JPS6115695A - Preparation of l-isoleucine by fermentation method - Google Patents

Abstract

PURPOSE:In production of the titled substance by cultivating a bacterium, to improve fermentation yield and accumulation, by using a bacterium belonging to the genus Brevibacterium or Corynebacterium, having alpha-ketomalonic acid resistance. CONSTITUTION:A bacterium belonging to the genus Brevibacterium or Corynebacterium, such as Brevibacterium lactofermentum, Corynebacterium glutanicum, etc., capable of producing L-glutamic acid is subjected to variation and induction, to collect a strain having alpha-ketomalonic acid resistance. Prepared Brevibacterium flavum AJ12152, Corynebacterium glutamicum AJ12153, etc. is cultivated, and L-isoleucine is collected from the medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] L-4 Soleucine is an important component of amino acid infusions and comprehensive amino acid preparations. The present invention improves the method for producing L-isoleucine by fermentation.

[Conventional technology]

In order for microorganisms of the genus Brevibacterium and Corynebacterium to have the ability to produce L-isoleucine, it is sufficient to impart resistance to α-amino-β-hydroxyvalerin (hereinafter abbreviated as AHV), etc. I know that. Furthermore, in addition to the drug resistance mentioned above, O-methylthreonine resistance,
It is known that L-isoleucine production ability can be improved by imparting resistance to β-hydroxyleucine or fd)lichloroalanine, and by imparting requirements for purine substances or lysine.

[Problem that the invention seeks to solve]

Improving L-isoleucine fermentation yield and accumulation is an important issue in industrial production.

[Means for solving problems]

The present invention has been made in order to solve the above-mentioned problems. As a result of research to find strains with improved fermentation yield, we found that α-ketomalonic acid (
Hereinafter, it will be abbreviated as α-KM. ) among the strains that have conferred resistance to
In addition to conventional isoleucine-producing bacteria, it has been discovered that there are strains that produce L-isoleucine at a high crystal yield.

That is, the present invention involves culturing a microorganism belonging to the genus Brevibacterium or Corynebacterium and having α-bitolerance and the ability to produce L-isoleucine in a liquid medium, and producing and accumulating L-isoleucine in the medium. Iso The microorganism used in the present invention belongs to the genus Brevibacterium or Corynebacterium, and is a mutant strain having α-store resistance and the ability to produce L-isoleucine.

To obtain the mutant strain of the present invention, the following wild strain may first be endowed with L-isoleucine production ability, and then alpha-KM resistance may be imparted, or alpha-KM resistance may be first imparted, then 4
The ability to produce leucine may also be imparted.

The wild strain that is the parent strain of this mutant strain is Corinepho A, Mu I, - of the genus Brevibacterium or Corynebacterium.
These are known as glutamic acid producing bacteria, such as the following.

Brevibacterium lactofermentum ATCC1
3869 Brevibacterium deipalicatum
ATCC14020 Brevibacterium calolyticum ATCC14066 Zorevibacterium flavum ATCC14067 Corynebacterium gullamium ATCC13032 Corynebacterium acetamidophyllum ATCC1
3870. To obtain the mutant strains of the present invention from these parent strains, conventional mutagenesis methods such as treatment with N-methyl-N'-nitro-N-nitrosoguanidine can be applied.

The method for isolating the mutant strain of the present invention from a bacterial solution subjected to mutation treatment is carried out by collecting a strain that grows in a medium containing α- cells.

Specific mutation induction method of the mutant strain shown in the present invention and α-KM
The relationship between the growth rate of the strain and the strain is shown below.

[Mutation induction method]

Brevibacterium flavum AJ 3686FERM grown on broth agar slant at 30°C for 24 hours
-P 2433 (ATCC 14067, 1: !J-induced AI (v-resistant strain) and Corynebacterium glutamicum AJ12150 FERM-P r76r7
4 (ATCC13032, an AHV-resistant strain induced by liniculture) was suspended in M/30') acid buffer, and 500 μg/ml was added to a cell suspension with a cell concentration of 108 to 109/ml.
N-methyA-N'-nido-N-nitrosoguanidine of rni was added and kept at 30°C for 20 minutes. The cells were then collected by centrifugation, washed well with M/30 phosphate buffer, and then inoculated into a medium with the following composition [cultured at 2,315°C for 2 to 10 days, 7, glucose 1.
0 f/-/diurea
0.2KH2PO40,1 MgSO4・7. H2O0,I P7dlF e
SO4・7H200-002”MnSO4・7H20
0,002# Biotin 100 μ9-/l Sian hydrochloride 100 # α-KM O,2';! /di agar 2.0 Among the strains grown on agar medium, Brevibacterium 7 Lava Aki 12152 was selected as a seedling with high L-isoleucine production ability.
FERM-P Wa 6r7g (AJ (v resistant α- resistant) and Corynebacterium glutamicum A AJ
12153, FERM-P r76 Wa 6 (AIrV
Resistance a, α-number resistance) was obtained.

Like this! The α1 resistance of the mutant strain was compared with that of the parent strain.

Glucose 0.5 F//di, Urea 0.2 field
i, ammonium sulfate 0,15'9/di, K2HO4'0.
39/de, K2HPO40,19/dl,
MgSO4・7H200,01g/de, C
aC42'2H200, 1m9/de, Biochi y100
ttl/l, fi7myyhydrochloride100fig/11
, FeSO4-7H200,0021/di.

MnSO4'7H20o, o O21! /dll, and the amount shown in the table, and the medium adjusted to PI (7,0) was mixed with a natural medium (-!! Momopton 1 g/de, yeast extract 11i/di,
NaC60.5Ii/dl, pH 7.0) Cells cultured in a slant for 24 hours were suspended in sterilized water and inoculated.
After culturing for 24 hours, the degree of growth was measured at once.

Table 1 Addition of properties known to improve the productivity of L-isoleucine, such as O-methylthreonine resistance, β-hydroxyleucine resistance, or trifluoroalanine resistance, to the above-mentioned mutant strains. In particular, the yield is often improved.

[Effect]

The medium used for culturing such mutant strains is a normal medium containing a carbon source, a nitrogen source, inorganic ions, substances that should satisfy the above-mentioned requirements, and other organic micronutrients such as vitamins as necessary. .

Carbon sources include carbohydrates such as glucose and sucrose, organic acids such as acetic acid, etc., and nitrogen sources include aqueous ammonia and
Ammonia gas, ammonium salts, etc. are suitable. Potassium ion is an inorganic ion.

Sodium ions, magnesium ions, phosphate ions, and others are added to the medium as needed.

It is desirable to cultivate under aerobic conditions, and the pH of the medium should be kept at 4 during cultivation.
Preferable results can be obtained by controlling the temperature between 25°C and 37°C. Thus, if the culture is continued for 1 to 7 days, a significant amount of L-isoleucine will be produced and accumulated in the medium. pL-inoleucine can be collected from the culture solution by a conventional method such as using an ion exchange resin.

This will be explained below using examples.

Example 1 Glucose 10g/di, (NH4)2S0471
1/di, KH2PO40,111/di, MgSO
4.7H200,04g/di, FeSO4.7H2
01m9/d1. MnSO4'4H201m9/dl
, Thiamine-HCL100μ! i/l, bithion 10
A medium containing 0μI/l, soybean protein acid hydrolyzate 60 da/dj (as total silica) calcium carbonate 5yZdec separate sterilization) was adjusted to PI (7,0), and the 20rnl was sterilized to 500
The mixture was placed in a shoulder-length flask and sterilized by heating. A loopful of the bacterial strains shown in Table 1 was inoculated into this, and the mixture was shaken for 4 days while being kept at 31.5°C. The amount of L-isoleucine shown in Table 2 was accumulated in the culture solution of each strain. AJ12152
was cultured by the above method to obtain culture solution 11, from which the bacterial cells were removed by centrifugation, and the supernatant was treated with a strongly acidic ion exchange resin [
It was passed through Diaion J 5K-IB (N1 (4+ type). After washing the resin with water, it was eluted with 2N aqueous ammonia, and the eluate was concentrated. From this, crude crystals of L-isoleucine 16.0.9 I got it.

Table 2 Bacterial Strain Characteristics
- Isoleucine (9Ah Brevibacterium
Flavum AJ 3686 (FERM-P 2433) AHt
” ' 0.6 Previbacterium flavum

Claims (1)

[Claims] A microorganism that belongs to the genus Brevibacterium or Corynebacterium and has α-ketomalon resistance and is capable of producing L-isoleucine is cultivated in a liquid medium, and L-isoleucine produced and accumulated in the medium is collected. Characteristic method for producing L-isoleucine.