JPS63198995A - Production of l-threonine, collection of bacterium and cell of bacterium - Google Patents

Abstract

PURPOSE:To obtain L-threonine efficiently and inexpensively, by treating 5- methyl-2-oxo-oxazolidine-4-carboxylic acid with a cell of Alcaligenes XY-207, a culture solution or a treated material thereof. CONSTITUTION:5-Methyl-2-oxo-oxazolidine-4-carboxylic acid obtainable industrially and relatively inexpensively as a raw material is treated with a bacterium capable of producing L-threonine, a culture solution or a treated material of the cell. Alcaligenes YY-207 strain (FERM P-9190) belonging to the genus Alcaligenes may be cited as the bacterium capable of producing L-threonine. the bacterium can be cultivated in a medium containing 5-oxoproline as only one carbon source and a nitrogen source.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is a biochemical method for producing L-threonine, which is one of the essential amino acids for humans and is useful as a pharmaceutical, food additive, etc. , a method for obtaining a microorganism effective for the production method, and a microorganism strain of the genus Alcaligenes YY-207.

(Prior art and problems) Because threonine has two asymmetric carbon atoms in its molecule,
There are four types of optical isomers, but among these isomers, L-threonine is currently the only physiologically active compound, that is, one that has industrial value.

Traditionally, methods for producing L-threonine have been known, including isolation from protein hydrolysates and synthesis methods, but compared to other amino acids, it is easier to isolate L-threonine from protein hydrolysates. It is said to be relatively difficult. on the other hand,
Synthesis methods are roughly divided into organic chemical methods and biochemical methods such as fermentation methods and enzyme methods.The former method usually yields DL-threonine (racemic form of threonine), so it is difficult to produce L-threonine. Optical resolution and racemization techniques are indispensable for this, and in most cases, it is unavoidable that a certain amount of physiologically inactive allothreonine (DL itself) is produced as a by-product in addition to DL-threonine. , separation from them,
The production cost, including the labor involved in refining, is high, and even if efforts are made to reduce the cost, there are limits, resulting in many economic problems.

Various biochemical methods have been studied as an effective method for selectively producing only L-threonine from among the four optical isomers. Conversion from L-homogenine using a culture solution is well known, and various other methods have been proposed, including direct fermentation using various microorganisms, precursor fermentation, and methods for converting synthetic compounds to L-threonine through enzymatic reactions. However, the threonine biosynthesis system is subject to so-called metabolic control, and for this reason, it is difficult for normal microorganisms to accumulate threonine above a certain level in the direct fermentation method, and methods such as genetic recombination are indispensable. . Furthermore, in the method using a precursor, there are economical difficulties in obtaining the precursor itself. On the other hand, methods using an enzymatic reaction include a method using a 2-oxazolidine derivative as a raw material (JP-A-58-237130) and a biochemical optical resolution method of a mixture of threonine isomers. However, even though it is a biochemical conversion method using synthetic compounds, in reality it simply replaces organic chemical reactions such as hydrolysis and decarboxylation with enzymatic reactions, and therefore, the compounds used as raw materials are pre-prepared with L. - In many cases, it is necessary to prepare an L monolith that can be converted into threonine, and this method cannot necessarily be said to be economically advantageous. In particular, in the case of threonine, as mentioned above, two adjacent carbon atoms are both indispensable. Because it is a homogeneous carbon, four types of isomers exist, and when each isomer is racemized, 1 For example, D-threonine usually produces a mixture with L-allothreonine, and even if it is optically resolved, L - Threonine cannot be obtained; to obtain L-threonine by optical resolution of isomers, it is necessary to racemize D-70 threonine and resolve it, etc. Other asymmetric amino acids with only one carbon atom It is often very troublesome compared to optical separation.

