JPS62195293A - Production of l-isoleucine by fermentation method - Google Patents

Abstract

PURPOSE:To obtain L-isoleucine in high yield inexpensively, by using a bacterium belonging to coryneform glutamic acid-forming mold, showing resistance to threoninehydroxamate, capable of producing L-isoleucine. CONSTITUTION:A strain having resistance to threoninehydroxamate can be obtained as a mold growing in an agar medium containing >=1g/l threoninehydroxamate by varying a coryneform glutamic acid-forming mold (a group of glutamic acid-forming mold belonging to the genus Corynebacterium, Brevibacterium, Microbacterium or Arthrobacter) by an ordinary variation means. Corynebacterium glutamicum H-4260 may be cited as the preferable example. It is cultivated in a medium containing a carbon source, a nitrogen source, an inorganic salt, etc., L-isoleucine is accumulated in a culture mixture and collected.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention exhibits resistance to threonine hydroxamate;
The present invention relates to a method for producing L-isoleucine using a microorganism belonging to coryneform glutamic acid-producing bacteria having the ability to produce L-isoleucine.

L-isoleucine is used in fields such as the pharmaceutical industry as an amino acid preparation.

Conventional technology Conventionally, as a method for producing L-isoleucine by a fermentation method, L-isoleucine belonging to Micrococcus glutamicus, threonine,
A method using microorganisms having auxotrophy for methionine, homoserine or leucine (Japanese Patent Publication No. 38-7091),
Belongs to Brevibacterium flavum, α-amino-β
- Method using microorganisms having hydroxyvaleric acid resistance and adenine or lysine auxotrophy (Japanese Patent Publication No. 51-623
7), a method using a microorganism resistant to α-amino-β-hydroxyvaleric acid and ○-methylthreonine (Japanese Patent Publication No. 51-21077), which belongs to the genus Brevibacterium or the genus Corynebacterium, Amino
β-hydroxyvaleric acid resistant and ethionine, 2-thiazolealanine and S-(2-aminoethyl)-L
- A method using a microorganism resistant to at least one of cysteine (Japanese Patent Application Laid-Open No. 101582/1982),
A method using a microorganism that belongs to the genus Corynebacterium and is resistant to isoleucine analogs and has a methionine or lysine auxotrophy (Japanese Patent Publication No. 1983-32070); A method that uses a microorganism that belongs to the genus Corynebacterium and has vitamin-P resistance Method (Japanese Unexamined Patent Publication No. 57-2687),
A method using a microorganism belonging to the genus Corynebacterium and having an arginine auxotrophy (JP-A-59-106295), a method using a microorganism belonging to the genus Corynebacterium and having fluoropyruvate sensitivity (JP-A-59-1989) 1
No. 06294) and the like are known.

Problems to be Solved by the Invention There is a need for a method for producing L-isoleucine, which is useful as an amino acid preparation, with higher yield and at lower cost.

Means for Solving the Problems The present inventor conducted research on better L-isoleucine producing bacteria in order to produce L-isoleucine with high yield and at low cost. As a result, when using a microorganism belonging to the coryneform glutamate-producing bacteria that is resistant to threonine hydroxamate and has the ability to produce L-isoleucine, L-isoleucine can be produced with good yield.
- It was discovered that isoleucine could be obtained, and the present invention was completed.

The present invention will be explained in detail below.

The present invention involves culturing a microorganism belonging to coryneform glutamate-producing bacteria, showing resistance to threonine hydroxamate, and having an ability to produce L-isoleucine in a medium, producing and accumulating L-isoleucine in the culture, and culturing the microorganism in a medium. L- than things
Provided is a method for producing L-isoleucine by a fermentation method characterized by collecting isoleucine.

As an example of the microorganism used in the present invention, any microorganism that belongs to the coryneform glutamic acid-producing bacteria, exhibits resistance to threonine hydroxamate, and has the ability to produce L-isoleucine can be used. A strain resistant to threonine hydroxamate can be obtained by mutating a coryneform glutamate-producing bacterium by conventional mutation methods and growing on an agar medium containing 1 g/(2 or more) of threonine hydroxamate. A suitable example is Corynebacterium glutamicum H-42.
I can give you 60.

As used herein, coryneform glutamate-producing bacteria are
A group of glutamic acid-producing bacteria belonging to the genus Corynebacterium, Brevibacterium, Microbacterium, or Arthrobacterium [see "Fermentation and Industry" Vol. 40, 102, 1982].

A specific example of the method for obtaining the above-mentioned threonine hydroxamate-resistant strain is shown below.

Corynebacterium glutamate H-3501 [arginine auxotrophic, S-(2-aminoethyl)-L-cysteine resistant, rifampicin resistant, fluoropyruvate sensitive, this strain was approved by the Agency of Industrial Science and Technology on July 16, 1985. FERM to the Microbial Technology Research Institute (hereinafter referred to as FERM)
It has been deposited as BP-848. ] N-methyl-N' ~ nitro N-nitrosoguanidine NTG)
After mutagenesis treatment (200x/ml, 30°C, 30 minutes), collect a strain that grows and vines on the following minimal agar medium supplemented with 2g/β threonine hydroxamate. I can do it. The obtained 50 threonine hydroxamate resistant strains were transformed into L-
A strain was selected which was subjected to an isoleucine production test (method in the example) and had significantly better L-isoleucine productivity than the parent strain. One of the strains was Corynebacterium glutamicum H-4260 (arginine auxotrophy, 5-(2-aminoethyl iL-cysteine resistant, rifampicin resistant,
Sensitive to fluoropyruvate, resistant to threonine hydroxamate].

This strain was FERMed to the Microtech Institute on February 13, 1986.
It has been deposited as BP-986.

Medium composition Nigelcose 0.5%, (NH4)2S0゜0
．． 15%, K H2P O* 0.15%, K2HP0
゜0.05%, NaCJ! 0.01%, Mg5C)
s ・7H200,05%, CaC#z'2HzO1
x/m15MnCl1z・4H207xr/ml, Fe
50<1H2010g/m+, thiamine hydrochloride Q,
1B/ml, biotin Q, 03■/ml, arginine hydrochloride 0.01%, agar 1.5%, pH 7.2 The above microorganisms were placed in a synthetic medium containing carbon sources, nitrogen sources, inorganic salts, growth factors, etc. L-isoleucine can be produced by accumulating L-isoleucine in the culture by culturing in a natural medium and collecting it.

Carbon sources include glucose, sucrose, molasses,
Sugars such as starch hydrolysates, acetic acid, propionic acid,
Organic acids such as formic acid, fumaric acid, and malic acid, alcohols such as methanol, ethanol, and propatool, and hydrocarbons can be used.

Nitrogen sources include ammonium sulfate, ammonium nitrate,
Ammonium chloride, ammonium phosphate, urea, ammonia, etc. can be used. Furthermore, in the case of mutant strains exhibiting auxotrophy, nutritional substances can be added as pure products or in the form of natural nutrients containing them.

Cultivation is performed under aerobic conditions such as shaking culture or deep aeration agitation culture. The culture temperature is usually in the range of 24 to 37°C, preferably 28 to 32°C, and the pH of the medium is in the range of 5 to 9.
It is desirable to maintain the temperature near neutrality. The pH of the medium is adjusted using calcium carbonate, an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia gas, or the like.

The culture period is usually 2 to 7 days, and L-isoleucine is produced and accumulated in the culture.

After culturing, L-isoleucine can be recovered from the culture solution by removing precipitates such as bacterial cells from the culture solution and using ion exchange treatment, concentration method, adsorption method, salting out method, etc. .

Examples are shown below.

Example 1 Corynebacterium glutamicum H-4260 [arginine auxotrophy, 5-(2-aminoethyl)-L-cysteine resistance, fluoropyruvate sensitivity, rifampicin resistance, threonine hydroxamate resistance] (FER
M BP 986) in 23 ml of seed medium (glucose 5%, yeast extract 1%, peptone 1%, urea 0.3%).
, NaC 10.25%, Corn Steep Liquor 0.
5%, biotin 50ttx/l, pH 7,2)
Inoculated into a 00ml Erlenmeyer flask and incubated at 28°C for 24 hours.
Shaking culture was performed on a rotary shaker at 21 rpm.

300ml of this seed culture containing 20ml of fermentation medium
It was inoculated into a 1 m Erlenmeyer flask and cultured for 72 hours in the same manner as the seed culture. The amount of L-isoleucine accumulated in the culture solution after completion of the culture was 13.4 g/#. As a result of culturing in the same manner using the parent strain H-3501 [arginine auxotrophic, S-(2-aminoethyl)-L-cysteine resistant, fluoropyruvate sensitive, rifampicin resistant] as a control, L-inleucine accumulation was observed. The amount is 11.6g/f
It was f.

The composition of the fermentation medium is as follows.

Waste ng < glucose conversion 3iE) 70g/C corn steep liquor 5g/L ammonium chloride 20g#, urea 2g/j! , KH2PO12g/11Mg5O4-7H
200,5g/j2SFeSO4・7H200,01g
//, MnCj! 2.4HzO0.01g#2, Cu5
O,・5H200,01g#,CaCff1z・2t(
to O, OXg/l, ZnSO4・7H7H2O1
/n, N 1 (1221 mg/n, % Ammonium ribdate tetrahydrate 1 mg/I2, Co C (12.6 Hz O1
mg/f, calcium pantothenate 10 mg/C2:+
Tinic acid 1 mg/#, biotin 50 μ/1, arginine hydrochloride 0.5 g/I2, pH 7, 200 ffll of a culture solution containing linoin obtained using the 4H-4260 strain was centrifuged (3,00 rpm, 10 minutes). ) to remove bacterial cells and other impurities.

The obtained supernatant liquid was passed through a column of strongly acidic cation exchange resin Diaion 5KI (H" type) (manufactured by Mitsubishi Chemical Industries, Ltd.) to adsorb L-isoleucine, and after washing with water, 0.5 N ammonia water was added. The L-isoleucine fraction was collected by elution.The collected fractions were concentrated and crystallized using an isoelectric point of pH 6.02 to obtain 99% pure L-isoleucine 1.4.
I got g.

Effects of the Invention According to the method of the present invention, L-isoleucine can be obtained in good yield.

Claims (1)

[Claims] A microorganism belonging to the coryneform glutamate-producing bacteria, showing resistance to threonine hydroxamate, and having the ability to produce L-isoleucine is cultured in a medium, L-isoleucine is produced and accumulated in the culture, and L-isoleucine is produced and accumulated in the culture. A method for producing L-isoleucine by a fermentation method, characterized by collecting isoleucine.