JP3415218B2 - Method for producing D-α-amino acid - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention has the general formula:

[Chemical 6] [In the formula, R represents a phenyl group, a phenyl group substituted with a hydroxyl group, a substituted or unsubstituted, preferably an alkyl group having 1 to 5 carbon atoms or a thienyl group. ] Represented by]
An enzyme having the ability to remove a carbamyl group (hereinafter referred to as “decarbamylase”) is allowed to act on N-carbamyl-α-amino acid to give a compound represented by the general formula:

[Chemical 7] [In the formula, R has the same meaning as described above. ] Represented by]
It relates to a method for producing an α-amino acid.

Such optically active D-α-amino acids are important compounds as pharmaceutical intermediates, and particularly D-phenylglycine and D, which are intermediates for the production of semisynthetic penicillin and semisynthetic cephalosporins. Examples of industrially useful compounds include-(4-hydroxyphenyl) glycine (hereinafter referred to as "D-HPG").

[0003]

2. Description of the Related Art As a method for producing D-α-amino acids,
It is known to remove the carbamyl group of the corresponding DN-carbamyl-α-amino acid to obtain the carbamyl group, and the carbamyl group can be removed by a chemical method (Japanese Patent Publication 58-58).
-4707) and methods utilizing the enzymatic reaction of microorganisms (JP-B-57-18793 and JP-B-63-2).
No. 0520 and Japanese Patent Publication No. 1-48758).

However, many of these microbial enzymes have an optimum pH in the vicinity of neutrality. DL-5-substituted hydantoin corresponding to D-α-amino acid is selectively hydrolyzed in the D-form by the action of an enzyme to form N-carbamyl-D-
In the reaction for obtaining an α-amino acid and subsequently for acting a decarbamylase to obtain a D-α-amino acid, the optimum pH of the hydantoin hydrolase in the first step is at pH 8 to 9,
It had to be carried out in a reaction system different from the step decarbamylase.

[0005]

DISCLOSURE OF THE INVENTION The present inventors
-For a microorganism having the ability to act on carbamyl-α-amino acids to enzymatically remove the carbamyl group and convert it to D-α-amino acids (hereinafter referred to as "decarbamyl"), the presence of decarbamylase has been conventionally known. As a result of various studies centered on genus for which the
The inventors have found that the bacteria belonging to the genus onas, the genus Blastobacter or the genus Rhizobium, and the genus Bradyrhizobium have a decarbamyl activity, and completed the present invention.

[0006]

That is, the present invention provides a compound represented by the general formula (I):

[Chemical 8] [In the formula, R represents a phenyl group, a phenyl group substituted with a hydroxyl group, a substituted or unsubstituted alkyl group having preferably 1 to 5 carbon atoms, or a thienyl group. ] N-carbamyl-D-α-amino acid represented by
A general formula (II):

[Chemical 9] [In the formula, R has the same meaning as described above. ] Represented by]
In the method for producing an α-amino acid, as an enzyme decarbamylase for converting an N-carbamyl-D-α-amino acid into a D-α-amino acid, Comamonas
Genus, Blastobacter genus, Rhizobium genus or Brady Rhizobium (Brady
There is provided a method for producing a D-α-amino acid, which comprises using an enzyme that can be produced by a microorganism belonging to the genus rhizobium).

The decarbamylase used in the present invention is D
The D-form, even if it acts specifically on the isomer,
It does not matter in fact whether the enzyme acts on any of the L-forms. An enzyme having severe stereoselectivity for DN-carbamyl-α-amino acid is DN-carbamyl-
It is sometimes called α-amino acid amide hydrolase.

Among these microorganisms, strains having relatively high decarbamyl activity include Comamonas sp. E222C
(FERM BP No. 4411), Comamonas sp. E2
06a, Comamonas sp. E217a, Blast Bactor s
p. NA88-b, Blastobacter sp. A17p-4
(FERM BP 4410), Rhizobium sp. KN
K1415 (FERM BP No. 4419), Bradyrhizobium japonicu
m) IFO 14783, Bradyrhizobium sp. IFO
15003 and so on.

As a typical example of the above-mentioned bacteria, Comamonas sp.
E222C, Blast Bactor sp. A17p-4 and Rhizobium sp. The mycological properties of KNK1415 are shown below.

Comamonas sp. E222C (a) Morphological properties ・ Bacillus (bent) ・ Gram stainability Variable (Variable) ・ No spores ・ No colony morphology Light yellow, slightly translucent, round, regular, round, bright , Low convex, smooth, diameter 1mm (48hr)

(B) Physiological properties Catalase + Oxidase − Glucose fermentability − NO ₃ reduction − Indole production − Acid production from glucose − Arginine dehydrolase − Urease − Eesculin Hydrolysis- ・ Gelatin hydrolysis- ・ β-galactosidase- ・ Cytochrome oxidase + (weak)

(C) Assimilation Glucose- ・ Arabinose- ・ Mannose- ・ Mannitol -N-acetyl-glucosamine- ・ Maltose- ・ Gluconic acid + ・ Caproic acid + ・ Adipic acid- ・ Malic acid + ・ Citric acid + ・ Phenyl acetate ＋

