JPH0822231B2 - Method for producing D-alanine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing D-alanine, which is extremely useful as a modifier for antibiotics, a raw material for synthetic sweeteners, and the like.

(Prior Art) Conventionally, as a method for producing D-alanine by an enzymatic method, a method of allowing D-hydantoinase to act on 5-substituted hydantoin (Japanese Patent Publication No. 56-1909) and N-acetyl-
Method for allowing D-aminoacylase to act on D-alanine (Japanese Examined Patent Publication No. 53-36035), D-amino acid transaminase (D-alanine aminotransferase; EC2.6.1.21; hereinafter, D-ATA) for pyruvate and D-amino acid. And the like) is known.

(Problems to be Solved by the Invention) However, these methods cannot be said to be economically excellent because the substrates used are expensive.

For example, when D-alanine is produced using D-ATA, pyruvic acid and an amino group donor D-amino acid are used as a substrate for the reaction, and the amino group donor D-amino acid is used for pyruvic acid. Excess amount is required.

(Means for Solving the Problems) The present inventors utilize D-ATA to convert D-ATA into inexpensive D
As a result of earnest research to establish a method for producing alanine, the present invention has been completed, focusing on the fact that DL-aspartic acid can be used as one having two functions of an amino group donor and a pyruvic acid source.

That is, the present invention provides D-ATA in the presence of a trace amount of pyruvic acid.
Is a method for producing D-alanine from DL-aspartic acid.

 The present invention can be represented by the following first formula.

That is, D-aspartic acid of the substrate DL-aspartic acid reacts with a small amount of pyruvic acid by the action of D-ATA to produce D-alanine and oxaloacetic acid. The oxaloacetic acid produced is chemically unstable and is naturally decarboxylated to give pyruvic acid. Therefore, even though the equilibrium constant of D-ATA is about 1, the product oxaloacetate does not accumulate, so the reaction proceeds toward the production of D-alanine. Further, pyruvic acid generated by decarboxylation of oxaloacetate is reused as a raw material keto acid.

The D-ATA used in the present invention can be prepared from microorganisms, plants and the like having D-ATA producing ability, and the origin thereof is not particularly limited, but specifically, Bacillus sp. YM. D-ATA (Japanese Unexamined Patent Publication No. 61-187786) and the like obtained from the -1 strain (Microtechnology Research Institute, Microbiology No. 8057).

In order to prepare D-ATA from these sources and use it in the reaction of the present invention to obtain D-alanine with high optical purity,
Of the enzymes produced by microorganisms and plants other than D-ATA, those that cause a decrease in the optical purity of D-alanine, for example, aspartic acid that decarboxylates L-aspartic acid to produce L-alanine. β-decarboxylase and D-
It is necessary to remove alanine racemase that racemizes alanine by purification, or to inactivate it by utilizing the difference in sensitivity to inhibitors, heat, pH and the like. For example, a plasmid pICT113 (Japanese Patent Application No. 62-39173) containing a gene encoding D-ATA derived from Bacillus sp. YM-1 strain.
No.) transformed into Escherichia coli (Esherichia c
When oli) is used, this D-ATA is highly thermostable, and therefore alanine racemase, which causes the optical purity of D-alanine produced by Escherichia coli, to be inactivated by heat treatment is preferable. is there.

In the reaction of the present invention, the substrate DL-aspartic acid is 50-1000 mM, preferably about 200 mM, pyruvic acid 0.1
~ 10mM, preferably about 1mM, D-ATA 0.1 ~ 10U / ml,
It is preferably added at a concentration of about 1 U / ml. Further, pyridoxal 5'-phosphate (hereinafter referred to as PLP), which is a coenzyme, is added in an amount of about 50 µM as needed.

The reaction temperature and the pH of the reaction solution are adjusted according to the optimum temperature and the optimum pH of D-ATA to be used, but are 25 to 60 ° C,
A pH range of 5-9 is preferred.

The reaction is usually carried out for 4 to 48 hours, but can be appropriately changed according to other conditions.

Collection of D-alanine from the reaction solution can be performed, for example, by deproteinizing the reaction solution with trichloroacetic acid or the like, then eluting D-alanine by cation exchange chromatography, and crystallizing by isoelectric focusing. .

