoinase includes, for example, Arthro-bacter SP DK-200. The above-mentioned Arylobacter DK-200 is a strain newly obtained by the present inventors from soil, and its mycological properties are as shown below. Mycological properties of Arylobacter DK-200 (a) Morphological microscopic observation (1) Cell shape and size: 0.3-0.5μ x 0.8-
It is a rod of 5.0 μm. (2) Presence or absence of cell pleomorphism: Pleomorphism is observed (3) Presence or absence of movement: No motility (flagellates are not observed) (4) Presence or absence of spores: None (5) Gram staining: Negative ~ Weakly positive but with Gram-positive particles (6) Acid-fast: Negative (b) Growth status in each medium (1) Broth agar plate culture: Colonies are circular in shape, the ridges are convex, and the periphery is entirely It is edge-shaped and the color of the colony is pale white. (2) Broth agar slant culture: shows filamentous growth under moderate growth conditions. Glossy (3) Meat juice liquid culture: The degree of turbidity is uniform, and there is no particular growth on the liquid surface. There is a precipitate (4) Meat juice gelatin puncture culture: Liquefies. (5) Litmus milk culture: Neutral and non-liquid. (c) Physiological properties (1) Reduction of nitrate: No reduction. (2) Denitrification reaction: Positive (3) MR test: Negative (4) VP test: Negative (5) Indole production: Not produced. (6) Generation of hydrogen sulfide: Not generated. (7) Starch hydrolysis: Not hydrolyzed. (8) Use of citric acid: Do not use either. (Using Kosev's medium and Christensen's medium (9) Utilization of inorganic nitrogen sources: Utilize (nitrates and ammonium salts) (10) Pigment production: No production (11) Urease: Negative (12) Oxidase: Negative (13 ) Catalase: Positive (14) Attitude towards oxygen: Aerobic (15) Growth range: Temperature: 17.8-33.2℃ PH: 5-10 (16) O-F test: Oxidation (17) Acids and gases from sugars Whether or not to generate:

〔Example〕

The present invention will be specifically described below with reference to Examples, but the present invention is not limited thereto. Example 1 Production example of hydantoinase 1.0 g of polypeptone, 1.0 g of yeast extract, 1.0 g of glucose, 1.0 g of sodium chloride, and 0.2 g of N-carbamoyl tryptophan were dissolved in tap water, the pH was adjusted to 7.5, and the total amount was dissolved. 200ml 500
The mixture was dispensed into two 100 ml Sakaguchi flasks. After sterilizing this medium at a temperature of 120°C for 15 minutes, it was inoculated with Arylobacter DK-200 (Feiko Kenboku Yo No. 7472) and cultured with shaking (100 s.pm back and forth) at a temperature of 30°C for 20 hours. After culturing, 200 ml of the culture solution was centrifuged, the resulting bacterial cells were washed once with physiological saline, and then added to 0.02M Tris-HCl buffer (PH8.0, containing 1mM manganese chloride).
The suspension was made into a liquid volume of 20 ml. This bacterial suspension
The mixture was divided into 10 ml portions, each treated once for 3 minutes using a 20K Hz ultrasonic crusher, and centrifuged to remove solids to obtain a crude enzyme solution. The crude enzyme solution was collected and made up to 30 ml with the above Tris-HCl buffer, and then
% protamine sulfate aqueous solution was added with stirring, and stirring was continued for 30 minutes. The precipitate was removed by centrifugation to obtain a supernatant. Add ammonium sulfate to the supernatant until the saturation is 0.30, and remove the resulting precipitate by centrifugation. Ammonium sulfate is further added to the obtained supernatant until the saturation is 0.60, and a precipitate is obtained by centrifugation. This precipitate was dissolved in 0.02M Tris-HCl buffer (PH8.0, containing 1mM manganese chloride).
This solution was dialyzed against the same buffer for 24 hours,
10 ml of enzyme solution was obtained. As described above, 5 ml of the ammonium sulfate fraction was adsorbed onto an anion exchange resin column for protein purification (manufactured by Mono Q Famacia) equilibrated with 0.02 M Tris-HCl buffer (PH 8.0, containing 1 mM manganese chloride). let
Next, by high-performance liquid chromatography, the buffer solution was used to thoroughly wash the solution, and the salt concentration was adjusted to 0 to 0.
Elution was performed by continuously increasing the concentration to 1.0M, and the active fraction was obtained by fractionation using a fraction collector. By performing this high performance liquid chromatography twice, an anion exchange resin purified enzyme solution was obtained from 10 ml of the ammonium sulfate fraction. This enzyme solution was salted out by adding 0.80 saturated ammonium sulfate to obtain 2 ml of concentrated enzyme solution. Next, it was applied to a gel filtration column (G3000SW x 2, manufactured by Toyo Soda) equilibrated with 0.05M Tris-HCl buffer containing 0.2M sodium chloride and 1mM manganese chloride, and eluted with the same buffer using high performance liquid chromatography. and collected the active categories. This active fraction was applied to the same gel filtration column again.
The active fraction collected by high performance liquid chromatography was used as a purified enzyme solution. The protein concentration of this enzyme solution is
When the activity was measured using a protein assay (manufactured by Bio-Rad), the specific activity was 34.2 units/mg. Example of using the hydantoinase of the present invention Using 0.5 units of the present enzyme solution, 2 mg of 5-benzylhydantoin, 0.02M Tris-HCl buffer (PH
8), 35 in 1 ml of solution containing 1mM cobalt chloride
℃ for 15 hours. After stopping the reaction by boiling, the generated N-carbamoyl-phenylalanine was developed by thin layer chromatography with n-butanol:acetic acid:water=4:1:1, and colored with p-dimethylaminobenzaldehyde reagent. When quantified by densitometry, it was found that 2.1mg/
ml of N-carbamoylphenylalanine was produced. The enzyme of Pseudomonas DK-910 FERM P-7473, which reacts specifically with N-carbamoyl-L-amino acids to produce L-amino acids, was added to this reaction solution in 0.05 M Tris/HCl. The reaction was carried out in a buffer solution (PH8.0) at 35°C for 15 hours.
When the produced L-phenylalanine was measured by a bioassay method using Lactobacillus arabinosus ATCC8014, 0.83 mg of L-phenylalanine was produced (molar yield 49.8% vs. N-carbamoylphenylalanine). Example 2 Example of using the hydantoinase of the present invention Using an enzyme solution obtained in the same manner as in Example 1, 5-
A reaction was carried out in the same manner as in Example 1 except that 5-indolylmethylhydantoin was used instead of benzylhydantoin. The generated N-carbamoyl-
Tryptophan was quantified by thin layer chromatography in the same manner as in Example 1 and found to be 2.1 mg/ml.
of N-carbamoyltryptophan was produced. This reaction solution was treated with Pseudomonas DK-910 (FERM Bacteria No. 7473) in the same manner as in Example 1.
P-7473) enzyme was reacted. When the produced L-tryptophan was measured by a bioassay method, 0.86 mg of L-tryptophan was produced (molar yield 49.6% vs. N-carbamoyltryptophan). [Effect of the invention] It is possible to efficiently produce N-carbamoyl-L-amino acids from 5-substituted hydantoin produced by organic synthetic chemistry using the present enzyme under mild conditions at room temperature and normal pressure. It is. Furthermore, this enzyme is effective in producing L-amino acids useful as pharmaceuticals, food additives, etc. by combination or sequential reaction with N-carbamoyl-L-amino acid hydrolase.

Claims (1)

[Scope of Claims] 1. N-carbamoyl-
A novel hydantoinase having the activity of producing L-amino acids. Substrate specificity: Does not act on 5-carboxymethylhydantoin. Optimum pH: Hydrolysis reaction from 5-substituted hydantoin PH8.0-9.0 Reverse reaction from N-carbamoyl-amino acid
PH5.0 to 7.0 Stable PH range; PH5.0 to 10.0 Suitable temperature range for action; 30 to 40℃ Temperature stability range; Up to 35℃, influence of stability inhibitors and metal ions; p-chloromercuryuribenzoate, Cobalt ion improves thermal stability, activity promoted by cobalt, manganese, and zinc ions, inhibited by ethylenediaminetetraacetic acid.