Blast Bactor sp. A17p-4 (a) Morphological properties-Bacillus (egg type) -Gram stainability Negative-Spore presence / absence-Colony morphology White, slightly translucent, round, regular, round, shiny, low convex , Smooth, mucoid formation, diameter 1.0 ~ 1.5mm (48hr)

(B) Physiological properties Catalase + Oxidase + (weak) Glucose fermentability-NO ₃ reduction-Indole production-Acid production from glucose-Arginine dehydrolase-Urease + -Esculin (Aesculin) hydrolysis + ・ Gelatin hydrolysis-・ β-galactosidase + ・ Cytochrome oxidase +

(C) Assimilation ・ Glucose + ・ Arabinose + ・ Mannose + ・ Mannitol + ・ N-acetylglucosamine + ・ Maltose + ・ Gluconic acid- ・ Caproic acid- ・ Adipic acid- ・ Malic acid- ・ Citric acid- ・ Phenyl acetate-

[0016]

[0017]

[0018]

[0019]

Rhizobium sp. KNK-1415 (FER
( MBP-4419) (a) Morphological properties-Bacilli (somewhat rod-shaped short bacilli) -Negative Gram stainability-No spores-Ben hair poles or peripheries-Colony morphology, slightly yellowish white, gloss Round, smooth, smooth, slightly convex,
Diameter 5mm (5 days), mucoid formation, growth at each temperature 30 ℃ + 37 ℃ (±)

(B) Physiological Properties Catalase + Oxidase + Glucose fermentability − NO ₃ reduction + Indole production − Acid production from glucose − Arginine dehydrolase − Urease + · Esculin Hydrolysis + · Gelatin hydrolysis − · β-galactosidase + · Cytochrome oxidase + · 3-ketolactose production − · H ₂ S production −

(C) Assimilation ・ Glucose + ・ Arabinose + ・ Mannose + ・ Mannitol + ・ N-Acetylglucosamine + ・ Maltose + ・ Gluconic acid- ・ Caproic acid- ・ Adipic acid- ・ Malic acid + ・ Citric acid-・ Phenylacetate-GC content 63.4% Quinone analysis Q-10 95.7% Q-9 3.3% Q-11 0.6% Q-8 0.4% Bacterial cell fatty acid analysis (total microbial cell fatty acid) Ingredient name Area ratio (%) 3-OH C14: 0 4.6 C16: 1 5.9 C16: 0 14.0 3-OH C16: 0 0.1 C18: 1 70.7 C18: 0 3.3 Unknown 1.4 (3-OH fatty acid) component name area ratio (%) 3-OH C14: 0 88.8 3-OH C16: 0 6.6 3-OH C18: 0 4.6 (2-OH fatty acid) detection not

[0023]

[0024]

These enzyme systems exhibiting decarbamyl activity can be prepared by culturing a microorganism by a usual method. Cultivation is usually carried out in liquid nutrient medium, but it can also be carried out by solid surface culture. The medium contains a carbon source, a nitrogen source, and inorganic salt nutrients essential for the growth of each microorganism, which can normally be assimilated. Further, D-p-hydroxyphenylglycine, D-phenylglycine and other D-
Amino acid; N-carbamyl-DL-methionine, N-carbamyl-D-phenylalanine and the like N-carbamyl-
α-amino acids; 5-substituted hydantoins such as DL-5-p-hydroxyphenylhydantoin and DL-5-phenylhydantoin; uracil, dihydrouracil, β-
Pyrimidine metabolites such as ureidopropionic acid; Fe ²⁺ ,
Fe ³⁺ , Be ²⁺ , Co ²⁺ , Al ³⁺ , Li ⁺ , Mn ²⁺ , Mg ²⁺ , C
It is preferable to add a small amount of metal ions such as s ⁺ ; urea or the like to enhance and accumulate decarbamylase. The concentration of these decarbamylase production enhancing substances in the medium is selected from the range of 0.1 to 10 mM for metal ions and 0.01 to 1% by weight for other substrates.

The temperature during culture is in the range of 20 to 85 ° C., and the pH is in the range of 4 to 11, and the growth of microorganisms can be promoted by aeration and stirring.

In the decarbamyl reaction of DN-carbamyl-α-amino acid, the culture solution of the microorganism, bacterial cells or treated product of bacterial cells obtained as described above is used as an enzyme source. The microbial cells can be used as they are as they are, but they can also be dried microbial cells such as freeze-dried microbial cells, crushed microbial cells or microbial cell extracts, surfactants or ultrasonic waves. The treated bacterial cell product can be used in the reaction of the present invention as a microbial enzyme source. Furthermore, the enzyme can be used in the state of an enzyme obtained by purifying or partially purifying an enzyme from a crushed product, an extract or a treated product of these bacterial cells or a crude enzyme. In addition, these enzymes or crude enzymes can be immobilized and used as immobilized enzymes. As the immobilization method, for example, the immobilization method of International Patent Application PCT / JP91 / 01696 can be used.

