JPS61177993A - Production of l-isoleucine - Google Patents

Abstract

PURPOSE:To produce efficiently L-isoleucine, by cultivating a microorganism of the genus Brevibacterium having a special activity aerobically in a culture medium containing ethanol as a main carbon source and DL-alpha-aminobutyric acid, etc. CONSTITUTION:A culture medium is prepared by incorporating 1-5vol% ethanol with 0.1-5wt% DL-alpha-aminobutyric acid or D-alpha-aminobutyric acid, ammonia, etc., as a nitrogen source, KH2PO4, etc., as an inorganic salt and other peptone, vitamin, etc. A precultivated Brevibacterium flavum which is an ethanol assimilating microorganism having enhanced D-alpha-aminobutyric acid deaminase activity is inoculated into the above-mentioned culture medium, and cultivated at 20-40 deg.C and 5-10pH under aerobic conditions of spinner culture or shaking culture with aeration for 2-9 days to produce and accumulate the aimed L- isoleucine in the culture fluid. The microbial cells, etc., are removed by centrifugation to collect L-isoleucine by the ion exchange resin treatment method, etc.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention is directed to the production of L-isoleucine by a fermentation method,
L'-isoleucine, which is characterized in that an ethanol-assimilating microorganism belonging to the genus Brevibacterium and having increased D-α-aminobutyric acid deaminase activity is cultivated aerobically, and L-isoleucine in the culture solution is collected. This is the manufacturing method.

(Prior art and issues) L-isoleucine is an essential amino acid that plays an important role in the nutrition of humans and animals, and is used as a medical, food, and feed fortifier. The number of J-types has been rapidly decreasing in recent years. As for the industrial production method of L-isoleucine, it is difficult to produce only the L-isomer by chemical synthesis method because stereoisomerism exists as in the case of other amino acids, and production is mainly carried out by fermentation method. ing. FiDL- as a fermentation method
Ia 5T such as α-amino butybean and threonine! II Direct Fermentation Method (Special Publication No. 38-7091. %hJ 1977-935
86 etc.).

Among them, the method for producing L-isoleucine using α-amino vI11rR as a precursor is based on the fact that the DL form of α-amino butylene is most easily and inexpensively produced by chemical synthesis. -α-aminobutyric acid is preferably used.

The present inventors have already reported that the ethanol-utilizing microorganism MJ-233 (FERM-
proposed a method for producing L-isoleucine by adding DL-α-aminobutyric acid to a medium using P4O10 (Japanese Patent Publication No. 57-26755).

In the production of L-isoleucine using MJ-233 mentioned above, the L-aminobutybean is quickly consumed, and the L-isoleucine is quickly consumed.
- Although accumulation of isoleucine was observed, the production rate of L-isoleucine from aminobutyric acid was found to be very low. Therefore, 1) When using L-α-aminobutyric acid as a precursor, L from D-α-aminobutyric acid
- By increasing the production rate of isoleucine, it is expected that L-isoleucine can be produced more efficiently as a complete stop.

(Structure and Effect) Here, the present inventors conducted intensive research with the aim of improving the rate of inleucine production from D-α-aminobutyric acid, and found that increasing D-α-aminobutyric acid deaminase activity Additionally, D-α-aminobutyric acid was also utilized relatively quickly, leading to accumulation of L-isoleucine.
As a result, it was found that the production rate was also improved when OL-α-aminobutyric k was used, leading to the present invention.

This can also be applied to effectively utilizing the D-α-aminobutyric acid remaining when the L form of fl and DL-α-aminobutyric acid is used for other purposes.

The gist of the present invention is that “D-α belongs to the genus Brevibacterium.
- Ethanol-assimilating microorganisms with increased aminobutyrate deaminase activity that use ethanol as the main carbon source and DL-α-
Fermentation characterized by culturing aerobically in a medium containing aminobutyric acid or D-α-aminobutyric rR to produce and accumulate L-isoleucine in the culture solution, and collecting L-isoleucine from this culture solution. ``Production method of L-isoleucine by method''. The modifier "l)-α-aminobutyric acid deaminase high activity" used as an adjective in the present specification is hereinafter referred to as rL)-α-
It has the same meaning as the modifier "aminobutyric acid deaminase activity increased."

The D-α-aminobutyric deaminase-high activity mutant used in the present invention can be obtained, for example, by the following procedure.