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.

(Example) Example (1) Construction of plasmid pICT111 containing a gene encoding D-ATA Bacillus sp. YM-1 in the medium shown in Table 1 55
After culturing at 6 ° C for 6 hours, the cells were collected to obtain 6.0 g of cells.

Next, from the obtained bacterial cells, according to the Saito-Miura method, the chromosome DN was obtained.
A was extracted, and this was digested with Hind III (Takara Shuzo) for 3 hours to obtain a DNA fragment of 1 to 10 Kb.

Subsequently, 3 μg of the plasmid pBR322 was completely digested with Hind III, and this was ligated with the DNA fragment from the chromosome obtained previously by T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.), and the reaction solution was concentrated by ethanol precipitation.

Using this concentrate, Mandel-Hig (Mandel-Hig
Escherichia coli C600 was transformed by the method (a) (Journal of Molecular Biology (J. Mol, Biol.), 53 , 159 (1970)).

In order to select a strain having a plasmid containing a gene encoding D-ATA from among the transformants, the transformants were cultivated on agar of a minimal medium having the composition shown in Table-2. Since the target strain acquires D-glutamic acid assimilation as a nitrogen source, only the target strain can grow on this medium, and the grown strain was about 100 colonies.

Next, to confirm D-ATA activity in situ,
The method of Raetz et al. (Proceeding of
National Academy of Sciences USA (Proc.Natl.Acad.Sci.USA), 72 , 2274 (1975))
Was subjected to activity staining according to the method described below, which was partially modified. That is, a replica was created on a filter from about 100 colonies grown on a plate and treated with lysozyme (30 minutes at room temperature in 2 ml of lysozyme solution (10 mg / ml)).
After removing excess water, 1 ml of respiratory chain inhibitor (20 m
M NaN ₃ , 1 mM KF, 1 mM Na ₂ HAsO ₄ , 4 mM Tris-hydrochloric acid buffer (pH 7.4)) was added, and the mixture was twice frozen and thawed to lyse.

Subsequently, this was treated at 70 ° C. for 20 minutes, 1.5 ml of the reaction solution shown in Table 3 was added, and the reaction was carried out at 50 ° C. for 10 minutes. As a result, the strain having D-ATA activity exhibits a blue color,
One colony showing a strong blue color was selected. The plasmid contained in this strain was designated as pICT111.

Next, the method of Oka the PICT111 (Journal of Bact (J.Bacteriol.), 133, 916 (1978))
Isolated according to Pst I, EcoR I, Ban III, Hind III, Sa
Mapping was performed by using I (manufactured by Takara Shuzo). pICT111
Fig. 1 shows the restriction enzyme digestion map of E. coli.

(2) Sub-cloning of pICT111 H. pICT111. C. HC Birnboin and J. Doly Method (Nucreic Acids Research
Research), 7 , 1513 (1979)) and EcoR
It was completely digested with I (manufactured by Takara Shuzo Co., Ltd.), and after agarose gel electrophoresis, a gel containing an EcoR I-EcoR I fragment of about 1.7 Kb was cut out and the DNA fragment was eluted.

The plasmid pUC18 was completely digested with EcoR I, ligated with the purified pICT111EcoR I-EcoR I fragment by T4 DNA ligase, Escherichia coli JM103 was transformed with this ligated plasmid, and the transformant was transformed with isopropyl-1.
The cells were cultured on L medium containing -thio-β-D-galactoside, 5-chloro-4-bromo-3-indolyl-β-D-galactose and ampicillin. Here, a strain having D-ATA activity does not exhibit a blue color, so a strain not having a blue color was selected. The plasmid contained in this strain was designated as pICT113.

Next, pICT113 was isolated according to Oka's method, and PstI, EcoR
Mapping was performed with I, BanI II, Hind III, and Sal I. A restriction map of pICT113 is shown in Fig. 2.

H. pICT113. C. It was isolated according to the method of Burnboyne & Jay Dawley.

(3) Escherichia coli containing pICT113; Creation of Escherichia coli HB101 (pICT113) and D from the cell
− Collection of ATA enzyme solution Journal of Molecular Biology (J.
Mol. Biol.), 53 , 159-162 (1970), and Escherichia.
E. coli HB101 was transformed by contacting it with the plasmid pICT113.

