JPS599156B2 - Method for producing L-glutamic acid by fermentation method - Google Patents

Description

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

In more detail, microorganisms that can assimilate ethyl alcohol and n-propyl alcohol and have the ability to produce L-glutamic acid are cultured in a medium containing ethyl alcohol and n-propyl alcohol as carbon sources. The present invention relates to a method for producing glutamic acid, which comprises producing and accumulating L-glutamic acid in a culture solution, and collecting L-glutamic acid from the culture solution.

Conventionally, microorganisms capable of assimilating ethyl alcohol or n-propyl alcohol and capable of producing L-glutamic acid were cultured in a medium containing ethyl alcohol or n-propyl alcohol as a carbon source to produce L-glutamic acid in the medium. There are many reports on methods for producing and accumulating L-glutamic acid and collecting L-glutamic acid from the culture solution (for example, Japanese Patent Publication No. 45-26711, Japanese Patent Publication No. 47-1834, Japanese Patent Publication No. 48
-19952, Japanese Patent Publication No. 49-34833, Japanese Patent Publication No. 49-
117683 etc.).

However, no report has been found on the use of a mixture of ethyl alcohol and n-propyl alcohol as a carbon source when producing L-glutamic acid by fermentation.

The present inventors have proposed an L-
In the production of glutamic acid, various studies were conducted with the aim of improving the amount of L-glutamic acid produced and accumulated and the yield. As a result, it was found that L-glutamic acid was added to a medium containing a mixture of ethyl alcohol and n-propyl alcohol in an appropriate ratio as a carbon source. - By culturing a glutamic acid-producing strain to produce and accumulate L-glutamic acid in the medium, the amount of L-glutamic acid produced is remarkable compared to when ethyl alcohol or n-propyl alcohol is used alone as a carbon source. The present invention has been completed based on the discovery that the

The present invention will be explained in detail below.

Examples of microorganisms used in the present invention include microorganisms that have the ability to assimilate ethyl alcohol and n-propyl alcohol and to produce and accumulate L-glutamic acid. Among these, particularly suitable microorganisms require piotin for growth. Corynebacterium glutamicum, Previbacterium ammoniagenes, Previbacterium flavum, Microbacterium ammoniafilm, and Microbacterium flavum are known to often produce L-glutamic acid from carbohydrate raw materials. Microorganisms such as

As the medium used in the present invention, a medium generally used for fermentative production of L-glutamic acid is used.

That is, as shown in the Examples, either a synthetic medium or a natural medium can be used as long as the medium contains a sufficient amount of a nitrogen source, inorganic substances, and other nutrients in addition to the main carbon source.

Carbon sources include not only a mixture of ethyl alcohol and n-propyl alcohol, but also carbohydrates, hydrocarbons,
Carbon sources mixed with organic acids, sugar alcohols, etc. are also used.

Nitrogen sources include ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphorus,
Organic and inorganic nitrogen compounds such as urea, ammonium acetate and ammonium citrate are used.

Contains some or all of other natural nutritional sources such as peptone, casamino acids, meat extract, cornstap liquor, soybean meal decomposition liquid, phosphoric acid source, potassium source, magnesium source, sulfur source, biotin, thiamin, etc. things are used.

Calcium carbonate may also be used as a pH lowering inhibitor.

The concentration of biotin or biotin-like active substances in the medium must be kept below the maximum growth amount, as in normal L-glutamic acid fermentation. When used for this purpose, a substance that inhibits the excessive growth of L-glutamic acid producing bacteria, such as an antibiotic or a surfactant, is added according to a conventional method.

Cultivation is performed under aerobic conditions such as shaking culture or deep agitation culture.

The culture temperature is generally 20-40°C, especially 25-35°C.
is suitable.

It is preferable to adjust the pH of the medium to slightly alkaline using ammonia or urea at the start of culture or during the culture period.

The concentration (total) of ethyl alcohol and n-propyl alcohol to be used cannot be determined unconditionally as the degree of inhibition differs depending on the microorganism, but growth tends to be inhibited at 31 liter/dl or higher, so the initial concentration is 37711/dl or higher. d
It is recommended that the alcohol be less than 1 ml (during cultivation, all alcohols are consumed almost completely and then added in such a way that the total concentration does not exceed 2 to 3 ml/dl).