(Problem to be solved by the invention) Therefore, in the biochemical production method of L-threonine using synthetic compounds, it is necessary to use compounds as cheap as possible as raw materials and to generate and accumulate only L-threonine from these compounds. In other words, it is important to find microorganisms (enzyme systems) that have the ability to discriminate between threo-allo and L-d forms.

(Means for solving the problem) The present invention provides 5-methyl-2 which can be obtained industrially at relatively low cost.
-oxo-oxazolidine-4-carboxylic acid (hereinafter MO
The present invention aims to provide a method for selectively converting L-threonine into L-threonine by biochemical means, a method for obtaining microorganisms effective for this method, and a novel microorganism. .

That is, the present invention provides a method for producing L-threonine, which is characterized by allowing 5-methyl-2-oxo-oxazolidine-4-carboxylic acid to act on a microorganism capable of producing L-threonine, a culture solution, or a treated bacterial cell product. and 5-methyl-2-oxo-oxazolidine-4-carboxylic acid is cultured in a medium containing 5-oxoproline as the sole charcoal and nitrogen source, and the grown strain is collected. The present invention relates to a method for obtaining L-threonine-producing microorganisms from 5-methyl-2-oxo-oxazolidine-4-carboxylic acid. In addition, as a representative microorganism isolated from soil by this method, a new bacterium belonging to the genus Alcaligenes, Alcaligenes YY-207 strain (1982
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as February θ, 2013, as Microtechnology Research Institute No. 8180. ) is provided.

The method of the present invention will be explained in more detail below.

When chemically converting MODN to threonine, L-)
L-threonine is obtained from lance MODN, but D
-) D-threonine, L-cis M from Lance MODN
L-70 threonine is obtained from ODN, and D-70 threonine is obtained from D-cis MODN. At the beginning of the research on the present invention, the present inventors obtained L-70 threonine from each isomer of MODN.
- In order to collect microorganisms that have the ability to selectively decompose only trans-MODN, microorganisms isolated from soil are cultured in a synthetic medium containing L-trans-MODN.
I investigated the presence or absence of threonine production and accumulation, MODN
Although several microorganisms having the ability to decompose L-threonine have been found, they all accumulate a small amount of L-threonine, and are therefore far from being of practical use.

Therefore, the microorganism was further cultured in a medium containing 5-oxoproline, and a strain that grew in a medium containing only 5-oxoproline as a carbon and nitrogen source was collected, and this was suspended in physiological saline and combined with MODN. It has been found that the reacted product produces threonine in high yield.

A typical strain thus obtained is the Alcaligenes strain YY-207, and its acquisition method and mycological properties are shown below.

[How to obtain bacteria]

The soil suspension was inoculated into a liquid medium containing MODN shown in Attached Table 1, and cultured at 30°C for several days. Furthermore, one drop of this culture solution was transferred to the same liquid medium and cultured at 30°C for several days. After repeating this operation several times, the culture solution was spread on the same agar medium and cultured, and colonies that were observed to grow were isolated.

Each of these isolated bacteria was inoculated into 5 sets of media shown in separate if, and after shaking culture at 28°C for 48 hours, bacteria were removed and MODN in the supernatant was quantified.
0 strains were isolated.

Next, we searched for bacterial cells that can grow in a medium containing 5-ocitubroline as the sole carbon source shown in Attached Table 2, and found two strains. Collect these bacteria and add 70 mmol of phosphorus Sa to each
When suspended in a buffer solution (pH 7) and reacted with MODN, strain YY-207 was found to decompose MODN and accumulate threonine.

Table 1 L-) 5 MODN 0.5%
KH2P0. 0.0
5 to 2HP0. 0.
05Mg504-7H200,05 (pH7) Table 2 5-oxoproline 0.5%KH2P
O40,05 to 2HPO40,05 Mg5O4-71 (200,05 (pH 7) [Bacteriological properties] (A) Morphology (1) Size of vegetative cells: 0.8-0.7 X 1.4-1. 5μzen (2
) Cell pleomorphism: None 3) Durham staining: Negative (4)! f! Mobility: Yes (5) Flagella: Perifelgate (6) Spores: Not formed (B) Culture findings (1) Peptone medium pH: 9.4-3.2 Growth. Especially p)+7.
Growth is good at temperatures between 2 and 4.3 Growth temperature: Grows at an upper limit of 40°C, but does not grow at 42°C.

