JPS63209593A - Production of l-threonine by fermentation method - Google Patents

Abstract

PURPOSE:To produce L-threonine useful as amino acid preparation, etc., efficiently and inexpensively, by cultivating a bacterium belonging to the genus Escherichia, etc, capable of producing L-threonine in a nutrient containing acetic acid or a salt thereof. CONSTITUTION:A bacterium (e.g. Escherichia coli ATCC21530) capable of producing L-threonine is aerobically cultivated in a medium containing a carbon source, a nitrogen source, inorganic salts etc., and 0.2-5g/l acetic acid or a salt thereof such as sodium acetate, ammonium acetate, potassium acetate, etc, at 20-40 deg.C, preferably 28-38 deg.C at pH 5-9, preferably pH of approximately neutrality for 2-7 days. After the culture is over, a cell or other impurities are removed and L-threonine is isolated from the supernatant liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing L-threonine by culturing a microorganism capable of producing L-threonine in a medium supplemented with acetic acid.

L-threonine is an amino acid that is not only useful as a medicine such as amino acid preparations (but also can be used as a feed additive).

Conventional technology Conventionally, the method for producing L-threonine by fermentation method is as follows:
A method using a microorganism belonging to the genus Escherichia that is sensitive to borrelidin (Japanese Patent Publication No. 51-6752); a microorganism that belongs to the genus Escherichia and exhibits a requirement for diaminopimelic acid and methionine; (Japanese Patent Publication No. 56-10037), a method using a microorganism belonging to the genus Serratia that is deficient in threonine dehydrogenase, and is resistant to threonine antimetabolites (
(Japanese Patent Publication No. 52-48195), belongs to the genus Corynebacterium, and contains α-amino-β-hydroxyvaleric acid and S
A method using a microorganism resistant to -(2-aminoethyl)-L-cysteine and auxotrophic for methionine (Japanese Unexamined Patent Publication No. 19087/1987), which belongs to the genus Brevibacterium and has α-amino-β- Hydroxyvaleric acid and 5-
A method using a microorganism resistant to (2-aminoethyl)-L-cysteine and auxotrophic for leucine (Japanese Unexamined Patent Publication No. 50-31093), which belongs to the genus Brevibacterium and is an α-amino-β-hydroxy Valeric acid and 5-(2
-aminoethyl)-L-cysteine and L-
A method using microorganisms that require isoleucine and lysine (Japanese Unexamined Patent Publication No. 58-224684), a method using microorganisms capable of producing L-threonine with one or more organic acids selected from fumaric acid, maleic acid, and malic acid. A method of culturing in a medium containing 1 mol of organic acid and saccharides at a ratio of 1 to 8 mol of saccharides (Japanese Patent Laid-Open No. 14969/1983)
Publication No. 0) and the like are known.

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

Means for Solving the Problems The present inventor conducted research on better L-threonine production conditions in order to obtain L-threonine in high yield. As a result, they discovered that L-threonine productivity could be improved by culturing microorganisms capable of producing L-threonine in a medium containing sugar as the main carbon source and acetic acid added, and completed the present invention. .

The present invention will be explained in detail below.

The present invention involves culturing a microorganism capable of producing L-threonine in a medium supplemented with 0.2 to 5 g/l of acetic acid or its salt, producing and accumulating L-threonine in the culture, and producing L-threonine from the culture. - Provides a method for producing L-threonine by a fermentation method characterized by collecting threonine.

As an example of the microorganism used in the present invention, any microorganism can be used as long as it has the ability to produce L-threonine. Specifically, Escherichia Co'JATCC2
Examples include 1530.

Threonine-producing bacteria of the genus Escherichia have the advantage of having a small amount of by-product amino acids.

By culturing L-threonine-producing bacteria in a synthetic medium or natural medium containing carbon sources, nitrogen sources, inorganic salts, growth factors, etc., and acetic acid or its salts added, L-threonine can be produced in the culture. (1) 7-threonine can be produced by accumulating M and collecting this.

As the carbon source, sugars such as glucose, fructose, molasses, and starch hydrolyzate are used as the main carbon source.