Next, the strain was treated with ampicillin-containing YT medium (peptone 1 g, yeast extract 0.5 g, NaCl 0.5 g, ampicillin 5 g / 100 ml, pH 7.
2) Cultured in 750 ml at 37 ℃ overnight, and collected cells by centrifugation.

2.2 g of the recovered bacterial cells was added to 10 mM potassium phosphate buffer pH 7.
Suspended in 5 ml of 2,0.01% 2-mercaptoethanol, 50 μM PLP.

The cells were disrupted by ultrasonic waves and then centrifuged at 15,000 rpm for 20 minutes to collect the supernatant, thereby preparing a cell-free extract.

The obtained cell-free extract was heat-treated at 65 ° C. for 30 minutes and centrifuged to obtain a supernatant, which was used as an enzyme solution.

The D-ATA activity of this enzyme solution was measured by the following method.

Tris-HCl buffer (pH 8.1) 50 μmol, PLP 50 nmol, α
-Ketoglutarate 10 μmol, D-alanine 25 μmol, lactate dehydrogenase (Boehringer Mannheim Yamanouchi) 5U, NADH
A 1 ml reaction solution containing 0.2 μmol and enzyme was reacted in a cuvette at 50 ° C., and the decrease in absorbance at 340 nm due to the decrease in NADH was measured. Here, 1 U is the amount of enzyme that catalyzes the decrease of 1 μmol of NADH in 1 minute under these conditions.

As a result, the specific activity of D-ATA in the enzyme solution was 23.4 U / mg-protein.

(4) Production of D-alanine using the obtained D-ATA enzyme solution D-ATA10U, HEPEP-KOH buffer solution (pH 7.0) 50 μmol, DL-
200 μmol aspartic acid, 1 μmol pyruvic acid, 50 nmol PLP
1 ml of the reaction liquid containing the mixture was reacted at 50 ° C. for 48 hours.

The reaction was stopped by adding 100 μ of 12% trichloroacetic acid to the reaction solution, and the amount of alanine produced was measured by an amino acid automatic analyzer for a part of the reaction solution. as a result,
The amount of alanine produced was 79.4 μmol, and the conversion rate from the substrate D-aspartic acid to alanine was 79.4%.

The optical purity of the obtained alanine was determined by selectively decomposing L-alanine mixed in alanine with L-alanine dehydrogenase and measuring the amount of NADH produced. As a result, 99% or more of the produced alanine was the D-form.

Table 1 0.5% of polypeptone 0.2% yeast extract 0.25% meat extract Glycerol _{0.5% KH 2 PO 4 0.2%} K 2 HPO 4 0.2% NaCl 0.05% MgSO 4 · 7H 2 O 0.01% DL- alanine 0.3% L-glutamic acid 0.2% biotin 4 × 10 ^-7% 700ml pH 7.2 table 2 thiamine 1 mg L-leucine 10 mg L-threonine _{20 mg MgSO 4 · 7H 2 O} 50 mg MnCl2 · 4H2O 5 mg FeSO4 · 7H2O 0.25ng CaCl2 0.5 mg yeast extract 0.5 mg Potassium phosphate buffer (pH7.2) 20 mmol Ampicillin 25 mg D-glutamic acid 4 g Pyruvate 1 g 1 Table-3 Tris-hydrochloric acid buffer (pH8.3) 150 μmol D-cysteine sulfinic acid 1.5 μmol α- Ketoglutaric acid 15 μmol PLP 75 nmol Phenazine methosulfate 150 nmol Nitroble-tetrazolium 900 nmol (Effect of the invention) The present invention makes it possible to produce D-alanine by an economically excellent method.

[Brief description of drawings]

FIG. 1 is a restriction enzyme digestion map of plasmid pICT111. FIG. 2 is a restriction enzyme digestion map of plasmid pICT113. In the figure The part represents the inserted part.

Claims (1)

[Claims] 1. A method for producing D-alanine from DL-aspartic acid by the action of D-amino acid transaminase in the presence of a trace amount of pyruvic acid.