JPH01285193A - Production of d-aspartic acid - Google Patents

Abstract

PURPOSE:To obtain D-aspartic acid in high efficiency by conjugating malic acid dehydrogenase, alanine dehydrogenase, alanine racemase etc. to L-malic acid and ammonia in the presence of a small amount of alanine and NAD<+>. CONSTITUTION:The objective D-aspartic acid can be obtained by conjugating malic acid dehydrogenase (MDH), alanine dehydrogenase (AlaDH), alanine racemase (AlaR), and D-amino acid transaminase (D-ATA) to L-malic acid and ammonia in the presence of a small amount of alanine and NAD<+>. The concentrations to be used are as follows: L-malic acid as a substrate: 50-1,000mM, ammonia: ca. double the amount of L-malic acid, NAD<+>: 0.1-10mM, alanine D, DL, or L strain: 0.1-10mM, D-ATA, AlaR, AlaDH and MDH: ca. 0.1-10U/ml, respectively.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention provides D
-Regarding a method for producing aspartic acid.

(Prior art and problems to be solved by the invention) Conventionally,
Methods for producing D-aspartic acid by enzymatic methods include a method in which D-hydantoinase is allowed to act on 5-substituted L-dantoin (Japanese Patent Publication No. 1909/1983), and a method in which D-aminoacylase is added to N-acetyl-D-aspartic acid. A method of making the agent act (Japanese Patent Publication No. 53-36035) is known.

However, these methods cannot be said to be economically superior, as they use expensive substrates and, in principle, only half of the raw material can be converted into the target D-aspartic acid.

Furthermore, as a method to solve the above problem, we used an enzyme system that regenerates the amino group donor RI-amino acid.
D- from oxaloacetic acid and amino group donor RI monoamino acid
Amino acid transaminase (D-alanine aminotransferase; EC2,6,1,21, hereinafter referred to as D-A
We have discovered a method for producing D-aspartic acid by the action of TA (denoted as TA) (Japanese Patent Application Laid-Open No. 62-205790), and are currently conducting further research.

(Means for Solving the Problems) The present inventors utilized D-ATA to obtain D-ATA from inexpensive raw materials.
- As a result of intensive research to establish a method for producing aspartic acid, we discovered that malic acid and malate dehydrogenase (hereinafter referred to as MDI) are an NADH regenerating system that accompanies the amino group donor enzyme system that regenerates amino acids. ) system, even oxaloacetic acid, which is a raw material keto acid, can be produced and utilized, and the present invention was completed.

That is, the present invention provides MDH, alanine dehydrogenase (hereinafter referred to as A
1 aDH), alanine racemase (hereinafter referred to as Al
This is a method for producing D-aspartic acid from malic acid and ammonia in the presence of trace amounts of alanine and NAD+ by conjugating D-ATA (denoted as aR) and D-ATA.

The present invention can be expressed by the following first equation.

In the first equation, the substrate L-malic acid is converted to oxaloacetate by the action of MDI, and at the same time, NAD+ changes to NAD
It is reduced to H. The generated oxaloacetic acid reacts with D-alanine, which is an amino group donor, by the action of D-ATA to generate D-aspartic acid and pyruvic acid. D-alanine is regenerated from the generated pyruvate by the action of Al aDH%A1aR, and at the same time, NAD+ is converted from NADH.
is played.

MDH used in the present invention, A1 aDH, AlaR,
D-ATA can be prepared separately or simultaneously from microorganisms, plants, animals, etc. that have the ability to produce them, and the origin is not particularly limited. For example, MDH is derived from Escherichia coli
), Thernus fIavu
It can be obtained from microorganisms such as S), pig myocardium, and commercially available products can also be used.

AlaDH and AlaR can be obtained from microorganisms and plants including the genus Bacillus.
Bacillus stea
(rothermophilus) I F O125
50 can be cited as an enzyme that is excellent in terms of stability and productivity.

D-ATA' can also be obtained from microorganisms and plants including the genus Bacillus, and specifically, D-A' obtained from Bacillus sp. ATA (Unexamined Japanese Patent Publication No. 187786/1986)
can be mentioned.