(2) Other cultivation j1! I: Meat extract, good growth on agar slanted medium, spreading shape, light yellowish-white glossy meat extract, agar plate medium diameter 2 to 3 servings ■ 48 hours 1 circular ridge shape, pale yellowish-white glossy meat extract on all edges, good growth on liquid medium , turbidity, precipitate, fungal membrane Meat juice Gelatin puncture culture Filamentous, not liquefied Lidomus milk alkaline (3) Attitude towards oxygen Aerobic (C) Physiological properties (1) Nitrate reduction + (2) VP test - (3) Generation of indole
- (0) Hydrolysis of starch (5) Utilization of citric acid + (6) Production of pigment - (7) Urease
・-(8) Oxidase +
(9) Catalase + (10) O-
F test Oxd, ± (11) denitrification reaction
+(12) MR test
-(13) Generation of hydrogen sulfide
+ (10 Utilization of inorganic nitrogen sources + (15) Production of acid and gas from sugars Sugar Acid production Gas production L-arabinose ± -D-xylose -〇-glucose ±
-D-mannose ± -D-fructose -D-galactose ± -maltose
- sucrose - lactose - trehalose ± -D-sorbitol ± -D-mannitol ±
- Inozite - - Glycerin ± - Starch ± - (1B) Other effects Does not hydrolyze gelatin and casein.

Poly-β-hydroxybutyrate is present.

There are no particular restrictions on the cultivation of microorganisms capable of producing L-threonine from MODN, such as the above-mentioned Alcaligenes YY-207 bacteria, but 5-oxtubebroline is generally cultured in a synthetic or natural medium containing a carbon source, nitrogen source, inorganic salts, etc. 0
．． About 1 to 2 t%, preferably about 0.5 to 1 wt%, is added and cultured aerobically. Glucose as a carbon source,
There is no particular restriction as long as it can be assimilated by bacteria, such as sugars such as sugars, starch saccharified liquid, and cellulose decomposition products, organic acids such as acetic acid, alcohols such as ethanol, etc. Nitrogen sources include ammonia, ammonium sulfate, and salts. Ammonium salts such as ammonium ammonium, ammonium nitrate, ammonium phosphorus, urea, nitrates, etc. are used as appropriate. Examples of inorganic salts include salts such as phosphoric acid, potassium, magnesium, iron, and manganese; for example, common industrial chemicals such as ammonium phosphate, potassium phosphate, potassium sulfate, caustic potassium, magnesium sulfate, ferrous sulfate, and manganese sulfate; and others. Salts such as calcium, sodium, zinc, boron, copper, cobalt, and molybdenum may be added as trace elements, and vitamins and amino acids as trace organic nutrients are not particularly required for the growth of bacteria. , these can be added, corn steep liquor, meat extract, yeast extract, peptone,
You may also add organic substances such as

Culture is carried out under aerobic conditions such as shaking culture 1 aeration stirring culture, culture temperature is 25-45°C, p)I is 5.0-9
．． 0, preferably in the range of 8 to 8.

Although there are no particular limitations on the manner in which L-threonine is produced by the method of the present invention, MODN is usually added to a solution in which bacterial cells isolated from a culture solution are suspended in physiological saline and reacted. However, depending on the case, the bacterial cell culture solution may be used as it is, or a bacterial cell-treated product may be used.

Although there are no particular restrictions on the reaction conditions, it is generally appropriate to carry out the reaction under the following conditions.