The preferred mixing ratio of ethyl alcohol and n-propyl alcohol used in the present invention, expressed as the ratio of n-7'propyl alcohol to the total alcohol used, is approximately 60% or less, several % or more, Preferably about 50% or less, about 1
It is 0% or more.

L-glutamic acid accumulates in the culture medium during a culture period of usually 1 to 5 days.

Various known purification methods can be applied to collect L-glutamic acid from the culture solution, but for example, the culture solution is passed through a centrifuge, the pH of the supernatant is lowered to 320, and the L-glutamic acid is centrifuged again. Glutamic acid crystals can be easily obtained.

Next, we investigated the production and accumulation of L-glutamic acid from ethyl alcohol and n-propyl alcohol in several strains selected from the strains belonging to the microorganism Corynebacterium glutamicum, which is one of the most representative L-glutamic acid producing bacteria. are shown in Tables 1 and 2.

The medium composition and culture method used here are as follows.

1. Medium composition ■ Preculture medium composition Ethyl alcohol 2l71l/dl (NH4) 2 SO4 0. 2 f?
/d. lKH2PO4 0.1 /
/K2HPO40.1 // Mg So 4・7H20 0.05 //Urea 0.05 tt FeSO4・7H20 0.001 〃MnSO4・
7H20 0.001 // Corn staple
0.2〃 Liquor Kyokuto Ehrlitsu 0.2〃 Meat extract biotin 10 μf-/1 Thiamine hydrochloride 1.0 ■/l Phenol red 0.
0 0 1? /dlpH 7.20 @ Main medium composition Ethyl alcohol each concentration * n-Propyl alcohol each concentration * Nore (NH4)2SO4 0.2 1? /cll
KM2PO4 0.1 //K2H
PO4 0.1 〃MgSO4・7H
20 0.05? /ctlurea
0.05 (initial concentration) FeSO4・7H20 0.
001 〃MnSO-7H20 0.001 /
/4 Corn Staple 0.2〃 Kerr Far East Ehrlitsu Meat 0.2〃 Exbiotin 2.5 μ? /1 thiamine hydrochloride 1.0 immediately /l phenol red
1.001v/dlpH 7.20 *Ethyl alcohol and n-propyl alcohol were used at the mixing ratio shown in Tables 1 and 2, but the initial total concentration of both alcohols was 2.0ml/dl. And so.

2. Culture method: Dispense 10 ml of pre-culture medium into 70 TLl large test tubes. After sterilization, prepare Corynebacterium glutamicum ATCC15.
455, ATCC10389, Summer ATCC2101 1
and one platinum loop of ATCC21158 was inoculated, and the temperature was 30°C.
Culture with shaking for 24 hours.

Add 17nl of this pre-culture solution to 10TLl of the above main medium to 70ml.
Add to the test tube dispensed into a ml large test tube, and heat at 30°C.
Shaking culture was performed.

Note that ethyl alcohol and n-propyl alcohol were added after sterilization using a Millipore filter.

During the culture period, the concentrations of ethyl alcohol and n-propyl alcohol in the culture solution were measured by gas chromatography at appropriate times, and the total concentration of both alcohols was determined according to the amount consumed. The feed was adjusted so as not to exceed 1 ml/dl.

The pH in the culture solution was adjusted by adding 25% urea water.
The temperature was adjusted to 7 to 8, and a nitrogen source was supplied.

The culture was terminated after 72 hours, and the L-glutamic acid produced and accumulated was quantified using E. coli L-glutamic acid decarboxylase.

As shown in Tables 1 and 2, the amount of L-glutamic acid produced is much larger when each alcohol is used in combination than when used alone, and the appropriate mixing ratio depends on the strain. It has been found that the production and accumulation of L-glutamic acid is promoted by selecting

Next, examples will be shown.