In order to prepare the above four enzymes from these sources and use them in the reaction of the present invention to obtain D-aspartic acid with high optical purity, it is necessary to prepare the enzymes produced by the microorganism, plant, or animal in addition to the above enzymes. Among them, substances that cause a decrease in the optical purity of D-aspartic acid are removed by purification, or inhibitors.

For example, A 1 aDH and AlaR derived from Bacillus stearothermophilus IFO12550, which need to be inactivated by taking advantage of differences in sensitivity to heat, pH, etc.
Plasmid pICDRT111 (Japanese Patent Application No. 1407-1981) simultaneously contains the gene encoding D-ATA derived from Bacillus sp. YM-1 strain.
When Escherichia coli that produces MDI is transformed with 07), the transformed strain Escherichia coli HBI
01 (PICDRTIII) is suitable for the present invention because it can simultaneously produce the above four enzymes.

Monomalic acid, which serves as a substrate in the reaction of the present invention, has a concentration of 50 to
1000mM, ammonia is about twice the amount of malic acid,
NAD+ is 0.1-10mM, alanine is D, DL, or L-form is 0.1-10mM, D-ATA, AlaR
, AlaDH and MDH are each 0°1~10U
/ml. Further, pyridoxal 5°-phosphate (hereinafter referred to as PLP), which is a coenzyme, is added at about 1 to 100 μM as necessary.

The reaction temperature and pH of the reaction solution are adjusted according to the optimum temperature and optimum pH of the four enzymes used, but generally a range of 20 to 50°C and a pH of 6 to 10 is preferred. A buffer solution can be used, or ammonia, which is one of the substrates, can also be used.

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

To collect D-aspartic acid from the reaction solution, for example, add a trichloride to the reaction solution to give a final concentration of 5% (14/V).
Add acetic acid and add Alberlite IR-120 (
D-aspartic acid is passed through Dowex 1 (acetic acid type), passing through Dowex 1 (acetic acid type), allowing alanine, etc. to pass through, and then eluting D-aspartic acid with 2N acetic acid. be able to.

The present invention will be explained below with reference to Examples, but the present invention is not limited thereto.

(Example) Example 1 (1) Creation of plasmid PICTIII containing the gene encoding D-ATA Bacillus sp.
After culturing at ℃ for 6 hours, the bacteria were collected and the bacterial cells were collected. Obtained Og.

Next, the chromosomal DNA was extracted from the obtained bacterial cells according to the Saito-Miura method.
Extract A and use Hindl (manufactured by Hindshuzo) to
DNA fragments of 1 to 10 Kb were obtained by time digestion.

Subsequently, 3223 μg of plasmid pBR3 was added to ind I
This was completely digested with [ and ligated with the previously obtained DNA fragment from the chromosome using 74 DNA ligase (manufactured by Hinshuzo), and the reaction solution was concentrated by ethanol precipitation.

Using this concentrate, Mandelhiga
-Higa) method (Journal of Molecular Biology (J, Mol, B111.), 5
3°159 (197G)) was used to transform Escherichia coli C600.

In order to select a strain having a plasmid containing the gene encoding D-ATA from among the transformed strains, the transformed strains were cultured on agar in a minimal medium having the composition shown in Table 2. Since the target strain acquires the ability to assimilate D-glutamic acid as a nitrogen source, only the target strain could grow on this medium, and the number of colonies that grew was about 100.

Next, to confirm D-ATA activity in 5 situ, we used the method of Raetz et al. (Proc. Natl.

Acad, Sci, USA), 72.2274
Activity staining was carried out according to the following method, which was partially modified from (1975), that is, replicas were made on a filter from about 100 colonies grown on a plate,
Lysozyme treatment (lysozyme solution <io■/ml) 2m
After removing excess water, add 1 ml of respiratory chain inhibitor solution (20 mM NaN3.10
mMKF, 1mM Na HASO4, 4mM Tris-HCl MWI solution (pH 7,4)) were added, frozen twice, thawed, and lysed.