UV irradiation, or chemical agents (e.g. N-methyl-
Brevibacterium flavum MJ-233 (FF
After inducing mutations in ERM-P4O10), this bacterial suspension was placed in a plate culture medium (urea 0.2%, ammonium sulfate 0.7%, K
HxP040.05chi, 0゜0.0541Mg5O
n ・7Hs00.05%, NaCA! 2yq/73
゜ZnSO4・7H202 Cape/l, Biotia 200
pf/l, thiamine hydrochloride 100. uf/l,l)-α
- 0.2% aminobutyric acid as the basic medium, to which 2.0% agar and 3 volumes of ethanol were added) [,
After culturing at approximately 30°C for several days and separating the resulting large colonies, each colony was inoculated into a 24φ dog-shaped test tube containing 10 volumes of the above basic medium, and after adding 2 volumes of ethanol, the cells were incubated at 30°C.
Culture with shaking for 48 hours.

The culture was collected by centrifugation, washed twice with 100mM potassium phosphate buffer (pH 7.0), and then D-
Reaction solution for measuring α-aminobutyrodeaminase activity (D-α
- 25 μmoles of aminobutyric acid + 100 pmoles of potassium phosphate buffer (pH 7,5).

Pyridoxal 5syg O,025pmoles,
) Ridone X-100, 1% by weight, deionized water 1-)
1d, and a shaking reaction is carried out at 30°C for 18 hours. After the reaction is completed, centrifugation (400 rpm).

The concentration of D-α-aminobutyric acid in the supernatant after removing the bacterial cells was measured, and the D-
The amount of decrease in α-aminobutyric acid is defined as D-α-aminobutyric acid deaminase activity. In the present invention, the D-α-aminobutyric acid deaminase highly active strain (microorganism) refers to the D-
It means a mutant strain in which α-aminobutyric deaminase specific activity is increased by 30% or more compared to the specific activity of Brevibacterium flavum MJ-233. A typical allogene obtained by the above procedure is Brevibacterium aeroflavum MJ-233-ABD-21 (hereinafter abbreviated as mJ-
233-ABD-21). This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, with accession number No. 8.
It has been entrusted as No. 055 (January 21, 1985). This mutant strain is Brevibacterium kistoflavum 1 old-
233 (FERM-P 3068) as a strain with high D-α-aminobutyric deaminase activity. The D-α-aminobutyric acid deaminase specific activity of MJ-233-ABD-21 is different from that of its parent strain MJ-23.
The specific activity is increased by 35% compared to No. 3.

The method for producing L-isoleucine of the present invention will be specifically explained below.

The medium composition used for the culture of the present invention is such that it uses ethanol as the main carbon source and contains DL-α-aminobutyric acid or D-α-aminobutyric acid, nitrogen source inorganic salt tl
'ith. Ammonia as a nitrogen source,
Ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. can be used alone or in combination.

As the inorganic salt, phosphoric acid-frozen potassium, phosphoric acid potassium dihydrogen, magnesium sulfate, etc. are used. In addition, nutrients such as peptone, meat extract, yeast extract, cornstarch liquor, Casaminoba, and various vitamins may be added to the medium if necessary for bacterial growth and L-isoleucine production.

The culture is carried out under aerobic conditions such as aeration and shaking, and the culture temperature is preferably 20 to 40°C, preferably tli 25 to 35°C. The pH during the cultivation is maintained at around 5-10, preferably around 7-8, and the pH during the cultivation is adjusted by adding acid or alkali.

The addition and preparation of OL-α-aminobutyric acid or FiD-α-aminobutyric acid is 0.1 to 5 parts per ton, preferably 0.25 to 3
It is heavy '1tts. Ethanol quality at the start of culture is 1
~5 answers, preferably 2 to 3 answers. The culture period tr is 12 to 9 days, and the optimal period is 4 to 7 days. To recover L-isoleucine from the culture solution, the culture Vk solution is centrifuged, etc. to remove the m-condensation, and then it can be easily carried out by known methods, i.e., the ionizing resin treatment method, the sedimentation method, etc. I can do it. , carried out below? Indicates lJ.

In addition, the properties of L-isoleucine, the Rf value of Beher chromatography, the mobility of electrophoresis, and the value of bioactivity were confirmed by bioactivity assay according to Dentsu.