Furthermore, the enzymes produced by these microorganisms by genetic engineering are basically equivalent to the enzymes that the microorganisms can produce.

The decarbamyl reaction is carried out in an aqueous medium, and its pH is in the range of 6 to 11,
Particularly preferably, the pH is 7 to 9.5. Furthermore, pH8
Some enzymes have optimum pH of ~ 9. The temperature for the decarbamyl reaction is usually in the range of 20 to 85 ° C, and a temperature suitable for the enzyme system of the strain to be used is adopted.

In the present invention, examples of the N-carbamyl-D-α-amino acid compound serving as a substrate for decarbamylase include DN-carbamylalanine, DN-carbamylmethionine and DN-carbamylvaline. , D-N-
Carbamyl leucine, DN-carbamylphenylglycine, DN-carbamyl- (4-hydroxyphenyl)
Glycine, DN-carbamyl- (2-thienyl) glycine, DN-carbamylphenylalanine and the like can be mentioned. In the general formula, R is a substituted alkyl group, and examples of the substituent include a phenyl group, an indolyl group, an alkylthio group, a hydroxyl group, an amino group and a carboxyl group.

The isolation of the D-α-amino acids produced by the decarbamyl reaction by the microbial enzyme as described above can be carried out by a known method such as concentration, neutralization or treatment with an ion exchange resin. That is, relatively hydrophobic D such as D-phenylglycine, D- (4-hydroxyphenyl) glycine, D-leucine and D-phenylalanine.
In the case of -α-amino acids, the desired D-α-amino acid can be obtained as a precipitate by removing the insoluble matter in acid or alkaline and then concentrating, neutralizing, etc. When D-thienylglycine, D-serine, D-alanine and the like are relatively hydrophilic, the target substance is adsorbed by an ion exchange resin, which is eluted with ammonia water or the like, and then neutralized. Then, the desired product can be obtained by concentrating.

By the way, these N-carbamyl-D-amino acid compounds, which are substrates for decarbamylase, are 5-substituted hydantoin derived from culture solutions of various species of microorganisms, cells, treated cells or enzymes extracted from cells. It can be produced by acting on a class. Such a manufacturing method is disclosed in JP-A-53-44690 and JP-A-53-69884.
JP-A-53-91189, JP-A-53-
It is described in JP-A-133688, JP-A-54-84086 and JP-A-55-7001.
These D-carbamyl-α-amino acids are converted to the corresponding optically active D-α-amino acids by the method of the present invention.

[0033]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 A soil sample (collection site name: collected from all over Japan) was used to prepare 2 ml of medium A (1 g / l KH ₂ PO ₄ , 1 g / l K ₂ HPO ₄ , 0.
3 g / l MgSO ₄ .7H ₂ O, 0.1 g / l yeast extract,
1 g / l NH ₄ Cl, 1.5 g / l compound used as carbon source (pH 7.0) described below, or medium B (1 g / l
l KH ₂ PO ₄ , 1 g / l K ₂ HPO ₄ , 0.3 g / l Mg
SO ₄ .7H ₂ O, 0.5 g / l glucose, 0.1 g / l yeast extract, 1.5 g / l compound described below (pH 7.0) used as a nitrogen source) and inoculated at 28 ° C. The cells were aerobically shake-cultured, subcultured in the same medium and cultured until growth of microorganisms was observed. Then, it was spread on a plate medium (similar medium containing 2% agar) and cultured at 28 ° C. for 2 to 6 days to isolate the microorganism.

Compound N-carbamyl-Dp-hydroxyphenylglycine used as a carbon source or a nitrogen source
(CD-HPG) N-carbamyl-D-phenylglycine (CD-PG) N-carbamyl-D-alanine (CD-Ala) DL-5-methylhydantoin (DL-Ala-hyd) DL -5-phenylhydantoin (DL-PG-hyd) DL-5-p-hydroxyphenylhydantoin (DL-)
HPG-hyd) Citrulline phenylureacarbamic acid-n-butyl ester

Each of the strains on the plate medium was suspended in 100 μl of the reaction solution (35 mM CD-HPG, 200 mM potassium phosphate buffer (pH 7.0), and the suspension was added at 28 ° C. for 1 to 3 times.
After reacting for a day, carbamyl D-HPG and D-HPG were analyzed by thin layer chromatography. For thin layer chromatography, 60F ₂₅₄ plate manufactured by Merck was used, developed with butanol: acetic acid: water, 3: 3: 1 (v / v / v), and UV lamp (254 nm) or p- (dimethylamino). Detected by spraying with cinnamaldehyde and ninhydrin. As a result, 1868 strain can be isolated as a microorganism that assimilates the above-mentioned carbon source or nitrogen source compound, of which 37 strains produce D-HPG, and 16 strains have good D-HPG productivity (2 mM. The above D-HPG was produced).