Subsequently, this was treated at 70°C for 20 minutes, and then 1.5 ml of the reaction solution shown in Table 3 was added and reacted at 50°C for 10 minutes. As a result, strains with D-ATA activity exhibit a blue color, and one colony exhibiting a strong blue color was selected. The plasmid contained in this strain was designated as plCTlll.

Next, plCTllll is a method of genius (Journal of Bacteriology (J, Bacteriol,
), L.B., 916 (1978)) and ps
Yu I, Fallen RI, Ban1l [, Hindlf [, 5al
I (manufactured by Hinshuzo) was mapped. plcT
The restriction enzyme cleavage map of 1ll is shown in FIG.

Table-1 Polypeptone 0.5% yeast extract 0.25% meat extract
0.2% glycerol 0.
5% KH2PO40.2% to 2HPO40.2% NaC10.05% MgSO4.7H200.01% DL-alanine 0.3% monoglutamic acid 0.2% biotin
4
50 ■ MnCl2-4H205ratz FeSO4・7H200,25n (I CaC120,5rst Yeast extract 0.5 ■ Potassium phosphate buffer (pt17.2) 20 ml
ol ampicillin 25 ■D-
Glutamic acid 4g Pyruvate
1gJ Table-3 Tris-HCl buffer (pH 8,3) 1'50
μmol D-cystine sulfinic acid 1.5μm
olα-ketoglutaric acid 15μ1oI
PLP 75 nmo
l Phenazine methosulfate 150 nmol
Nitro blue tetrazolium 900 nmol (
2) Subcloning of PICTl 11 PICTI
II to H, C, Burnpoint & G.A.
Dolly (H, C, Birnboin and J,
Doly) method (NIJCreiCACidS Re5earch)
').

E., 1513 (1979)) and isolated according to I.R.
After complete digestion with I (manufactured by Hinshuzo) and agarose gel electrophoresis, the gel containing the approximately 1.7 Kb 1 molar RI-fallen RI fragment was cut out and the DNA fragment was eluted.

Plasmid pUc18 was completely digested with EC0RI, and this was ligated with the purified L parallel RI-E mark RI fragment of pIcT112 using T4 DNA ligase, and Escherichia coli JM103 was transformed with this ligated plasmid.
The transformed strain was treated with isopropyl-1-thio β-D-galactoside, 5-chloro-4-bromo-3-indolyl-
Medium containing β-D-galactose and ampicillin (
1% polypeptone, 0.5% yeast extract, 0.5% Na
C1,0,1% glucose; pH 7,2).

Here, since strains having D-ATA activity do not exhibit blue color, strains that were not blue were selected. The plasmid contained in this strain was designated pIc7113.

Next, pIcT113 was isolated according to the method of Saito, and Ps
tI, EcoRI, Ban1ll, Hindl [
, 5atI was used for mapping. pIc711
The restriction enzyme cleavage map of No. 3 is shown in FIG.

PICT113 was isolated according to the method of H.C., Burnpoint and J.Dawley.

(3) Creation of plasmid pICD112 containing the gene encoding AlaDH The method described in JP-A-60-180590, pages 4-5 (Following this, we created pICR301 as referred to in the publication). (named pIcD112) was used to transform Escherichia coli C600.

Next, extract plcD112 from the transformed strain.

It was isolated according to the method of C., Burnpoint & G.A., Doley.

<4) Creation of plasmid pIcR113 containing the gene encoding AlaR Biochemistry
pICR401 was created (we named it plcR113) and transformed into Escherichia coli ceoo according to the method described in , 25° 3268-3274, (1986).

pIcR113 was isolated according to the method of H.C., Burnpoint & G.D., Doley.