Leuconostoc mesenteroides ATCC
The microbioassay method using 8042 was carried out in combination with Cosmos chromatography (C-5A). In addition, in the following implementation, ``chi'' means ``Ndochi''.

Actual 7m? lJ1 preculture medium (urea 0.4%, ammonium sulfate 1.4%)
%, KH2PO40.05%, K2HPO40.0
5%, MgSO4.7H200.05%, CaCA'
@2'fis02 ppm.

Fe50+ "7H202ppm %Mn5O<'
4-6 H2O2pPm%Znb(Ja” 7
H2O2ppm %NaCl2 ppm-Biotin 20
0μy/l, thiamine/HCl 100μs, Casamino I
No, 1%, yeast extract 0.1%) 1〇-24φ
After applying partial pressure to a large test tube and sterilizing it (pf (7,0) after sterilization),
No.), add 0.3 - ethanol aseptically, and
The culture was incubated with shaking at 0°C for 2 days.

Next, the main culture medium (urine X 0.4%, ammonium sulfate 1.
4%, K2HO40.05%, K2HPO40.05%
, MgSO4@7)ho O,05%, CaC1z
@2H2O2ppm.

Fe5Oi @ 7 fbo 2 ppm s Mn5
O+ ・4~6H202ppm%ZnSO4*7)
ho2ppm% NtaCl2ppm.

Biotin 200μt/l, Thiamine H (J100μt/l)
t/l, corn staple liquor 10 go/l.

DL-α-aminobutyric acid (0.5%) was dispensed into a 24φ large test tube, and after sterilization (pH 7.0 after sterilization), 0.1% of the preculture solution was inoculated, and ethanol was added aseptically. 0.3- and carry out weight culture at 30°C for 7 days. Ethanol is added as it is consumed.

(At this time, the ethanol should not exceed 3 volumes tS.

Also, I lost all ethanol in 11 Fi 6.5 answers J1 Chi. ) Cultured for 7 days
, 32 were accumulated.

Cultivate! The filtrate, which has been removed from the liquid by removing sulfuric acid and other impurities, is passed through a column of strong cation exchange m fat (H1 type) to adsorb L-isoleucine, washed with water, and eluted with 0.5N aqueous ammonia. The L-isoleucine fraction was separated, L-isoleucine crystals were precipitated with cold ethanol, and culture solution 1
Crude crystals of 92.5F per portion were obtained.

In addition, when using the parent strain (ivfJ-233) under the same conditions, the accumulation of L-isoleucine in the medium,
That's 3.59 tfc.

Example 12 Preculture was carried out under the same conditions as those for Sei mushio+41. Next, the main culture medium (urea 0.4%, ammonium sulfate -1.4%,
K2HPO40.05%, K2HPO40.05chi, M
g S04 ・7 HxO0,05To, CaC1z
#2 Hzo 2 PPm%FeSO4・7H202
ppmSMn5O+ ・4~6 HsO2ppm%Zn
SO4” 71 (202ppm, N&C12ppm,
Biotin 200 μfl/l, Thiamin 1 (CA'10
0 μf/l, corn stable liquor 10 d/l, D-α
-Amino-number 0.25%) 10-, after partial pressure sterilization in a 24φ large reclining tester (after sterilization pl (7,0), inoculate 0.1- Add 0,3 - ethanol and culture with shaking at 30°C for 7 days. Ethanol becomes saturated as it is consumed, so do not exceed U (3 volumes of ethanol at this time). Also, the total ethanol consumption T (was 6.5 volumes tS.) On the 7th day of culture, L-isoleucine was 1.1% per culture solution. It was hoarded.

When L-isoleucine crystals were precipitated by the same operation as in Example 1, the amount was 0.659 per 11 culture solutions.

When the parent strain (ivfJ-233) was cultured under the same conditions, 0.3 r of L-isoleucine was produced and accumulated per culture W1.11.

Claims (1)

[Claims] 1. Ethanol-utilizing microorganisms belonging to the genus Brevibacterium and having increased D-α-aminobutyric acid deaminase activity were grown in a medium containing ethanol as the main carbon source and containing DL-α-aminobutyric acid or D-α-aminobutyric acid. A method for producing L-isoleucine by a fermentation method, which comprises culturing aerobically to produce and accumulate L-isoleucine in a culture solution, and collecting L-isoleucine from the culture solution.