Example 2 For the 16 decarbamylase producing strains obtained in Example 1, 1 ml of medium C was used for secondary screening.
(1.5 g / l CD-HPG, 1 g / l KH ₂ PO ₄ ,
_{1g / l K 2 HPO 4,} 0.3g / l MgSO 4 · 7H
₂ O, 3 g / l yeast extract, 3 g / l meat extract, 10 g / l glycerol, 2 g / l polypeptone (pH 7.0)) were cultured at 28 ° C for 3 days. 1 ml of the culture solution was centrifuged, and 100 μl of the same reaction solution as used in the example was used for the bacterial cells.
Suspended in and reacted at 30 ° C or 50 ° C for 24 hours,
The produced D-HPG was quantified.

The amount of D-HPG was determined by Cosmocil (Cosmocil).
sil) 5C18 column (φ4.6 × 250 mm, manufactured by Nacalai Tesque, Inc.), water: acetonitrile: phosphoric acid (9
5: 5: 0.01 (v / v / v)) was used as an eluent for separation by high performance liquid chromatography (HPLC).
This was done by measuring the absorbance at 4 nm. The result is shown in FIG.

3 out of 16 strains, E206a, E22
2C and E215 were grown with CD-Ala as a single carbon source and showed high activity at 30 ° C. In E222C, 30.8 mM D-HPG was produced at 30 ° C., but only 3.5 mM D-HPG was produced at 50 ° C. The other three strains, A17p-1, A17p-3 and A17p-4, grew using CD-HPG as a single carbon source and showed high activity at 50 ° C. A17p-4 produced 28.8 mM D-HPG at 50 ° C, but produced only 2.5 mM D-HPG at 30 ° C.

Example 3 Substrate specificity of the obtained decarbamylase Comamonas sp. E222C and Blastobacter sp.
To examine the substrate specificity of the decarbamylase produced by A17p-4, the medium C described in Example 2 was used, and the temperature was 28 ° C.
Then, Comamonas sp. E222C for 1 day or Blastobacter sp. A17P-4 was shake-cultured for 7 days.
3 ml of the culture solution was centrifuged, and the cells were suspended in 300 μl of a reaction solution (200 mM potassium phosphate buffer, 1% (w / v) various reaction substrates), and Comamonas sp. With E222C, at 30 ° C, Blastobacter sp. 5 for A17P-4
The reaction was carried out at 0 ° C. for 24 hours each.

As shown in Table 1, both bacterial cells showed strong hydrolytic activity against N-carbamyl compounds of aliphatic and aromatic D-amino acids.
-Low activity for carbamyl form. Also, Comamonas sp. With E222C, β-ureidopropionic acid can be hydrolyzed, and Blastobacter sp. It was found that A17P-4 also has a hydantoinase activity.

[0041]

[Table 1]

Example 4 Comamonas sp. Increased production of E222C strain decarbamylase
Strong substance Comamonas sp. When culturing the E222C strain, an enzyme production-increasing substance that increases decarbamylase production was searched for. Nutrient medium, medium D (1 g / l KH ₂ PO ₄ ,
_{1g / l K 2 HPO 4,} 0.3g / l MgSO 4 · 7H
₂ O, 3 g / l yeast extract, 3 g / l meat extract, 10 g / l glycerol, 2 g / l polypeptone (pH 7.0)),
The compound shown in FIG. 2 was added at a concentration of 0.15% (w / v),
The bacteria were planted and cultured at 28 ° C. for 2 days with shaking.

1 ml of this culture broth was centrifuged to obtain 10 cells.
0 μl of reaction solution (200 mM potassium phosphate buffer (pH
7.0), suspended in 35 mM CD-HPG), 30
The reaction was performed at 24 ° C. for 24 hours, and the produced D-HPG was quantified by HPLC according to the method shown in Example 2. As a result, as shown in FIG. 2, it was found that the activity was enhanced by urea, β-ureidopropionic acid, D-phenylglycine, and N-carbamyl-DL-methionine.

Example 5 Comamonas sp. Decarbamylase produced by E222C
Purification of Comamonas sp. E222C was cultured at 28 ° C. for 3 days with shaking in the medium D described in Example 4 to which β-ureidopropionic acid (0.15% (w / v) was added, and a total of 3.6 1 of the medium was centrifuged. The cells were separated by suspending them in 100 ml of 10 mM potassium phosphate buffer (pH 7.0),
It was disrupted by sonication and the residue was removed by centrifugation. While adding ammonium sulfate to this crude enzyme solution, a saturated concentration of 20
The ammonium sulphate-precipitated fraction at concentrations of 40% to 40% was separated by centrifugation and dissolved in the above buffer.

177 ml of this enzyme solution was dialyzed for 12 hours against 10 l of the above buffer solution to obtain DEAE-Sephacel (Sepha).
cel) column (φ5 × 15 cm) and adsorbed on a linear gradient of NaCl concentration of 0 to 1 M to collect active fractions.
The NaCl concentration of this eluate was adjusted to 4 M, adsorbed on a Phenyl-Sepharose CL-4B column (φ1.5 × 15 cm), and eluted with a 4-0 M linear NaCl concentration gradient to obtain the active fraction. Collect YM-10
It was concentrated by ultrafiltration using a membrane (Amicon).