(5) EcoRI-3ac of plasmid plcT113
Plasmid plcT11330μrt obtained in I fragment preparation (2)
MWI solution for 5acI containing 1300 U of sac (10
mM Tris-HCl buffer (pH 8,0), 7mM
MgCl, 7mM 2-mercaptoethanol (
2-ME), 0.01% bovine serum albumin (BSA))
After reacting and digesting in 400 µO at 37°C for 5 hours,
Add 3 μj of M NaCl and 850 μm of cold ethanol, and collect the DNA as ethanol precipitate by centrifugation. The recovered DNA pellet was dissolved in 20 μj of TE, and EcoRI buffer containing 1150 U of EcoR (5
0mM Tris-HCl buffer (1) H7,5), 7
mM MgCI, 100mM NaCl, 7mM
After reaction and digestion in 400 μm (2-ME, 0.01% BSA) for 5 hours, 800 μm cold ethanol was added and the DNA was recovered as an ethanol precipitate by centrifugation.

(6) EcoRV-Hln of plasmid pIcD112
dll [40 μg of pICD112 obtained in fragment preparation (3) was added to Tsuji Ichidan dl buffer (10 nH) containing 40 U of restriction enzyme H1ndI [(manufactured by Takara Shuzo Co., Ltd.)] Lis-hydrochloric acid buffer (+) H7
゜5), 7mM MgCI, 60mM Na'CI
) Reacted for 3 hours in 30Gμm, 5M NaC13
μj, 650μ of cold ethanol was added, and the DNA was recovered as an ethanol precipitate by centrifugation.
The NA pellet was dissolved in 20 μm of TH, and then mixed with RV buffer (10 mM Tris-HCl buffer (pH 7,5), 7
mM MgCI, 150 mM NaCI, 71N
2-ME, 0.01% BSA) 37 in 300 μm
The reaction mixture was incubated at ℃ for 3 hours, and the DNA was recovered as an ethanol precipitate by adding 600 μm of cold ethanol and centrifuging. This DNA was dissolved in 20 μιι TF.
．． 7%LM P (low meltino point
t) Agarose (Bethesda Re5ear
electrophoresis was performed at 100 V for 5 to 6 hours at 4°C using a
1.4 Kb Hi containing the gene encoding DH
The ndl-EcoRV fragment was separated, the agarose containing this fragment was cut out, 5 times the volume of TE was added, and the agarose was dissolved by heating at 65° C. for 5 minutes. This solution was extracted with phenol extraction, phenol-chloroform <1:1) extraction,
After performing chloroform extraction once each, DNA fragments were recovered as ethanol precipitation. Add this DNA fragment to a small amount of T.
Dissolved in E and subjected to reverse phase liquid chromatography (NENS
ORB"20, manufactured by DuPont), eluted with 50% ethanol, dried, and then dissolved in 20 μO TH.
Purification of plasmid plcD112 EcoRV-H1nd
l! Prepare 7r pieces.

(7) EcoRI-3ac of plasmid pIcR113
40 μg of plcR11 obtained in I fragment preparation (4) was treated with restriction enzyme 5
After reaction and digestion for 5 hours in 400 μm of 3 cl buffer containing 300 U of acI (manufactured by Takara Shuzo Co., Ltd.), 5M
Add 3 μm of NaCl and 850 μm of cold ethanol,
DNA was recovered as ethanol precipitate by centrifugation. The recovered DNA pellet was dissolved in 20 μm TH,
The DNA was reacted at 37° C. for 5 hours in 400 μm of IIWI solution for EcoRI containing 150 U of restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.), and 800 μm of cold ethanol was added to recover the DNA as an ethanol precipitate. This D
NA was dissolved in 20 μ m TH and electrophoresed using 0.7% LMP agarose at 100 V for 5 to 6 hours at 4°C.
The Kb EcoRI-3ac I fragment was isolated, the agarose containing this fragment was cut out, and 5 times the volume of TE was added.
The agarose was dissolved by heating at 5°C for 5 minutes, and the solution was extracted with phenol and phenol-chloroform (1:
1) Extraction and chloroform extraction were performed once each, and DNA fragments were recovered as ethanol precipitation. This DNA fragment was dissolved in a small amount of TH, purified by reverse phase liquid chromatography, eluted with 50% methanol, and dried to prepare a purified EcoRI-8ac I fragment of plasmid PICR113.

