JPS58129990A - Racemization process of amino acid - Google Patents

Abstract

PURPOSE:To racemize methionine, alanine, or homoserine efficiently, by cultivating a strain belonging to the genus Aeromonas, capable of racemizing methionine, alanine, or homoserine, using its culture solution, mold, or extract of mold. CONSTITUTION:Newly separated Aeromonas punctata subspecies caviae MT- 10243 (FERM BP-21) or MT-10244 (FERM BP-22) is cultivated in a medium such as one consisting of 1wt% meat extract, 0.5wt% peptone, and 0.1wt% yeast extract at about 7 initial pH at 20-40 deg.C for 1-3 days. Its culture solution, separated mold, or extract of mold is reacted with d-methionine, d-alanine, d-homoserine, so that it is nearly completely racemized.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a method for racemizing phosphorus. More specifically, a strain belonging to the genus Aeromonas that has the ability to racemize methionine, alanine, or homoserine is cultured, and methionine is produced in the presence of the culture, cultured cells isolated from the culture, or extracts of the cultured cells. , relates to a method for racemizing alanine or homoserine.

Methionine, alanine and homoserine are all types of amino acids, and methionine is important as a medicine and feed additive, alanine as a medicine and food additive, and homoserine is important as a pharmaceutical raw material and biochemical reagent.

These amino acids have two types of optical isomers due to differences in tertiary structure: L-type and D-type. Amino acids obtained by fermentation and enzymatic methods are usually L-type, and amino acids obtained by organic synthesis are DL-type.

Amino acids as constituent components of proteins are L-type, and many physiologically active amino acids are also L-type. However, a small amount of D-type amino acids also exist in biological components and play a significant role in the living body.

Amino acids used in infusions and the like are L-type. D-type amino acids are used as biochemical reagents and are expensive. DL-
Some amino acids are used in larger quantities as feed additives than, for example, methionine. Furthermore, L-type and D-type amino acids become 1) L-type by racemization. DL amino acids can be separated into D-type and L-type by known optical resolution methods. Therefore, L-type,
The required type of amino acid can be obtained by racemization and optical resolution, whether it is D type or 1) L type. As understood from the above description, racemization of amino acids is one of the important steps for the amino acid industry.

As a method for racemizing amino acids, a method in which an aqueous solution of amino acids is treated at high temperature and high pressure is known.

However, this method requires equipment that can withstand high temperature and high pressure treatment, requires pre-cutting energy to achieve high temperature and high pressure, and tends to cause side reactions other than racemization reactions during high temperature and high pressure treatment. It has its drawbacks.

As a result of investigating various racemization methods that do not have these drawbacks, the present inventors have found that a culture of a bacterial strain belonging to the genus Aeromonas,
It was discovered that cultured bacterial cells isolated from the culture or extracts from the cultured bacterial cells catalyze the racemization reaction of methionine, alanine, and homoserine, and the present invention was completed based on this discovery. .

In the method of the present invention, a strain belonging to the genus Aeromonas is used, and the strain used in the Examples described below is a strain isolated from nature by the present inventors, and is called Aeromonas bunctata subspice caviae (A4 boar). -ta=Saku 1JiptcL Ou Sandandan) MT-1o243 (
pp; FLMBP-21) and MT-10244 (3
ERM BP-22).

The taxonomic properties of these strains are as follows, and the properties of both strains are slightly different from each other and also slightly different from ATOO15468, the type strain of Aeromonas ebunctata subspice caviae, but taxonomically. are considered to belong to the same taxonomic group. Next, we will explain the taxonomic properties of these strains.

Morphological properties: Colonies of both strains MT-10243 and MT-10244 are smooth, gold-rimmed and opaque after 24 hours on nutrient agar and plain vert infusion agar. On sheep blood agar, it is smooth and shines all over, with gray pigment present in areas of active proliferation. After 24 hours on rabbit and sheep blood agar, MT-10243 showed very weak hemolysis around the colonies, but MT-10244
does not show any hemolytic phenomenon. Both strains are highly motile;
It is a short bacillus that is Durham negative and also has a polar or pure polar uniflagellate.

