JPS5813151B2 - Production method of L-norleucine by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-norleucine by fermentation.

Norleucine is an amino acid well known as an antimetabolite of methionine [Genetics, 58,473
-492 (1968)], and is also useful as an analog of branched chain amino acids such as isoleucine, parine, and leucine [see Biotechnology and Bioengineering, 7, 245-268 (1965)].

The present inventors previously reported that a mutant strain of Serratia marsetuscens that is auxotrophic for isoleucine and valine and in which the metabolic regulation mechanism by leucine has been released has the ability to generate and accumulate L-norleucine from norvaline. [Collection of lecture abstracts from the 1971 Japan Fermentation Engineering Society Conference, 71-72
See page].

However, this strain did not grow at all unless methionine was added to the medium, and was unable to produce and accumulate L-norleucine from norvaline.

Therefore, the present inventors conducted a wide search for microorganisms that can produce and accumulate L-norleucine without adding methionine and norvaline to the culture medium, and found that they belong to the genus Serratia.
A mutant strain that is auxotrophic for isoleucine and valine (including partial auxotrophy, the same shall apply hereinafter) and in which the metabolic regulation mechanism by leucine and the metabolic regulation mechanism by methionine is abolished exhibits L- even in a medium that does not contain both methionine and norvaline. They discovered that they have the ability to produce and accumulate norleucine, leading to the completion of the present invention.

Examples of microorganisms used in the present invention include, for example, Serratia marsetuscens [Bergie's Manual of Determinative Bacteriology, 8th edition,
[See page 362] Mutant strains that require isoleucine and valine and have a metabolic regulation mechanism by leucine and a metabolic regulation mechanism by methionine are mentioned.

Such mutant strains can be obtained, for example, as follows.

First, mutations are induced in the original strain by conventional mutation induction operations, such as ultraviolet irradiation or treatment with mutagenic agents (e.g., N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, etc.), and this induces leucine metabolism. Metabolic regulation by leucine can be investigated by culturing for 2 to 3 days in a plate medium (e.g., Davis basal medium) supplemented with antagonists such as α-aminobutyric acid and 4-azaleucine at approximately 10 mg/ml each, and isolating the resulting large colonies. It is possible to obtain mutant strains in which the mechanism has been released.

Next, mutations were induced in this mutant strain in the same manner as described above, and this was cultured for 1 to 2 days on a plate medium (e.g., Davis basal medium) supplemented with approximately 2 μg/ml of each of isoleucine and valine, resulting in small to medium colonies. By isolating this strain and confirming the auxotrophy of this strain by the replica method, it is possible to obtain a mutant strain that is auxotrophic for isoleucine and valine and in which the metabolic regulation mechanism by leucine has been abolished.

Next, mutations were induced in this mutant strain in the same manner as described above, and the mutant strain was cultured in a plate medium supplemented with a small amount of the required amino acid and approximately 10 mg/ml of a methionine antimetabolite antibody (e.g., norleucine, methionine hydroxamate, α-methylmethionine, etc.). For example, isoleucine,
It is possible to obtain an L-norleucine-producing bacterium that requires valine and has a leucine-based metabolic mechanism and a methionine-based metabolic control mechanism released.

Specific examples of L-norleucine-producing bacteria obtained in this way include Serratia marsetuscens NLV-
11 (FER No. 3498) (isoleucine and valine auxotrophic mutant strain and resistant to α-aminobutyric acid, 4-azaleucine and norleucine), Serratia marsetuscens NLG-21 (FER No. 3499) ) (a mutant strain that requires isoleucine and valine and is resistant to α-aminobutyric acid, 4-azaleucine, and norleucine).

The method for inducing L-norleucine-producing bacteria according to the present invention is not particularly limited. For example, first, a mutant strain in which the metabolic regulation mechanism by leucine has been released is obtained, and then this mutant strain is subjected to metabolic regulation by methionine. An L-norleucine-producing bacterium can also be obtained by imparting a property in which the mechanism is released and finally imparting isoleucine and valine auxotrophy to this mutant strain.

In addition, among strains that can be transduced, L-norleucine producing bacteria can also be obtained by separately obtaining mutant strains having any of the above three properties and transducing these properties with each other. can.

The thus obtained microbial L-norleucine production medium contains 2 to 15% of sugars such as glucose and starch hydrolyzate, organic acids such as fumaric acid and citric acid, and alcohols such as glycerol as carbon sources; 0.5 to 3% of organic ammonium salts such as asmonium acetate, inorganic ammonium salts such as ammonium sulfate and ammonium chloride, urea, etc. as nitrogen sources; 0 to 2% of corn steep liquor, peptone, yeast extract, etc. as organic nutrients;
Furthermore, a medium containing a small amount of required amino acids such as isoleucine and valine is preferably used.

In addition to these, a small amount of potassium phosphate, magnesium sulfate, etc. may be added, and calcium carbonate or ammonia may be added if necessary to maintain the pH of the medium at 6 to 9.