(8) EcoRI-3ac of plasmid plcT113
MuoRI-8a of I fragment and 1 lasmid pIcR113
c EcoRI of PICT113 prepared in I fragment ligation (5).

The Sac I digest was dissolved in 20 μm TH, and the BcoRI, 5ac I digest of pIcR113 prepared in (7) was dissolved in 20 μm TE. Eco of pIcT113
RI-8ac I fragment solution 1. uj, 8 μm of EcoRI-3ac I fragment solution of pICR113 was added to T4 DNA.
T4 DNA ligase buffer containing 700U of ligase (
66mM Tris-hydrochloric acid solution (DH7,6), 6.
The DNA fragments were ligated by reacting overnight in 20μ of 6mM MgCl, 10mM dithiothreitol (DTT), 1nHATP.

(9) Transformation of Escherichia coli HBOI with the DNA ligated in (8) Escherichia coli HBOI was transformed into 100 ml of YT medium (1 t of polypeptone, 0.!zr of yeast extract, 1 ml of NaCl).
G, 5 g/l GG ml, pH 7,2) for 2 to 3 hours, collect the bacterial cells by centrifugation, and place in a cold 100 m
After washing with M MgCl2, cold 100mM CaCl
Suspend in 2 and leave on ice for 1 hour! After removing the supernatant by centrifugation, the cells were resuspended in 125 ml of cold 100 mM CaC to obtain competent cells.

Next, 10 μm of the ligated DNA solution obtained in (8) and 200 μm of the competent cell suspension were mixed at 0°C, left on ice for 60 minutes with occasional stirring, and then heated at 42°C for 2 hours.
Let it stand for a minute and then quickly cool it on ice. Then add 1 ml of this suspension.
YT medium was added and cultured with shaking at 37°C for 1 hour, then routed to ampicillin-containing (50 μg/ml) YT-agar medium (agar 20t/j), and cultured overnight at 37°C, forming colonies. The cells were replated onto YT-agar medium containing ampicillin, and the colonies obtained by culturing at 37° C. overnight were used as transformed strains.

(10) Selection of transformants harboring the desired plasmid Ten of the 15 transformants obtained in (9) were cultured overnight in 5 ml of YT medium containing ampicillin (100 μg/ml). One lasmid of each was isolated on a small scale from. These plasmids were converted into EcoR according to method (5).
When digested with Sac I and Sac I, all 1 lasmids were cut at one point each, resulting in a single band of approximately 4.5 Kb.

Furthermore, double digestion with EcoRI and Sac I produced two bands of approximately 2.7 Kb and approximately 1.9 Kb, indicating that all plasmids were Ec of plcT113, which is derived from the vector pUC18.

It was found that one R1-3acI fragment and one EcoRI-3acI fragment of pIcR113 containing the gene encoding AlaR were fused. One of these lasmids was designated pIcR114.

(11) EcoRI-Ec of plasmid plcT113
40 ut of plCT113 obtained in preparation of oRI fragment (2) was first coR
3 in 400 μm of EcoRI buffer containing 11 sOU.
After reacting and digesting at 7°C for 5 hours, add cold ethanol 800
This DNA was dissolved in 20 μ'' of TH and separated by LMP agarose electrophoresis in the same manner as in (9).
Approximately 1.7 Kb DNA containing the gene encoding ATA
The A fragment was excised and purified in the same manner as in (7), and 20μ
Eco of plasmid pIcT113 was dissolved in TE of m.
A RI-EcoRI fragment solution was obtained.

(12) A portion of pIcR114 (approximately 4 ug) prepared by digestion of plasmid plcR114 with EcoRI (10)
, wIw liquid 5 for BcoRI containing IOU of EcoRI
After reacting and digesting in 0 μm at 37°C for 5 hours,
μm of cold ethanol was added, centrifuged to collect as ethanol precipitate, and dissolved in 10 μm of TE to obtain a solution of F, c and RI digests of plasmid plcR114.

