JPH04169187A - Simultaneous fermentative production of neutral amino acid and glutamic acid - Google Patents

Abstract

PURPOSE:To obtain the subject amino acids at an industrially lower cost by adding a surfactant to a culture medium, culturing a neutral amino acid- producing bacterium belonging to Brevibacterium genus in that culture medium and carrying out the subject simultaneous fermentative production of the neutral amino acids and L-glutamic acid. CONSTITUTION:A mutant (e.g. Brevibacterium FERM-1769) belonging to Brevibacterium genus or Corynebacterium genus and capable of producing neutral amino acids selected from L-Leu, L-ILe, L-Val, L-Thr, L-TrP, L-Phe, L-Tyr, L-citrulline and L-homoserine is cultured in a culture medium containing a surfactant (e.g. polyoxyethylene sorbitan monopalmitate).

Description

Detailed Description of the Invention (Industrial Application Field) When fermenting neutral amino acids by culturing neutral amino acid-producing mutant strains belonging to the genus Brevibacterium or Corynebacterium in a medium, a surfactant is added to the medium. The present invention relates to a method for co-fermenting and producing a neutral amino acid and glutamic acid, in which L-glutamic acid is simultaneously fermented by adding L-glutamic acid.

(Prior art) Conventionally, L-leucine (Leu),
L-isoleucine (Il+, l, L-valine (Vat), L-threonine ITh・i, L-tryptophan (Tap), L-phenylalanine (Phe), L-tyrosine (D7+),
L-citrulline (cit) and L-homoserine (use
) and other neutral amino acids are industrially produced.

However, there is still a strong desire to further reduce production costs.

Incidentally, it is known that a surfactant is added to the culture medium in the fermentation production of neutral amino acids, but this is only as a means to improve the yield of a single target amino acid. Co-production of glutamic acid (Glu) is not observed (see JP-A-62-288).

(Problems to be Solved by the Invention) An object of the present invention is to provide a method for industrially producing amino acids at a lower cost by co-producing Glu in the production of neutral amino acids by fermentation.

(Means for Solving the Problems) As a result of intensive research in order to solve the above problems, the present inventors cultivated a neutral amino acid-producing mutant strain belonging to the genus Brevibacterium or the genus Corynebacterium in a medium. The present inventors have discovered that when a surfactant is added to a culture medium during the fermentation of sexual amino acids, Glu is simultaneously produced, leading to the present invention.

That is, the present invention is directed to the use of plants belonging to the genus Brevibacterium or Corynebacterium and L-leucine, L-isoleucine, L-noculin, L-threonine, L-tryptophan, L-phenylalanine, L-tyrosine, L- A method for fermentatively producing a neutral amino acid by culturing a mutant strain that produces either citrulline or L-homoserine in a medium, which method comprises adding a surfactant to the medium. The present invention relates to a method for simultaneously fermenting and producing L-glutamic acid and L-glutamic acid.

According to the method of the present invention, the total amount of amino acids produced from raw sugar can be increased by the co-production of glutamic acid, rather than producing each neutral amino acid individually. Incidentally, from an economic point of view, Glu that is co-produced is usually [1,
It is desirable to accumulate 05g/c11 or more.

The method of the present invention will be explained in detail below.

In carrying out the method of the present invention, the seed culture, main culture, isolation of the target amino acid from the fermentation liquid as needed, etc. can all be carried out by conventionally known fermentation production methods for each neutral amino acid. The difference is that a surfactant is added to the culture medium and that not only neutral amino acids but also glutamic acid are isolated from the fermentation solution if necessary. Separation of amino acids as a mixture or each individual amino acid, fractionation of an amino acid mixture into each amino acid, etc. can be carried out by methods conventionally known to those skilled in the art, so there is no need to add a special explanation here.

Therefore, the addition of a surfactant will be explained below.

Examples of surfactants that can be used in the method of the present invention include:
Anionic surfactants such as higher alcohol sulfates, alkylbenzene sulfonates, alkyl phosphates, dialkyl sulfosuccinates, cationic surfactants such as alkylamine salts and quaternary ammonium salts, betaine, somidazoline betaine, etc. Examples include amphoteric ionic surfactants, polyoxyethylene alkyl esters, and nonionic surfactants such as polyoxyethylene sorbitan monoalkyl esters. These surfactants may be used alone or in combination of two or more.

The effective amount of surfactant to be added to the culture medium varies depending on the neutral amino acid-producing bacteria, the surfactant, the amount of seed inoculated, etc.
Usually 0.00, which a person skilled in the art can very easily determine in a given case by simple preliminary experiments with varying amounts of addition.
It is within the range of 01 to 5 g/dl. In addition, please also refer to Examples described later. (Example) Hereinafter, the present invention will be further explained with reference to Examples.