This concentrated solution (5 ml) was added to Sephacryl S-2 in the above buffer containing 0.2 M NaCl.
Gel filtration was performed using a 00HR column (φ1.8 × 80 cm), and the active fraction was desalted by dialysis, and then adsorbed on a hydroxyapatite column (φ1.2 × 10 cm) to give 0-1.
Elution was performed with a linear concentration gradient of M potassium phosphate buffer (pH 7.0), and the active fraction (8.5 ml) was dialyzed against 500 ml of 10 mM potassium phosphate buffer (pH 7.0) for 12 hours. This was adsorbed to a Mono (Q) HR5 / 5 column and eluted with a linear concentration gradient of 0 to 1.0 M NaCl, and this active fraction (1.2 ml) was used as a purified enzyme solution for analysis.

By the above purification operation, as shown in Table 2,
The specific activity was improved by 108 times as compared with the cell disrupted supernatant, and the enzyme activity recovery rate was about 2%. Also, 10 mg of this purified enzyme was used according to the method of King and Laemley [King, J., L.
aemmli, UK, Journal of Molecular Biology 62
Vol., 465-477 (1971)] and subjected to SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel. As shown in FIG.
A single band was obtained around 8,000.

[Table 2]

Also, Sephadex G-
0.2M using 150 columns (φ1.5 × 85cm)
Gel filtration was performed with a 10 mM potassium phosphate buffer (pH 7.0) containing NaCl and 0.1 mM dithiothreitol (DTT), and as shown in FIG.
It was eluted around 000.

Example 6 Comamonas sp. Properties of E222C decarbamylase Using the enzyme solution obtained in Example 5, Comamonas sp. E222
The optimum reaction pH and the optimum reaction temperature of the decarbamylase produced by C were examined. The optimum reaction pH was measured by the following method. Reaction solution (500 μl) buffer (concentration 2
00 mM), at pH 4.2 to 5.9, acetic acid-hydrochloric acid buffer solution, at pH 5.0 to 8.8, potassium phosphate buffer solution, pH
H7.6-9.9, Tris-HCl buffer and pH8.
In 9 to 11.1, a glycine-NaOH buffer solution was used, and 10 mM N-carbamyl-D-phenylalanine and enzyme solution were added to the reaction solution, which was reacted at 30 ° C. for 20 minutes, and 500 μl ethanol was added. Then, the reaction was stopped.

The amount of D-phenylalanine produced was determined by
200 mM potassium phosphate buffer (pH 7.0), 1.5
mM 4-aminoantipyrine, 2.1 mM phenol,
2.25u peroxidase (derived from horseradish, Calzyme Laboratory (CALZYME Lab.))
And 1 part of the above enzyme reaction solution (final volume 500 μl) was added to the reaction solution containing 0.375 units of D-amino acid oxidase (manufactured by Sigma) and reacted at 37 ° C. for 60 minutes to increase the absorbance at 500 nm. Was measured.

The optimum reaction temperature is 10 ° C. to 80 ° C. for 20 minutes in the same reaction solution composition as described above using a potassium phosphate buffer solution (pH 7.0). Phenol method [Bollter,
W. T. ) Et al., Analytical Chemistry (Analy)
33, 592-594 (196)
1)).

The above results are shown in FIGS. Comamonas sp. Optimal reaction with E222C decarbamylase
It can be seen that the pH is pH 8 to 9 and the optimum reaction temperature is 40 ° C.

Example 7 Comamonas sp. E222C strain decarbamylase amino
Acid Sequence Using the enzyme solution obtained in Example 5, the amino sequence at the amino terminus of the decarbamylase protein was determined. The enzyme solution was desalted using Centricon-10 Microcentrator (manufactured by Amicon), charged in a pulse type liquid phase protein sequencer, and analyzed. As a result, the amino terminal sequence of the decarbamylase protein was

[0054]

[Chemical 10] It was found that the 20th and 26th Arg peaks were unclear and could not be determined.

Example 8 Blastobacter sp. A17p-4 strain decarbamylase
Productivity-enhancing substances blast Enterobacter sp. When culturing the A17p-4 strain,
We searched for substances that enhance enzyme production that increase decarbamylase production. To the same medium D as used in Example 4, 0.15% (w / v) of the compound shown in FIG. 7 was added, and the cells were inoculated and cultured with shaking at 28 ° C. for 3 days. D-HPG produced by the method shown in Example 4 at a reaction temperature of 40 ° C.
Was quantified by HPLC, as shown in FIG. 7, it was found that when uracil, dihydrouracil or β-ureidopropionic acid was added, the production amount of decarbamylase was increased.

Further, in order to investigate the effect of adding metal ions, 0.15% (w / v) uracil was added to the medium D, and the metal ions shown in FIG. 8 were further added at a concentration of 2 mM. The cells were inoculated and cultured with shaking at 28 ° C. for 4 days. Then, when the produced D-HPG was quantified, as shown in FIG. 8, Fe ²⁺ , Fe ³⁺ , Li ⁺ , Cs ⁺ , Be ²⁺ , Mg ²⁺ , M
By adding ions such as n ²⁺ , Co ²⁺ and Al ³⁺ ,
It was found that the production of decarbamylase was increased.