(13) EcoRI-F of plasmid pIcT113,
Eco of plasmid pICT113 prepared by ligating the coRI fragment with the BcoRI digest of plCR114 (11)
8 µm of the RI-EcoRI fragment solution and 0.5 µm of the plcR114 EcoRI digest solution prepared in (12) were reacted at 16°C overnight in 20 µm of T4 DNA ligase buffer containing 700 U of T4 DNA ligase.
The IJi pieces were ligated.

(14) Transformation of Escherichia coli HBIOI with the DNA ligated in (13) Competent cells 2GGμ prepared in the same manner as in (9)
m and 10 μm of the reaction solution containing the DNA linked in (13) were mixed, and a transformed strain was obtained in the same manner as in (9).

(15) Selection of transformed strains with D-ATA activity (1
168 out of approximately 2000 transformed strains obtained in 4)
Buffered lacquer (10mM potassium phosphate buffer (pH 7.2), 0.01% 2-ME
, 50 μM pyridoxal-5°-phosphate (PLP))
D-ATA activity was measured using the enzyme solution that was suspended in water and disrupted by ultrasonication for 30 seconds.

-Measurement method of D-ATA activity- Tris-salt vI buffer (t)H8,1) 50. tzm
o 1 , PLP 50 nmo 1 , a-ketoglutaric acid 10 μmol, D-alanine 25 μmol, lactate dehydrogenase (LDH, manufactured by Boehringer Mannheim Yamanouchi)
A 1 ml reaction solution containing 5 U, NADHo, 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.

Note that IU was defined as the amount of enzyme that catalyzes the reduction of 1 μmol of NADH in 1 minute under these conditions. As a result of measuring the D-ATA activity of 168 colonies according to the above method, D-ATA activity was observed in two colonies.

(16) Confirmation of 1 lasmid in a strain retaining D-ATA activity (1
Plasmids were isolated on a small scale from the two strains selected in 5). Digestion of these plasmids with 5acI resulted in a single band of approximately 6.2 Kb, and digestion with EcoRI resulted in a band of approximately 4.5 Kb, the same as the EcoRI digest of pIcR114, and the EcoR of pIcT113.
A band of approximately 1.7 Kb, identical to the I-EcoRI fragment, was generated. Also, a 1 Kb band, EcoR of pIcR114.
The same approximately 1.9 Kb band as the I-dan acl fragment and the p
Since these plasmids produced a 2.7 Kb band, these plasmids were added to the EcoRI digest of pIcR114.
It was found that the EcoRI-EcoRI fragment of T113 was combined.

These plasmids were named pICRTI11.

Therefore, the obtained plasmid plcRT111 is Ala
This is a plasmid that simultaneously contains a gene encoding R and a gene encoding D-ATA.

(17) Preparation of plasmid pIcRT111 Purified pICRTI1 was purified from Escherichia 3988101 strain transformed with pIcRTll by the method described above.
1 was prepared.

(18) Hlodl of plasmid pIcRT111 [[
- Preparation of 3maI fragment 5 μt of plasmid pIcRT111 and 20U of SmaI
HWI solution for SmaI (10mM) containing (manufactured by Hinshuzo) Lis-hydrochloric acid buffer 1pH8, O), 7mM MgC12,
20mM KG1, 7mM 2-MEO,0
After reaction and digestion at 37°C for 5 hours in 50 μl of 1% BSA, 11 μl of 5M NaC and 100 μl of cold ethanol.
μ'' was added and the DNA was recovered as an ethanol precipitate by centrifugation.

The recovered DNA pellet was dissolved in 20 μm TH and
H1ndllI buffer containing 20U of lndl[[50
After reaction and digestion in μm for 5 hours, 5M NaCl
0.5 μm and 100 μm of ethanol were added and centrifuged to recover the DNA as an ethanol precipitate.

The recovered DNA pellet was dissolved in 20 μm TB and p
It was made into a Hl ndl [-3maI fragment solution of IcRTlll.