Example 1 (Lea) 5 Qd of an aqueous seed medium having the composition shown in Table 1 was dispensed into a 500 d shaking flask and sterilized by heating at 120° C. for 15 minutes. This is combined with Brevibacterium lactofermentum (B+evibacterinmlxclof!+
Menuam) FERM P-1769 was taken from an agar slant culture and inoculated by one platinum loop, and incubated at 31.5°C for 24 hours.
A seed culture solution was obtained by shaking culture for hours.

Table 1 Ingredients Concentration Glucose 3 g/diKH2P04
G, 1 gldiM g S O40
,04g/d IDL-Methionine 40
■/dIFe++ 1
■／dIM n++ t
■/di “Mameko” (as total nitrogen) 200
■/diV B 1o, 2 wg/d 1 biotin 0,05■/di urea
0.3 g/djL-tyrosine
40 ■/dirGD 113 J
*0.005m1/di'Ca CO35N/d
, 1 pH 7.0 (N a OH) Polyoxyethylene polyoxypropylene glycerin ether On the other hand, polyoxyethylene sorbitan mono I lumitate was added as a surfactant to an aqueous medium having the composition shown in Table 2.
．． Add 15g of main fermentation medium to 500m shoulder flask 1
The solution was dispensed into 2 Od bottles each and sterilized by heating at 120° C. for 15 minutes.

Table 2 Ingredients Concentration Glucose 6 g/dj! solid
In 0
．． 1 g/d IM g S Oi
, o, rJ 4 g/d
j! (NH4)2S04 4 g/dl Biotin 0.05■/dlV B IO,3■
/d I DL-Methionine 70 ■/dlKH2
PO40, 1g/di F e S 04 1”g
/ d IM n SO41tag / d 1 "Mameko" (as total nitrogen) 625 ■/dirGD-
113J O,002d/alpH7,
0 (KOH) 5 g/cl of C a CO 3 that had been dry heat sterilized in advance was added to the medium in each flask, and 3 d of the above seed culture solution was added and cultured at 31.5° C. for 96 hours.

Combine the culture fluids from each flask and combine the Lea and Gl in the culture fluid.
When the accumulated amount of u was quantified, it was 0.54 g/
dj! and 1.91 g/dl for a total of 2.45
g/dl.

For comparison, the results of culturing without adding surfactant are as follows.
The accumulated amount of LeLl and Glu is 114g/
dl and Og/di, and no co-production of Glu was observed.

Example 2 (lit) Polyoxypropylene pentaerythritol stearate was added as a surfactant to an aqueous medium containing glucose as a carbon source and having the composition shown in Table 3 to give It! A production medium was prepared, and after adjusting the eH to 7.2, it was dispensed into 10 shaking flasks of 5 [10 m capacity each for 20 d, and sterilized by heating at 115° C. for 10 minutes.

Table 3 Glucose L5. Ogldi(NH4
)2SO41,Og/di KH2PO40,15g/di MgS0 ・7H200,04g/diF e S
0 ・4 H201,0”g/d j'Mn50
・4H201,O■/d1 Thiamine hydrochloride 3(lG, fl R/dl
.

Soybean protein acid decomposition solution 25.0 ■/di (as total nitrogen) Biotin 5.01197di
ERM BP-759 was inoculated and cultured with shaking at 315°C until glucose disappeared. The time required was approximately 72 hours.

Combine the culture fluids from each flask and
When the accumulated amount of u was quantified, it was 1.07 g/
dl and 1.5 (Ig/d/, total 2.57 g/dl
there were.

For comparison, the results of culturing without adding surfactant are as follows.
The accumulated amount of 11ε and Glu is 1.8[1g
/dj! and Og/d1, and no co-production of Glu was observed.

Example 3 (Vxl) 0.20 g/di of cationic MAJ (myristylamine acetate, manufactured by NOF Corporation) was added as a surfactant to an aqueous medium shown in Table 4 containing glucose as a carbon source to produce L-valine. After preparing a production medium and adjusting the pH to 7.0, 20-ml of the medium was poured into each of ten 500 td shaking flasks, and sterilized by heating at 115° C. for 10 minutes.