Example 1 Corynebacterium glutamicum ATCC as inoculum
15455 as the primary seed medium, KH2P0,
0.1 g/dl, K2HPO, 0. 3ff/d
l, MgSO, 7 H20 0.0 5 ? /dl
, FeSO4 + 7 H2 0 0. O O 2
f /dl, MnSO4・7H20 0.002f?
/dl1 corn staple liquor-0. 5? /dl,
Far Eastern Ehrlitsuhi Meat Extract 0. 5? /dl, thiamine hydrochloride o. 1ynti/dl, biotin 0.01
■/dl, urea 0.1 g/dl1 (NH4)2 S
O40. 5? /dl, CaCO3 1 P/dl
, ethyl alcohol2. A medium consisting of 5 TLl/dl was used.

A platinum loopful of the inoculum was inoculated into 10 ydo of the above-mentioned medium in a 79 mA test tube, and cultured with shaking at 30°C for 24 hours.

The total volume of this seed culture solution was put into a 2L Erlenmeyer flask.
The cells were inoculated into 0 ml of secondary seed medium.

The composition of the second seed medium is the same as that of the first seed medium.

This secondary seed culture was cultured with shaking at 30°C for 24 hours.

The entire volume of this secondary seed culture solution was mixed with KH2 PO4 0. 0
5f/dl, K2HPO40. 1 5 f/dl
, MgSO4 ・nH200.05? /dl,FeSO
4 ・7H200.0 0 1? /dl, MnSO
4-7 H20 0.0 0 1t/dl, corn staple liquor, 4g/dl, biotin 0.0
1~/dl, thiamine hydrochloride 0.1/dl, (N
H4) 2 SO40. 2? A 5 liter jar fermenter containing 3 liters of sterile medium consisting of 3 liters of sterile medium was inoculated with ethyl alcohol and n-propyl alcohol at a total concentration of 2% (V/V) each in the volume mixing ratio shown in Table 3. Culture concentration: 30°C, aeration volume: 3J/mil,
Main culture was carried out at a stirring speed of 60 rpm.

The pH during the culture was adjusted to 7.40 with 28% ammonia water.

About 7 hours after the start of culture, when the bacterial cells in each culture solution reached the specified concentration, penicillin G potassium salt was added to the culture solution 1.
Add 25 U per ml and add a mixture of ethyl alcohol and n-propyl alcohol at the respective ratios shown in Table 3 to the culture solution using gas so that the total concentration is 0.1% (V/V) or less. Feed was carried out continuously while the concentration was measured by chromatography.

Table 3 shows the amount of L-glutamic acid produced 48 hours after the start of the culture and the yield of L-glutamic acid based on the total weight of ethyl alcohol and n-propyl alcohol used.

After removing the bacterial cells, each culture solution was concentrated under reduced pressure and hydrochloric acid was added to adjust the pH to 3.2 to obtain crude crystals of L-glutamic acid.

Their yields are shown in Table 3.

Example 2 Corynebacterium glutamicum ATCC21011 was cultured in the same manner as in Example 1, and the results shown in Table 4 were obtained.

Example 3 Prepibacterium ammoniagenes AT as inoculum
CC13745, Previbacterium flavum ATC
C13826, Microbacterium ammoniafilm ATCC 15354 and Microbacterium flavum ATCC 1 0 3 4 0, respectively.
As the primary seed medium, KH2PO40.1 g/dl, K2HPO40.3 g/dl, MgSO4.7 H20 0.0 5? /
dl1FeSO4@7 H20 0.0 0 2
P/cll, MnS04・nH2 0 0. O
O2? /dl, -7 7s7Eprica 0.5
? /dl1 yeast extract (manufactured by Inugo Nutrition Co., Ltd.) 0.1 g/
dl, (NH4)2SO40.5g/dl, biotin 0
．． 0 1 m97dl, thiamine hydrochloride 0.1ynq
/dl, urea 0. 1? /di! , CaCO31
f? /di! , ethyl alcohol2. 5 TLl/d
1 of medium was used.

A platinum loopful of each seed culture was inoculated into 10 ml of the above seed medium in a 71-liter test tube, and cultured with shaking at 30° C. for 24 hours.

The total volume of each of these seed culture solutions is 2 liters! A secondary seed medium of 30 QTll in an Erlenmeyer flask was inoculated.

The composition of the second seed medium is the same as that of the first seed medium.