Reaction temperature: 20-35°C, preferably 25-30°C, p
H: so~9.0. Preferably 8.0 to 8.0,
Bacterial cell concentration: 20 to 200 layers g/ml, MODN: Proportional to bacterial cell concentration, but usually 50 to 500 mol/fL,
Reaction time: 10-24 hours, preferably 15-20 hours.

Known methods can be used for separation, recovery, and analysis of L-threonine from the reaction solution.

For example, after removing bacterial cells from the reaction solution by centrifugation,
According to a conventional method, it can be purified and concentrated by adsorption/philic treatment using a cation exchange resin and a 7-ion exchange resin, and if necessary, it can be isolated by performing isoelectric focusing crystallization.

L-threonine was identified using an amino acid analyzer.

This can be performed using IR spectrum, mass spectrum, or nuclear magnetic resonance spectrum.

The optical purity of L-threonine is measured using an optical rotation needle, but it can be analyzed with higher accuracy by using commercially available optical resolution column chromatography.

Example 1 Using the nutrient medium shown in Table a, 24 strains of YY-207 were grown.
The cells were cultured at 30°C for an hour. 160g of centrifuged bacterial cells
DL-trans/MODNIOG was suspended in a phosphate buffer at a ratio of
Time reacted.

Analysis of the liquid composition by high performance liquid chromatography revealed that unreacted DL-)lance-MODN and threonine 25
Millimoles were detected, and it was confirmed by optical resolution column chromatography that it was pure L-threonine. After bacterial cell separation, it was passed through a cation exchange resin, the adsorbed content was eluted with aqueous ammonia, and further treated with an anion exchange resin, and the aqueous solution was concentrated several tens of times to obtain white crystals.

The elemental analysis values, IR spectrum, mass spectrum, and magnetic resonance spectra (D20 solvent) of this white crystal were in good agreement with the L-threonine standard value.

Also, the specific optical rotation is [α) = -33,5° (C = 1H
2O).

Example 2 The reaction was carried out in the same manner as in Example 1, except that MODN in which the cis and trans forms were mixed at a ratio of 2 was used as the substrate. A concentration of 23 mmol was detected, and pure L was determined by optical resolution column chromatography (Chiral Pack WH type).
- It was confirmed that it was threonine, and no alloform was observed.

Comparative Example (Study of Culture Conditions) Using bacterial cells cultured in nutrient medium (Table a), MODN medium (Table b), and 5-oxoproline medium (Table C), (p)I was added in 1 to 11 JI of phosphate buffer, respectively. ? ) Millimoles of MODNIO were added and reacted at 30°C.

After the completion of the reaction, after sterilization by boiling for 2 minutes, the supernatant was analyzed. Even after 24 hours, no accumulation of threonine was observed in the reaction solutions derived from the first two, only those derived from the 5-oxytubroline medium. The presence of 4 mmol of threonine was observed.

Table a Peptone 1.0% meat extract
1.0 Mail 0.5 Table b L-) Lance MODN 0.25%KH2PO
40,l MgSO4・7H200,05 FeSOa ' 7H200,001 MnSO4・7H200,000? (pH 7) Table C 5-oxoproline 0.25%KH2PO4
0,l MgSO4・71(200,05 FeSOa・?H200,001 Mn504” 7H200,000?(pH7)

Claims (3)

[Claims] (1) 5-methyl-2-oxo-oxazolidine-4-
L-, which is characterized in that a carboxylic acid is applied to a microorganism capable of producing L-threonine, a culture solution, or a processed product of bacterial cells.
Method for producing threonine. (2) 5-methyl-2-oxo-oxazolidine-4-
5-Methyl-2-oxo-oxazolidine-4-carvone, which is characterized by culturing microorganisms capable of degrading carboxylic acids in a medium containing 5-oxoproline as the sole charcoal and nitrogen source, and collecting the grown strains. Method for obtaining L-threonine-producing microorganisms from acid. (3) Alcaligenes YY-207