Table 4 Ingredients Concentration Glucose 15.0 g/d 1(N
H4) 2So4 3. Og/dfKHPO 240,1g/di MgSO-7F(200,(14g/d1Fe50
・4H201, fl tar/diM n S 0
・4 H201゜D ■/dithiamine dihydrochloride 30. [l 119/di soybean protein acid decomposition solution 80.0 ■/d1 (as total nitrogen) DL-methionine 60. Ottg/di biotin
5. Brevibacterium lactofermentum (B + evibxcterium Iac + olerm) obtained by pre-culture in the medium of each flask with OIII/di (sterilization addition of calcium carbonate 5.0 gldi)
en+um) FERM P-1,845 (FERM
HF-1061 was inoculated and cultured with shaking at 31.5°C until glucose was consumed.

The accumulated amounts of Yal and Glu in the culture solution were determined by combining the culture solutions of each flask, and found that each was 0.87 g.
/di and 1.52 g/dl for a total of 2.39 g/di.
There was d1.

For comparison, the results of culturing without adding surfactant are as follows.
■! The accumulated amount of 1 and Glu is 1.80g/
cl and Og/d1 and no co-production of Glu was observed.

Example 4 (Tb+) Brevibacterium flavum (Br
ewibacje+ium [11vum)FERM
A seed culture solution of P-4164 was obtained.

On the other hand, polyoxyethylene sorbitan monopalmitate was added as a surfactant to an aqueous medium having the composition shown in Table 5.
Add 15g of main fermentation medium to a 500d shoulder flask lO
Each volume was dispensed into 20H1 volumes and sterilized by heating at 115°C for 15 minutes.

Table 5 Ingredients Concentration Glucose 6 g/diBetaine
0. l g / d IM g S O
4o, 04 g/d 1(NH4)2SO43g
/dl Biotin [05■/divB1
3 N/di.

KH2PO40, 1g/di F e S Oi, 1 ■
/ d IM n S Ot,
L ■/d 1 “Mameko” (as total nitrogen) 60
■/d/G D -1130,002d/d
/ pH 7.4 (KOH) Add 5 g/dl of C a CO3, which had been dry heat sterilized in advance, to the medium in each flask, and then add 3 d of the above seed culture solution and culture at 31.5°C for 96 hours. did.

Combine the culture fluids from each flask and add Thr and Gl in the culture fluid.
When the accumulated amount of u was quantified, each OJ1g/d
1 and 1.03 g/dl, for a total of 1.34 g/d1.

For comparison, the results of culturing without adding surfactant are as follows.
The accumulated amounts of Thr and Gin are each 0.95g/
cl' and Og/dN and no co-occurrence of Glu was observed.

In addition, bacterial cells and CaCo are removed from the fermentation liquid by centrifugation.
Except for 3, the supernatant was treated with an ion exchange resin according to a conventional method to obtain Thr and Glu, respectively.

Example 5 (Phel Brevibacterium e lactofermentum (B+eyibxctt+iumlactoie
A seed culture solution of ERM l'-5417 (+mentum) was obtained.

On the other hand, 0.15 g of polyoquinoethylene sorbitan monopalmitate was added as a surfactant to the aqueous medium shown in Table 6.
The added main fermentation medium was dispensed into 10 500-mem shoulder flasks, 20 times each, and sterilized by heating at 110° C. for 10 minutes.

Table 5 Ingredients Concentration Glucose 6 g/d 1 Betaine 0.1 g/dIMg S O4G
, l g/d 1(NH4)2SO41g/d
l Biotin 005■/diVB,
2 ■/diDL-methionine
40 ■/cilKH2PO40,1g/
di M n S 041 11g / d 1 "Taste liquid J
5 M1/diL-tyrosine 40 ■/diacetic acid
03 Me/di fumaric acid 1.2 g/di 5 g/di of CaCO3, which had been dry heat sterilized in advance, was added to the medium in each flask, and the above seed culture solution was added in 3 d portions at 96° C. at 31.5°C. Cultured for hours.

Phe and GI in the culture solution were combined by combining the culture solutions of each flask.
When the accumulated amount of + was quantified, IJOg/d
j and 0.10 g/dJ, for a total of 1.40 g/di.

For comparison, the results of culturing without adding surfactant are as follows.
The accumulated amount of Pbe and Glu is 1.30g/each.
d/ and Og/d/, and no co-production of Gla was observed.

Example 6 (TYr) An aqueous seed medium having the composition shown in Table 7 was dispensed for 50 days into a 5OOd volume shaking flask, and after sterilization by heating at 110°C for 5 minutes, Corynebacterium glutamicum (Cor.
ynebscterium glujxrAicam)
FERMP-5836 was inoculated and cultured with shaking at 31.5°C for 16 hours to obtain a seed mother culture.