(19) Hindl-Ec of plasmid pIcD112
oRV fragment and plasmid p ICRTI 11 Hi
prepared by ligation (6) of the ndlll-3maI fragment and fip
Hlndl of IcD112[-EcoRV fragment solution IQ
μj and Hlnd of pIcRTlll prepared in (18)
ll[-3maI fragment solution (10 μm) was reacted overnight in 30 μm of T4 DNA ligase M solution containing 700 U of T4 DNA ligase to ligate the DNA fragments.

(20) Transformation of Escherichia coli HBOI with the DNA ligated in (19) Escherichia coli HBOI was cultured in 100 ml of YT medium for 3 hours, and the bacterial cells were collected by centrifugation. After washing with mM Mg CI2, suspend in cold 100mM CaCl2 and leave on ice for 1 hour.Then, remove the supernatant by centrifugation, and incubate in cold 100mM CaCl2.
The cells were resuspended in 125 ml of 0 m M Ca C to form competent cells.

5 μm of the ligated DNA solution obtained in (19) and 200 μm of the competent cell suspension were mixed at 0°C, placed on ice for 60 minutes with occasional stirring, left at 42°C for 2 minutes, and rapidly cooled on ice. Then add 1 ml of YT to this suspension.
After adding the medium and incubating with shaking at 37°C for 1 hour, it was plated onto ampicillin-containing (50 μg/ml) YT-agar medium (20 t/Jl of agar), cultured overnight at 37°C, and the formed colonies were incubated with ampicillin. Contains YT
- The colonies obtained by re-plating on agar medium and culturing overnight at 37°C were used as transformed strains.

(21) Selection of a transformed strain carrying the desired plasmid 1) Create a replica of the transformed strain obtained in (20) on an A1aDH activity staining filter, and place it at the Proceedings of the Natural Academy of Sciences. -・Science of Ninisny,
72.2274-2278 (1975), a 50 m M glycine-KCI-KOHW solution [
pH 9,0), 50mM L-alanine, 1.3
2 ml of a reaction solution containing 0.128 mM phenazine methosulfate (PMS) and 0.48 mM nitro blue tetrazolium (NBT).
゛Transformed strains having A1aDH activity were identified and isolated by color development.

2) Confirmation of plasmid Six of the strains possessing Al aDH activity obtained in 1) were treated with ampicillin-containing (100 μt/ml) YT.
Each plasmid was isolated on a small scale by culturing overnight in 5 ml of medium.

These plasmids were transferred to Hindll according to the method (6).
When digested with [, all plasmids were excised at one point each, resulting in a single band of approximately 7.6 Kb.

Additionally, EcoRI digestion produced a band of about 5.9 Kb and a band of about 1.7 Kb, which is the same as the EcoRI-EcoRI fragment of pIcT113. Ec.

Double digestion of R1,5 acl yielded the 1.7 Kb band described above, a ˜1.9 Kb band identical to the EcoRI-3 acl fragment of plcR113, and a ˜4° I Kb band.

In double digestion of anti-L11, Hin d 1M, p
K of lcRTIII
! Approximately 6.2 Kb band same as the digest, and pIcD
112 coRV-H1ndl [approximately 1.4
A band of Kb was generated.

From the above, these plasmids are PICRTI
Hindll-3ma I of II, fragment and pICD
It was found that 112 EcoRV-HinduIIr fragments were combined.

This plasmid was named pIcDRTlll.

That is, the obtained plasmid p ICDRTI 11 is A
This is a plasmid containing a gene encoding laR, a gene encoding D-ATA, and a gene encoding A1 aDH.

(22) Escherichia including p ICDRTI 11
coli; Escherichia coli HBOI (pICDRT
III) Creation and collection of enzyme solution from the bacterial cells Journal of Molecular Biology (J
, Mat, Biol,), Record, 159-162
(1970), calcium chloride-treated Escherichia coli HBIOI was transformed by contacting with plasmid p ICDRTI 11 at around 0°C.