This secondary seed culture was cultured with shaking at 30°C for 24 hours.

The entire volume of the second seed culture was added to KH2 P0,0. 0 51
/d. l , K 2 HP 0 40.1 51/d
．． l, MgSO4 ・nH200.0 5'? /dl
,F e SO4 ・7H2 0 0- 0 0 1
S' /cil, MnSO, -nH2 0 0.
O O 1 f / dl, corn steep liquor - 0
．． 4? /dl, yeast extract (manufactured by Inugo Nutrition Co., Ltd.) 0.
1 f/dl, biotin 0.01 i/dl, thiamine hydrochloride 0. 1 mg7dl, (NH4)2S
O4 0. 5 containing 3 liters of sterile medium consisting of 27/dl
A 1-volume jar fermenter was inoculated with 2% (V/V) of ethyl alcohol and n-propyl alcohol in the volume mixing ratio shown in Table 4, and then Table 5 shows the results of culturing in a similar manner to produce guiretamic acid.

The fermentation broth was treated to obtain crude crystals in the same manner as in Example 1 using the strain that showed the best results for each strain, and the yields were as shown in the notes to Table 5.

Example 4 Corynebacterium glutamicum ATCC as inoculum
15455 and Corynebacterium. Glutamicum A
TCC21011 was used as the primary seed medium, and KH2PO40. 1 fl/dl, K2 HPO4
0. 3S'/cll, Mgso4H 7H
200.0 5 f/dl, FeSO4 ・7H20
0.0 0 2? /dl, MnSO4・nH200
．． 0029/dl,=r-en-steep liquor-0.5
? /dl, casamino acid 0.2? /dl, biotin 1
0 μf? /l, thiamine hydrochloride 0.1 instant/dl, (
NH4) 280, 0. 5g/di, urea 0. 1
? /dl, CaC031. O? /dl,
A medium consisting of 2.5 ml/dl of ethyl 7 alcohol was used.

A platinum loopful of each seed culture was inoculated into 10 volumes of the above seed medium in a 70 ml test tube, and cultured with shaking at 30°C for 24 hours.

The total volume of this seed culture solution was put into a 2J Erlenmeyer flask.
The cells were inoculated into a secondary seed medium of 0-.

The composition of the second seed medium is the same as that of the first seed medium.

The second seed culture was cultured with shaking at 30°C for 24 hours.

The entire volume of this secondary culture solution was mixed with KH2PO, 0. 0 5
? /dl, K2HPO40. 1 5? /dl,Mg
S04・7H, 20 0.05 g/dl, FeSO
4 ・7H20 0.0 0 1? /dl,MnS
04・nH2 0 0. 0 0 1? /dl, corn steeply force-〇. 2 g/dl, casamino acids 0.
5g/dl1 biotin 3.5μ? /l, thiamine hydrochloride 0.1g/dl, (NH4)2sO, 0.2g/dl
A 5 liter jar fermenter containing 3 liters of sterile medium consisting of ethyl alcohol and n-propyl alcohol at a volume mixing ratio shown in Table 6 at a total concentration of 2% each (V/V)
The culture concentration was 30°C and the aeration volume was 3, J.
/mm, and the number of stirring was 60 rpm.

During the cultivation period, the pH of the culture solution was adjusted to 7 with 28% ammonia water.
．． Adjusted to 40.

The mixture of ethyl alcohol and n-propyl alcohol was continuously fed while measuring the concentration using gas chromatography so that the total concentration was 1.0% (■/V) or less.

The culture was completed in 48 hours, and the amount of L-glutamic acid produced in the culture solution was measured. It looked like Table 6.

Crude crystals were collected from each culture solution in the same manner as in Example 1.

Claims (1)

[Claims] 1 Belongs to the genus Corynebacterium, Previbacterium, or Microbacterium, and has the ability to assimilate ethyl alcohol and n-7'ropyl alcohol, and
- Cultivating microorganisms capable of producing glutamic acid in a medium containing ethyl alcohol and n-propyl alcohol as carbon sources, producing and accumulating L-glutamic acid in the medium, and collecting L-daltamic acid from the culture solution. A method for producing glutamic acid, characterized by: