JPS5820194A - Preparation of l-glutamic acid by fermentation method - Google Patents

Abstract

PURPOSE:To collect the titled substance produced and accumulated in a liquid culture medium, by cultivating a microorganism of the genus Escherichia having the ability to produce the titled substance in the liquid culture medium containing lactose or galactose as a carbon source. CONSTITUTION:A microorganism of the genus Escherichia, e.g. Escherichia coli No.283-5(NRRLB-12295)(tolerant to p-fluorophenylalanine) or Escherichia coil EG-19(NRRLB-12282)[tolerant to S-(2-aminoethyl)-cysteine], having the ability to produce L-glutamic acid is cultivated in a liquid culture medium containing lactose or galactose or milk whey or soybean whey containing them as a carbon source and ammonium ions, phosphoric acid ions vitamins, etc. under aerobic conditions. The aimed L-glutamic acid is then collected from the culture medium by the conventional method.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-glutamic acid by fermentation.

L-glutamic acid is produced using Phrineform bacteria of the genus Brevibacterium and Corynebacterium.
It is manufactured from carbon sources such as sucrose and acetic acid. However, these conventionally used lineform bacteria cannot assimilate either galactose or lactose, and therefore L- It was not possible to produce glutamic acid.

In contrast to the conventional method for producing L-glutamic acid as described above, the present inventors developed a strain of Escherichia microorganisms that has the ability to produce L-glutamic acid in high yield using galactose or lactose as a carbon source. We succeeded in separating them and completed the present invention.

Specific examples of the microorganisms of the genus Escherichia having the ability to produce L-glutamic acid used in the present invention include Escherichia coli AJ11499 (FERM-P53
01, NRRLB-12286), Escherichia coli A J 11545 (FERM-P5407, NRR
LB-122 (+9) Cao AJ11499 and AJ115
No. 45 was bred and isolated as follows.

(1) Escherichia coli EG-19 strain (K-1
2 (ATCC10798).

) in 1e medium (peptone 1%, yeast extract 0.5
%, Glucose 0.1%, NaCl 0.5%
; After culturing with shaking at 37c in PT (7, 21 co-adjusted) for about 3 hours, collect the bacterial cells in the logarithmic growth phase, and then use the phenol method.
Chromosomal DNA was extracted and purified using a conventional DNA extraction method, and a final amount of 3.4 mg was obtained.

(2) D of plasmid pBR322 as a vector
NA was prepared as follows. First, we infected Escherichia fistula carrying pBR322 as a plasmid.
One strain was added to 1 g of glucose-casamino acid-mineral salt medium (glucose 2 t1NH4C11f, NagHPO
46f, KHIPo.

32, NaC1591MgSO4e7HqOO, 1f.

CaCIg-2T (20°C, 15°F, casamino acid 2°T dissolved in 1E pure water and adjusted to pH 7.2 was used as the basic medium, to which was added 100μ2 of thiamine hydrochloride) was inoculated. , 37c until late logarithmic phase, 170 pf/yxl of chloramphenicol was added, and the cells were further cultured overnight.

This operation increases the amount of plasmid DNA in the cell.
t is produced. Bacteria were collected 16 hours after addition of chloramphenicol, lysed by lysozyme/SDS treatment, and supernatant obtained by ultracentrifugation at 3o, oooxr, for 1 hour. After concentrating the plasmid DNA from this, cesium chloride-ethidium bromide equilibrium density gradient centrifugation was performed to obtain a final 480 μm pBR322° plasmid DNA.
A fraction was collected.

+31 (Take 10 pf of the obtained chromosomal DNA and treat it with restriction endonuclease, Hlnd [7 and give 3
7C for 5, 10, 20, 30, or 60 minutes to allow partial or complete cleavage.

After heat treatment at 65tZ' for 5 minutes, it was mixed with pBR322 that had been completely digested with T(ind[l), and DNA ligation was performed using DNA ligase derived from T4 phage in the presence of ATP and dithiothreitol for 24 hours using IOC. .

After heat treatment at 65C for 5 minutes, twice the volume of ethanol was added to the reaction solution to precipitate and collect the DNA after the completion of the ligation reaction.

(4) Histidine auxotrophic strain El (R-13) and p-fluorophenylalanine resistant strain Jf6283-5, which were induced from Escherichia coli by nitrosoguanidine mutation treatment, were each incubated at 0.1 ml under ice cooling.
So-called competent cells were prepared by sequentially suspending each solution of M MgCl, O-I M C* CIt. To this competent cell suspension, (3)
Add the DNA solution obtained in step 1 and react for 30 minutes under water cooling.
Immediately, the cells were subjected to a heat shock of z42C for 2 minutes, and then cooled with water again for 30 minutes to incorporate the DNA into the cells. Next, a certain amount of the cell suspension was inoculated into a new L medium, and cultured with shaking at 37C for 2 hours to complete the transformation reaction. plate (glucose 291 (NH4), SO41f,
T(PO47f, IK)I2PO422, MgSO4
・7H,OO,1? , sodium citrate A0.5F, and AEC-T (CIO, 5f were dissolved in 1e pure water and adjusted to pH 7.2, and agar 209 was added to sterilize the solid medium. Ampicillin was added at 20 μf/me. The plated sushi was added so that

When the resulting colonies were picked and their properties examined, they all showed AEC resistance and ampicillin resistance. Therefore, these strains were used as transformed strains.