Table 7 Medium composition On the other hand, an aqueous main fermentation medium having the composition also shown in Table 7 was used at 50%
Dispense 20 d into 10 0 ge capacity shoulder flasks, add 0.15 g/dl of polyoxyethylene sorbitan monopalmitate as a surfactant, and add 0.15 g/dl of polyoxyethylene sorbitan monopalmitate as a surfactant.
After sterilizing by heating for 3 minutes, 3 d of the above seed culture solution was added to the medium in each flask and cultured at 31.5° C. for 96 hours.

Combine the culture fluids from each flask and add TH and Gla in the culture fluid.
When the accumulated amount of each was quantified, it was 0.70 g/d.
j! and 0.25 g/d1, for a total of 0.95 g/dZ.

For comparison, the results of culturing without adding surfactant are as follows.
The accumulated amounts of T7 and Glu were 0 and 78 g, respectively.
/ d j and Og/d1 and no co-production of GIO was observed.

Example 7 (Cil Glucose 3g/di, K H2P O4
G, Ig/df, Mg50 ・7 H20G,
04 g/d'.

Fe50 ・7H201,0■/djl, MnSO4
・4H201, G■/d1. Urea 0.3g/dl, soybean protein acid hydrolyzate 60μ/di as total nitrogen.

Thiamine/HCl30γ/di, biotin20
An aqueous seed medium containing γ/di was adjusted to pH 6.0, and the 50Id was placed in a 500 d shoulder flask and sterilized by heat.

In addition to this, Corynebacterium glutamicum (Cor7n
eb*c+e+iom glulamicum) FE
Inoculated with RM P-5643 and kept at 31.5°C,
A seed mother culture solution was obtained by culturing with shaking for 13 hours.

On the other hand, glucose 13g/dl KH2PO40, Ig
/ di , M g S 0 ・7 H20o,
04 g/dl, F e S 0 ・7
H20t, Qmg/dl.

Mn S 0 ・4 H201,0■/di, ammonium sulfate 3.0g7di, soybean protein acid hydrolyzate as total nitrogen 65■/dl, thiamine・HC/20γ/d1
．． Biotin 10γ/di, L-arginine 70■/dl
A medium prepared by adding 0.15 g/dl of polyoxypropylene erythritol dimyristate as a surfactant to an aqueous medium adjusted to pH 7.0 and containing 20 dl of the medium was dispensed into ten 500 d shaking flasks, and heated to 115°C. te10
Sterilize by heating for minutes.

Add 3 d of the above seed culture solution to the medium in each flask,
Shaking culture was carried out at 1.5°C until glucose disappeared.

The time required was approximately 96 hours.

Combine the culture fluids from each flask and add Cil and Gl in the culture fluid.
When the amount of u accumulated was quantified, the results were as follows.

0.71g/d/ and 0.71g/dA, total 2.4
It was 2 g/dl.

For comparison, the results of culturing without adding surfactant are as follows.
The accumulated amounts of Cil and Glo were each 1.76g/
dl and Og/dl and no co-production of Glu was observed.

Example 8 (Trp) An aqueous seed matrix having the composition shown in Table 8 was dispensed for 20 days into a shaking flask having a volume of 50 (20 mL), and after sterilization by heating at 115° C. for 10 minutes, Brevibacterium flavum ( B+
evibacte+iI1m flsvum)FERM
P-2238 was inoculated and cultured with shaking at 30°C for 24 hours to obtain a seed culture.

Table 8 On the other hand, 50% of the aqueous main fermentation medium having the composition shown in Table 8
Dispense 20 d each into 10 0 m capacity shoulder flasks, add 0.20 g/dA of polyoxyethylene sorbitan monopalmitate as a surfactant, and sterilize by heating at 115°C for 10 minutes. The above seed mother culture solution was added to the medium for 3 days and cultured at 30°C for 72 hours.

Combine the culture fluids from each flask and add Trp and Gl in the culture fluid.
When the accumulated amount of u was quantified, it was 020 g/dl, respectively.
and 0.12 g/d12, for a total of 0.32 g/d1.

For comparison, the results of culturing without adding surfactant are as follows.
The accumulation amount of Tap and Glu is 0°25g/d, respectively.
12 and Og/di, and no co-occurrence of Glu was observed.

(Effects of the Invention) The present invention has reduced the production cost of amino acids.

Claims (1)

[Claims] (1) Belongs to the genus Brevibacterium or Corynebacterium and L-leucine, L-isoleucine, L-valine, L-threonine, L-tryptophan, L-phenylalanine, L-tyrosine, L-citrulline, and L-homoserine Neutral amino acids, L-glutamic acid, A method for simultaneously fermenting and producing.