Next, strain YT containing ampicillin (5g/100ml)
Cultured in 750 ml of medium at 37°C for one night, and collected cells by centrifugation. 2.2 g of collected cells were diluted with 10 mM
Potassium phosphate buffer pH 7°2, 0.01% 2-mercaptoethanol, 50 μM PLP 501
1.

After crushing the bacterial cells with ultrasonic waves, 2
The supernatant was collected by centrifugation for 0 minutes, and a cell-free extract was prepared.

Next, the enzyme activity of the obtained cell-free extract was measured by the following method.

- Method for measuring D-ATA activity - Method for measuring AlaR activity by the method shown in (15) - Glycine-KCI-KOHIli solution (pH 9,0) 10
0 μnol, PLP50nmol, D-alanine 50μ11゜1, NAD” 2.5μ1loI
, Al aDH (from Bacillus stearothermophilus IF012550) 10U and enzyme 1
ml of the reaction solution was reacted in a cuvette at 50°C, and the NA
The increase in absorbance at 340 nm resulting from the production of DH was measured. Note that IU is 1 minute per minute under these conditions.
The amount of enzyme that catalyzes the production of NADH in μ1101 was taken as the amount.

-A1 Method for measuring aDH activity- Glycine-KCI-KOHM buffer solution (pH 10,7) 10
0μm1G+, L-alanine 50μmol, N
AD” 2゜5μ11ol and 1ml containing enzyme
The reaction solution was reacted in a cuvette at 50°C, and NADH
The increase in absorbance at 340 nm resulting from the production of was measured.

Note that IU is 1 μsol of N per minute under these conditions.
This was defined as the amount of enzyme that catalyzes the production of ADH.

-Measurement method of MDI activity- Tris-HCl buffer (pH 8,0) 50 μsol
, L-malic acid 20 μlot, NAD” 2.5
1 ml of the reaction solution containing μsol and enzyme was heated at 30°C.
The reaction was carried out in a cuvette, and the increase in absorbance at 340 nm resulting from the generation of NADH was measured. Note that IU here was defined as the amount of enzyme that catalyzes the production of 1 μsol of NADH in 1 minute under these conditions.

As a result, D-ATA activity was 1040U/ml, A
laR activity is 8G, 7U/ml, Al aDH activity is 106 U/ml, MDH activity is 18.5LJ/m
It was l.

Next, this cell-free extract was treated at 50°C for 30 minutes to obtain a heat-treated enzyme solution.

(23) Production of D-aspartic acid using the obtained heat-treated enzyme solution Next, 10 μm of this heat-treated enzyme solution, 100 μm of ammonium malate buffer (pH 8,0),
L-alanine 1μ1IOl, NAD” 0.1
, tclloI, and PLP50n101 were reacted at 37° C. for 24 hours.

After the reaction is complete, use an amino acid analyzer and Chiral Pack WH (
The amount of D-aspartic acid produced in the reaction solution was measured by high performance liquid chromatography (manufactured by Daicel Chemical). As a result, the amount of D-aspartic acid obtained was 28°7 μsol.

Example 2 The reaction was carried out in the same manner as in Example 1, except that the amount of heat-treated enzyme solution was changed to 20 μm. As a result, the amount of D-aspartic acid obtained was 40.6μIIol.

(Effects of the Invention) The present invention has made it possible to produce D-aspartic acid using an economically superior method.

In addition, Escherichia coli HBIOI (pICDRT
By using III), the method of the invention can be carried out very simply.

[Brief explanation of the drawing]

FIG. 1 is a restriction enzyme cleavage map of plasmid plcT111. FIG. 2 is a restriction enzyme cleavage map of plasmid pIc7113. Patent applicant Daicel Chemical Industries, Ltd. Figure 1 Restriction enzyme cleavage map of pIcTlll Figure 2 Restriction enzyme cleavage map of pIc7113 (Kb)

Claims (1)

[Claims] By conjugating malate dehydrogenase, alanine dehydrogenase, alanine racemase, and D-amino acid transaminase, trace amounts of alanine and NAD^+
A method for producing D-aspartic acid from L-malic acid and ammonia in the presence of