Example 9 Blastobacter sp. Deca produced by A17p-4
Purification of Millase Blastobacter sp. A17p-4 was added to the same medium D used in Example 4 with 0.15% (w / v) uracil and 2 m.
FeSO ₄ .7H ₂ O of M was added and inoculated into this medium,
The cells were cultured at 28 ° C for 7 days with shaking. A total of 4.8 l of the culture solution was centrifuged, and the bacterial cells were mixed with 10 mM potassium phosphate buffer solution 12
Suspended in 0 ml, glass beads with a diameter of 0.25 mm [Dyno-Mill KDL]
(Switzerland)] for 5 minutes at 5 ° C., and the residue was removed by centrifugation. While ammonium sulfate was added to this crude enzyme solution, the ammonium sulfate precipitation fraction at a concentration of 20% to 40% of the saturated concentration was separated by centrifugation and dissolved in the above buffer solution.

41 ml of this enzyme solution was dialyzed against 5 l of the above buffer solution for 12 hours, and DE
Purification was done by AE-Sephacel column and Phenyl-Sepharose CL-6B column and concentrated by ultrafiltration. This concentrated solution (3 ml) was added with 0.2M NaCl.
Sephadex G-150 column in the above buffer containing
(φ1.5 × 80 cm) was subjected to gel filtration, the active fraction was desalted by dialysis, and then purified using the Mono QHR5 / 5 column by the method described in Example 5 to obtain the active fraction (2 ml).
Was used as a purified enzyme solution for analysis. By the above purification operation, as shown in Table 3, the specific activity was 37 times higher than that of the cell disruption supernatant, and the enzyme activity recovery rate at that time was 2.3%.

[Table 3]

When this purified enzyme was subjected to SDS-polyacrylamide gel electrophoresis, a single band was obtained at a molecular weight of around 39,000, as shown in FIG. Further, when gel filtration was carried out using a Sephadex G-150 column by the method shown in Example 5, as shown in FIG. 10, it was eluted at a molecular weight of about 120,000.

Example 10 Blastobacter sp. A17p-4 of decarbamylase
Properties Using the enzyme solution obtained in Example 9, Blastobacter sp. A
The optimum reaction pH and optimum reaction temperature of 17p-4 decarbamylase were investigated. In each case, the reaction was performed according to the method shown in Example 6. These results are shown in FIG. 11 and FIG.
Shown in. Blast Bactor sp. The optimum reaction pH of A17p-4 decarbamylase is 9, and the optimum reaction temperature is 50.
It was found to be ° C.

Example 11 Blastobacter sp. A17p-4 of decarbamylase
Amino acid sequence Using the enzyme solution obtained in Example 9, the amino-terminal amino acid sequence of the decarbamylase protein was determined by the same method as in Example 7. The amino-terminal amino acid sequence of the decarbamylase protein was:

[0062]

[Chemical 11] (The 38th Arg had an unclear peak and could not be determined.).

Example 12 For 7 strains of the genus Rhizobium and the genus Bradyrhizobium in Table 4, 1 ml of 805 liquid medium (1 g of yeast extract
/ l, mannitol 5g / l, K ₂ HPO ₄ 0.7g / l, KH ₂
PO ₄ 0.1 g / l, MgSO ₄ / 7H ₂ O 1 g / l, C-D-
HPG 1 g / l (pH 7.0)) with a final concentration of 1 g / l CD-
Inoculate into a medium supplemented with HPG or CD-Ala,
The cells were shake-cultured at 24 ° C for 24 hours. The bacteria were collected by centrifugation and 0.5 ml of 1% CD-HPG or CD-Ala,
It was suspended in 0.1 M K-phosphate buffer (pH 7.0), 0.1% Triton X-100. After reacting for 24 hours at 37 ° C. with shaking, analysis was carried out by thin layer chromatography by the method described in Example 1. As shown in Table 4, it was found that the strain of the genus Bradyrhizobium has decarbamyl activity.

[0064]

[Table 4] Strain name Decarbamylase activity Rhizobium loti ＋ (Rhizobium loti) IFO 14779 Rhizobium meliloti IFO 14782 Rhizobium fredii ＋＋ (Rhizobium fregaii ＋ Rhozobium FOV) Fukui ＋ (Rhizobium huakuii) IFO 15243 Bradyrhizobium japonicum ＋ IFO 14783 Bradyrhizobium sp. ＋ IFO 15003

Example 13 The soil was further screened by the method described in Example 1. Screening with N-carbamyl-
D-leucine (C-D-Leu), N-carbamyl-D-
Alanine (CD-Ala), N-carbamyl-D-phenylglycine (CD-PG) or DL-5-methylhydantoin (DL-Ala-hyd) was used as a carbon source or a nitrogen source. Of the grown bacteria, 9-
The decarbamyl activity was examined in the same manner as in Example 12 using D-Ala and CD-HPG. As shown in Table 5, the results show that strains with higher activity against CD-HPG and CD
-There were strains with higher activity against Ala.