In this way, ERR-13 was used as a recipient strain for AJ11.
499 and A283-5 as the recipient strain AJ11
545 were obtained respectively.

The medium used for culturing such a microorganism of the genus Escherichia that has the ability to produce monoglutamic acid does not contain lactose or galactose as a carbon source.
It's nothing special. As lactose and galactose, milk whey, soybean whey, etc. containing these may be used. In addition to these carbon sources, the medium may also contain glucose, sucrose, etc. as auxiliary carbon sources. In addition to carbon sources, the culture medium contains ordinary nitrogen sources such as ammonium ions, ammonia gas, and aqueous ammonia, inorganic ions such as phosphate ions, magnesium ions, and potassium ions, and organic trace nutrients such as vitamins and amino acids. contained within.

Cultivation is carried out under aerobic conditions. During the culture, the culture temperature should be maintained at an appropriate temperature within the range of 27 to 37R, and the pH of the medium should be adjusted.
It is desirable to maintain H at an appropriate pi (in the range of 5.5 to 7.5).Thus, if the culture is continued for 1 to 4 days, a significant amount of L-glutamic acid will be produced and accumulated in the medium. .

Collection of L-glutamic acid accumulated in the medium can be carried out by a conventional method.

Example 1 Galactose 3.6tt/dl, (NH,), So,
2.51/117! ,KH,PO40,2y/dt
, MgSO4・7H, OO, 1t/di, yeast extract o
, o s t /dl, L-tyrosine 10.0 mg
/ d11 thiamine hydrochloride 100 μt / dt 5
FeSO, -71 (,01vr9/di and Mn804
A medium containing tatami 4H2011 tg/dl and adjusted to pH 7 was placed in a 500-proof flask at 2 Oygl and sterilized. One platinum loop of the bacterial strains shown in Table 1 was planted on this, and 3
Cultured at 1C for 48 hours. In addition, during cultivation, 4597d
0.3 d of urea water was added at a time to prevent the pH from decreasing. At the end of the culture, the amount of monoglutamic acid accumulated in the tube was as shown in Table 1.

1st Table l6283-5 828 AJ11545 1109
EG-19720 AJ11499 1050
Example 2 Lactose 597dl, (NH4)s 5o42.5t/
ctt, K) L, PO40, 2f/di, MgS
O4-7) 1,0 0.1t/di.

Yeast extract o, o sy/dl, L-tyrosine 1
0.089/dl, thiamine hydrochloride 100μf/
dt.

FeSO4-7H, Olvrg/dl, MnSO4・
4H1011g/di, and calcium carbonate 2.5
A culture medium adjusted to 7.0 and containing t/lri (separately added for sterilization) was sterilized by adding 50011/1 flask and sterilizing it. The cells were cultured for 72 hours.The amount of monoglutamic acid accumulated at the end of the culture was as shown in Table 2.

2nd Table 283-5 1050 AJ11545 1325 EC-19976 Example 3 5 as the following acid precipitated milk whey lactose? /des(Nl
(4), So, 2.5 f/dl, KH, PO4
0.2f/dl.

Mg5O, -7H, OO, 1f/dl, yeast extract 0.
05t/de,, L-Chiq Shiv 1o, o mg
/(H1thia = 7 hydrochloride 1o o 'pt /d
e, Fe1on-7T (, o 11117/d/
! Contains xMn 804-4Ht O1g17/dl and calcium carbonate 2.5t/dl (separately, added),
Prepare a medium adjusted to pH 7, 2T1m with KO)I and add 50
One 0yd flask was placed in each 20w5e flask and sterilized. One platinum loop of the bacterial strains shown in Table 3 was inoculated and cultured at 31C for 72 hours.

The accumulated mt of L-glutamic acid at the end of the culture is
It was as shown in Table t.

The ingredients of acid-precipitated milk whey are 93.9% water and 4.5% lactose.
h, total solid content 6.1 parts, nitrogen compounds 0.80 parts, -
The ash content was 0.70 inch and the fat content was 0.1 inch.

Table 3 A283-5 875AJ
11545 1055EC, -
19830 AJ11499 11 90
Patent applicant Ajinomoto Co., Inc.

Claims (1)

[Claims] A fermentation method characterized in that a microorganism of the genus Escherichia capable of producing monoglutamic acid is cultured in a liquid medium containing lactose or galactose as a carbon source, and monoglutamic acid produced and accumulated in the medium is collected. Method for producing L-glutamic acid.