Nitrogen sources include various inorganic salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, ammonium salts of organic acids, amines, and other nitrogen-containing compounds, as well as peptone, meat extract, corn steep liquor, and casein hydrolysis. Soybean meal hydrolyzate,
Various fermented microbial cells and their digested products are used.

As the inorganic salts, potassium phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. are used.

The concentration of acetic acid added to the medium is preferably 0.2 to 5 g/β. It can also be added as a salt such as ammonium acetate, sodium acetate, potassium acetate, or the like. Both synthetic and natural acetic acids can be used, and any substance containing acetic acid can be used even if it is not a pure product.

Cultivation is performed under aerobic conditions such as shaking culture or deep aeration agitation culture. Culture temperature is 20=40℃, preferably 28℃
-38°C. The pH of the medium is in the range of 5 to 9,
Preferably, it is maintained near neutrality. The pH of the medium is adjusted using calcium carbonate, inorganic or organic acids, alkaline solutions, ammonia, pH buffers, and the like.

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

After completion of the culture, L-threonine can be recovered from the culture solution by removing precipitates such as bacterial cells from the culture solution and using an ion exchange treatment method, a concentration method, a salting out method, etc. in combination.

It has been known that acetic acid can be used as a carbon source and salts such as ammonium acetate can be used as a nitrogen source. However, the concentration of acetic acid added in this case is 10 g/β or more.

The present invention does not use acetic acid or ammonium acetate as a carbon source or nitrogen source, but instead uses 0.0% acetic acid in a synthetic medium or a natural medium (but not containing acetic acid) used for culturing L-threonine producing bacteria. It is shown that L-threonine can be produced with higher yield by adding 2 to 5 g/β.

Examples are shown below.

Example 1 A -threonine production test was conducted using Escherichia coli ATCC 21530 strain as the inoculum. ATCC2
1530 strain was grown in a seed medium (pH 7.4) with a composition of 2% glucose, 1% peptone, 1% yeast extract, Na (1! 0.25%, and diaminopimelic acid 0.1 g/j!) for 30 minutes.
The cells were cultured with shaking at ℃ for 16 hours. 2 ml of the obtained seed culture solution was inoculated into 25 Qml Erlenmeyer flasks each containing 23 ml of a production medium with the following composition and acetic acid added to the concentration shown in Table 1, and incubated at 30°C for 72 ml. Cultured with shaking for hours.

Table 1 shows the amount of L-threonine accumulated at that time.

Chapter 1 Table The composition of the production medium is as follows.

Glucose 7%, (NH4) a 3041.4%,
KH, PO40, 2%, M g S 04.7H, OO
,1%, diaminopimelic acid 0.3g/l, DL-,1
Onin 0.1g/C Corn Steep Liquor 〇, 2
%, CaC0a 3% (pH 7,4) Acetic acid 1 g/f
20 Qml of the L-threonine-containing culture solution obtained as above using the production medium containing
pm for 10 minutes) to remove bacterial cells and other impurities. The obtained supernatant liquid was passed through a column of strongly acidic ion exchange resin Diaion 5KI (H” type) (manufactured by Mitsubishi Chemical Industries, Ltd.) to adsorb L-threonine, and after washing with water, it was eluted with 0.5N ammonia water. The L-threonine fraction was collected.

Both collected fractions were concentrated, ethanol was added, and the mixture was stored under cooling to obtain 1.9 g of L-threonine crystals with a purity of 98% or higher.

Example 2 An L-threonine production test was conducted in the same manner as in Example 1, except that sodium acetate or ammonium acetate was added to the production medium instead of acetic acid.

IJ and ammonium acetate were added to the production medium in terms of acetic acid so that the concentrations were as shown in Tables 2 and 3.

The amount of L-threonine accumulated is shown in Tables 2 and 3.

Table 2 Table 3 Effects of the Invention According to the present invention, L-threonine can be obtained in good yield.

Claims (1)

[Claims] A microorganism capable of producing L-threonine is cultured in a medium supplemented with 0.2 to 5 g/l of acetic acid or its salt, L-threonine is produced and accumulated in the culture, and L-threonine is produced and accumulated in the culture.
L produced by a fermentation method characterized by collecting threonine
- A method for producing threonine.