[0066]

[Table 5] Strain name Growth substrate activity Rhizobium sp. KNK1415 C-D-PG +++ (C-D-HPG) Alcaligenes xylosoxidans subsp. Denitrificans CL66-2a C-D-Leu ++ (C-D-HPG) CL67-1 C-D-Leu ++ (C-D-HPG) CL85-1 C-D-Leu + (C-D- HPG) CA17-1 CD-Ala + (CD-Ala) Arthrobacter sp. CA17-2 C-D-Ala ++ (C-D-Ala) CA77-2 C-D-Ala ++ (C-D-Ala) AH71-1 DL-Ala-hyd + (C-D-Ala) AH57- 1 DL-Ala-hyd ++ (C-D-Ala)

Example 14 Rhizobium sp. 1 for KNK1415
0 l SE medium (sucrose 23 g / l, yeast extract 4
g / l, urea 2 g / l, KH ₂ PO ₄ 2 g / l, Na
_{2 HPO 4 2g / l, MgSO} 4 · 7H 2 O 1g / l,
MnCl ₂ .4H ₂ O (0.01 g / l (pH 6.5)) was inoculated and shake-cultured at 30 ° C. for 40 hours. After harvesting the cells by centrifugation, washing the cells with 0.9% saline solution, disrupting the cells with ultrasonic waves, removing the residue by centrifugation, and removing with protamine sulfate treatment (0.1 mg / mg protein) Nucleic acid.
Then, the centrifuged supernatant was heat-treated at 50 ° C. for 30 minutes, the denatured protein was removed by centrifugation, and ammonium sulfate was added to achieve 30% saturation. The precipitates were collected by centrifugation, dissolved in 500 ml of 20 mM Tris · HCl (pH 7.5), 2 mM DTT, dialyzed against the same buffer, adsorbed on a DEAE cellulose column, and adsorbed on a 10 mM Na-phosphate buffer (pH 7.
2), eluted with 0.15M NaCl, 1 mM DTT. This eluate was concentrated by ultrafiltration using a YM-10 membrane (Amicon). When this enzyme solution was analyzed by SDS-polyacrylamide gel electrophoresis, this decarbamylase was electrophoresed at around 35,000.

Example 15 SD in which the decarbamylase obtained in Example 14 was analyzed
The gel of the band portion of decarbamylase was cut out from the S-polyacrylamide gel, and the elution buffer (50 mM
Tris / HCl (pH 7.5), 0.1% SDS, 0.1 m
M EDTA, 150 mM NaCl, 5 mM DTT)
Homogenized in and eluted at room temperature. The extract was concentrated by ultrafiltration, and the reverse phase HPLC column (AP-
303; manufactured by YMC) and eluted with a concentration gradient of acetonitrile. The fraction containing this decarbamylase was added to a gas phase protein sequencer (Applied
(Made by Biosystems Inc.) and analyzed,
The amino terminal sequence of decarbamylase is

[Chemical 12] I found out.

[0069]

EFFECTS OF THE INVENTION D- which is important as an intermediate raw material of antibiotics
The discovery of a novel decarbamyl enzyme that produces α-amino acids has led to the discovery of new conditions such as pH8.
The reaction at ~ 9 and the reaction at 50 ° C can now be performed, and the D-amino acid can be efficiently produced.

[Brief description of drawings]

Figure 1: Five of the strains obtained by the new screening
The result of the decarbamyl reaction at 0 degreeC and 30 degreeC is shown.

FIG. 2 Comamonas sp. The effect of the compound added during the culture of E222C on the production of decarbamylase is shown.

FIG. 3 Comamonas sp. The result of having analyzed the purified decarbamylase of E222C by SDS-polyacrylamide gel electrophoresis is shown.

FIG. 4 Comamonas sp. The purified decarbamylase of E222C was purified by Sephadex G-150 column.
The result of gel filtration is shown.

FIG. 5: Comamonas sp. The result of having investigated the influence of pH at the time of reaction using the purified decarbamylase of E222C is shown.

FIG. 6 Comamonas sp. The result of having investigated the influence of the temperature at the time of reaction is shown using the purified decarbamylase of E222C.

FIG. 7: Blast Bactor sp. The effect of the compound added during the culture of A17p-4 on decarbamylase production is shown.

FIG. 8: Blast Bactor sp. The effect of metal ions added during the culture of A17p-4 on decarbamylase production is shown.

FIG. 9: Blast Bactor sp. The result of having analyzed the purified decarbamylase of A17p-4 by SDS-polyacrylamide gel electrophoresis is shown.

FIG. 10: Blast Bactor sp. The result of carrying out the gel filtration of the A17p-4 refined decarbamylase by the Sephadex G-150 column is shown.

FIG. 11: Blast Bactor sp. The result of having investigated the influence of pH at the time of reaction using A17p-4 purified decarbamylase is shown.

FIG. 12: Blast Bactor sp. The result of having investigated the influence of the temperature at the time of reaction is shown using purified A17p-4 decarbamylase.