Furthermore, a precursor for the biosynthetic system of L-norleucine, such as norvaline or α-ketovaleric acid, is added to such a medium.
If it is contained in an amount of up to 3%, the amount of L-norleucine produced and accumulated can be significantly increased.

According to the present invention, these mediums are inoculated with the L-norleucine-producing bacteria and incubated at 25-37° C. for 1 to 4 hours under aerobic conditions, for example, under conditions with good oxygen supply such as vigorous shaking.
By culturing for days, L-norleucine can be produced and accumulated in the medium.

The produced L-norleucine can be easily collected by a conventional separation and purification method using, for example, an ion exchange resin.

Hereinafter, the present invention will be specifically explained with reference to Examples. The ninhydrin reaction was carried out, and its quantification was carried out by cutting out and extracting the colored portion resulting from the reaction, and using a colorimetric method.

Example 1 2% glucose, 10% dextrin, 1% urea, DL-
Norvaline 2%, dibasic potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, corn steep liquor 0.7%, L-isoleucine 0.5~/ml, L-valine 1.0mg/ ml and 15 ml of a medium (pH 7.0) containing 3% calcium carbonate are poured into a 50 ml shaking Kolben and autoclaved.

However, glucose and dextrin are added after separate sterilization.

One platinum loop of Serratia marsetuscens NLV-11 (Feikoken Bibori No. 3498) (a mutant strain that requires isoleucine and valine and is resistant to α-aminobutyric acid, 4-azaleucine, and norleucine) was inoculated into this. , shake culture for 72 hours at 30° C., 140 revolutions/min, and an amplitude of 8 cm.

The amount of L-norleucine produced in the culture solution was 4.5 mg.
/ml.

Collect 1 liter of culture solution and filter after heat treatment.

The filtrate is strongly acidic ion exchange resin Amberlite 1R-12.
0 column.

After washing this column with water, it is eluted with 0.15M aqueous ammonia.

The eluted L-norleucine fraction was concentrated under reduced pressure, methanol was added, and the precipitated crystals were collected by filtration to obtain L-
Obtain 2.5 g of norleucine.

Example 2 Using the same medium as in Example 1, Serratia marsetuscens NLG-21 (FEI No. 3499) (isoleucine and valine auxotrophic and resistant to α-aminobutyric acid, 4-azaleucine 8 and norleucine) was grown. Example 1
It was cultured in the same way.

The amount of L-norleucine produced in the culture solution was 25 mg/
It was ml.

Example 3 Serratia marsetuscens NLG-21 (Feikoken Bacterial Serial No. 3499) was cultured in the same manner as in Example 1 using the medium from Example 1 except that DL-norvaline was removed.

The amount of L-norleucine produced in the culture solution is 0.8 mg
/ml.

Claims (1)

[Scope of Claims] 1 An L-norleucine-producing strain belonging to the genus Serratia that is auxotrophic for isoleucine and valine and in which the metabolic regulation mechanism by leucine and the metabolic regulation mechanism by methionine have been released is cultured in a medium, and L-norleucine is produced in the medium. - A method for producing L-norleucine by a fermentation method, which is characterized by producing and accumulating norleucine and collecting it. 2. The production method according to claim 1, wherein the bacterial cells belong to the genus Serratia and are L-norleucine-producing bacteria that require isoleucine and valine and are resistant to leucine antimetabolites and methionine antimetabolites. 3. The production method according to claim 1, wherein the bacterial strain belongs to the genus Serratia and is an L-norleucine producing bacteria that is auxotrophic for isoleucine and valine and resistant to α-aminobutyric acid, 4-azaleucine and norleucine. 4. The production method according to claim 1, wherein the strain is Serratia marsetuscens, which is isoleucine and valine auxotrophic and has an ability to produce L-norleucine in which the leucine-based metabolic control mechanism and the methionine-based metabolic control mechanism have been released. 5. The production method according to claim 1, wherein the bacterial strain is Serratia marsetuscens, which is isoleucine and valine fertile and has an ability to produce L-norleucine, which is resistant to leucine antimetabolite antibodies and methionine antimetabolite antibodies. 6 L strain whose strain is isoleucine, valine auxotrophic and resistant to α-aminobutyric acid, 4-azaleucine and norleucine
- The production method according to claim 1, which is Serratia marsetuscens having the ability to produce norleucine. 7 Culture under aerobic conditions, pH 5-9, temperature 25-37.
The manufacturing method according to claim 1, 2, 3, 4, 5 or 6, which is carried out at ℃. 8 Culture in a medium containing a precursor for the L-norleucine biosynthetic system under aerobic conditions at pH 5-9 and temperature 25-37°C.
Claims 1, 2, 3, and 4
5. The manufacturing method according to item 5 or 6. 9. The production method according to claim 8, wherein the precursor of the L-norleucine biosynthesis system is norvaline or α-ketovaleric acid.