Front page continued (72) Inventor Yukio Yamada 34-6 Minori Kakogawa-machi, Kakogawa-shi, Hyogo Prefecture (72) Inventor Hironori Namba 2-63 Kohama-cho, Takasago-machi, Takasago-shi, Hyogo (72) Inventor ▲ High ▼ Masayuki No, 299-3-106 Higashi-Futami, Futami-cho, Akashi-shi, Hyogo (72) Inventor ▲ Satomi Takahashi 1-13-13, Kawadai, Tarumi-ku, Kobe-shi, Hyogo (56) Reference JP-A-55-88697 (JP, 1988) A) JP 62-25990 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 13/00 -13/24 C12N 9/80 BIOSIS / WPI (DIALOG)

Claims (18)

(57) [Claims] 1. A general formula: [In the formula, R represents a phenyl group, a phenyl group substituted with a hydroxyl group, a substituted or unsubstituted alkyl group, or a thienyl group. ] An enzyme produced by a microorganism is allowed to act on an N-carbamyl-D-α-amino acid represented by the general formula: [In the formula, R has the same meaning as described above. ] Represented by]
In the method for producing an α-amino acid, as an enzyme decarbamylase for converting an N-carbamyl-D-α-amino acid into a D-α-amino acid, Blastobacte belonging to the genus Comomanas
r), D-α characterized by using an enzyme capable of being produced by a microorganism belonging to the genus Rhizobium or the genus Bradyrhizobium
-Amino acid manufacturing method. 2. A decarbamylase capable of being produced by the microorganism is used as a culture solution, a microbial cell, a crushed microbial cell, a microbial cell extract, a treated microbial cell, a crude enzyme, a purified enzyme, immobilized microbial cell or immobilization. The method according to claim 1, which is caused to act as an enzyme. 3. The microorganism is Comamonas sp. E222C
(FERM BP No. 4411), Blast Bactors
p. A17p-4 (FERM BP No. 4410), Rhizobium sp. KNK1415 (FERM BP No. 4419
Issue), Bradyrhizobium japonicum (Bradyrhiz
obium japonicum) IFO 14783 or bradyrizobium sp. The method according to claim 1 or 2, which is IFO 15003. 4. The method according to claim 1, wherein the microorganism is cultivated in a medium to which a decarbamylase production enhancing substance is added, and the produced decarbamylase is allowed to act on the microorganism. 5. The decarbamylase production enhancer is D-
The production method according to claim 4, which is an amino acid, an N-carbamyl-α-amino acid, a 5-substituted hydantoins, a pyrimidine metabolite or a metal ion. 6. The microorganism is a microorganism of the genus Comamonas, and the production-enhancing substance is urea, β-ureidopropionic acid,
The method according to claim 4, which is D-phenylglycine or N-carbamyl-DL-methionine. 7. The microorganism is a microorganism belonging to the genus Blastobacter and the production enhancer is uracil, dihydrouracil, β-ureidopropionic acid, Li ⁺ , Cs ⁺ , B.
The method according to claim 4, which is e ⁺ , Mg ²⁺ , Mn ²⁺ , Fe ²⁺ , Fe ³⁺ , Co ²⁺ or Al ³⁺ . 8. The method according to claim 1, wherein the pH of the aqueous medium is maintained at 6 to 11. 9. The method according to claim 1, wherein the pH of the aqueous medium is maintained at 7.5 to 9.5. 10. The method according to claim 1, wherein decarbamylase is allowed to act at 40 ° C. to 50 ° C. 11. A decarbamylase derived from a microorganism belonging to the genus Comamonas and having the following physicochemical properties: (a) Action N-carbamyl-D-α-amino acid is replaced with D-
(b) Molecular weight for converting into α-amino acid SDS-polyacrylamide electrophoresis 38,000, gel filtration 111,000 (c) optimum pH 8-9 (d) optimum temperature 40 ° C. 12. Comamonas sp. E222C (FERM
Decarbamylase according to claim 11, which is produced by BP4411). 13. The amino acid sequence on the amino terminal side is: The decarbamylase according to claim 12, which is 14. A decarbamylase derived from a microorganism of the genus Blastobacter and having the following physicochemical properties: (a) Action N-carbamyl-D-α-amino acid is replaced with D-
(b) Molecular weight for conversion into α-amino acid SDS-polyacrylamide electrophoresis 39,000, gel filtration 120,000 (c) optimum pH 9 (d) optimum temperature 50 ° C. 15. Blastobacter sp. A17p-4 (F
The decarbamylase according to claim 14, which is produced by ERM BP 4410). 16. The amino-terminal amino acid sequence is as follows: The decarbamylase according to claim 15, which is 17. Rhizobium sp. KNK1415 (FE
Decarbamylase derived from RM BP No. 4419) and having the following physicochemical properties: (a) Action N-carbamyl-D-α-amino acid is converted into D-
(b) Molecular weight converted into α-amino acid 35,000 by SDS-polyacrylamide gel electrophoresis. 18. The amino acid sequence on the amino terminal side is as follows: The decarbamylase according to claim 17, which is