Biochemical properties: ATOOMT MT 15468 10243 10244 Catalase + + Ten Oxidase +
+ + Oxidation - Fermentation medium Glucose oxidation + + + Glucose fermentation + Jushi alkaline glucose --- Indole raw 1 戊 + Ju Ju Methyl red room temperature + + + Karael room temperature Syrah biboxylase hydrogen sulfide (TSI) -- − Urease −−− Oxidation of aminated gluconic acid −−− Gelatinase + + + Reduction of nitrate + Mo + Hydrolysis of pectin −−− Utilization of citric acid −−− Utilization of monotartrate −−− Hydrogenation of esculin + 7 Acid generation from gas generation from gas generation from group acid generation Acid generation from sucrose 10 + 10 acid generation 0 NPG generation Momo ” - Casein hydrolysis + Mo + Phosphatase - Shi +7.54NdOf
Tolerance --- 05% Nar+J requirement --- Growth as sole carbon source (Stanniel's medium) DL-arginine mo + mo L-
Histidine + + +D-glucose + + +D-mannitol + 10 10-arabinose +
+ 10 controls --- Le culture medium) The above mycological properties are expressed as
sbo, l of D4-mui Asahikokuyt BαOmul L
Judging from the classification criteria of α1α9y 8th edition (1974), strains MT-10243 and MT-10244 are Aeromonas punctata subspice caviae (A ccymentzb pwrbc, 1tzJb
+Iw4ip40irLi cavQa< ).

Although the strain of Aeromonas maple used in the present invention is commonly called an anaerobic bacterium, its growth normally progresses more smoothly when cultured under aerobic conditions rather than anaerobic conditions. Culture temperature is 20-40°C
It is. It is desirable to maintain the pH of the medium during culture at neutral or slightly alkaline. The culture period is usually 1 to 6 days.

Any carbon source and nitrogen source used in the culture medium may be used as long as they are available to the bacteria used. That is, various carbohydrates such as glucose, glyceptol, fructose, sucrose, starch hydrolyzate, and molasses can be used as the carbon source.

Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, or meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fishmeal or its digested products. ,
Natural nitrogen sources such as defatted soybean meal or its digestate can be used.

Many natural nitrogen sources can be both a nitrogen source and a carbon source. Further, as inorganic substances, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, etc. may be used as necessary.

The enzyme source used in the present invention includes the culture of an Aeromonas strain capable of racemizing methionine, alanine, or homoserine as it is, live bacterial cells collected from the culture solution by a method such as centrifugation, and dried bacteria thereof. Processed microbial cells obtained by grinding the microbial cells or microbial cells, autolysis, sonication, etc., as well as extracts from these microbial cells and enzyme citrates obtained from the extracts, can be used. It is. Of course, these immobilized enzymes or immobilized microbial cells may also be used.

The racemization reaction of methionine, alanine and homoserine is carried out in an aqueous solution, but there are no particular restrictions on the concentration of these amino acids. The reaction temperature is preferably 20 to 50°C, and the pH of the reaction solution is preferably in the range of 5 to 10.

Next, the present invention will be explained with reference to Examples, in which the degree of racemization of these amino acids was measured by optical rotation and bioassay. All values are expressed in weight%.

Example 1 One platinum loop of Aeromonas φ Bunctata subspice caviae MT-10243 was inoculated into a Sakalo flask containing 50 ml of the following medium, and cultured with shaking at 30°C for 24 hours.

Medium composition Meat extract 10% Peptone 05% Yeast extract 01% Initial pH 7.0 Centrifuge the culture solution 11 to kill the bacterial cells and prepare for the next race.
A 1-minimization reaction was carried out.

D-) 50 g of thionine, 100 g of pyridoxal phosphate,
The bacterial cells obtained from 1 hr of a phosphate buffer solution containing the above were added, and the reaction was carried out at 35°C for 24 hours with gentle stirring under a nitrogen seal. After the reaction, we analyzed the reaction solution and found that
Haha! , "The reaction proceeded to 100%, and the reaction solution contained the same amount of D-methionine and L-methionine. D-
Similarly, in experiments using D-alanine and D-homoserine in place of methionine, the same amounts of D-form and L-form were obtained.

Example 2 MT-102 instead of strain MT-10243 in Example 1
A similar experiment was conducted using No. 44. Analysis of the reaction solution revealed that methionine, alanine and homoserine contained the same amount of D and L forms.

Example 3 An experiment similar to that in Example 1 was conducted using L-methionine, L-alanine, and L-homoserine in place of D-methionine, p-alanine, and D-homoserine in Example 1, respectively. The L-type and D-type amino acids were contained in equal amounts.

Patent applicant: Mitsui Toatsu Chemical Co., Ltd.

Claims (1)

[Claims] A bacterial strain belonging to the genus Aeromonas that has the ability to racemize methionine, alanine, or homoserine is cultured, and methionine, alanine, or A method for racemizing amino acids, comprising racemizing an amino acid selected from the group consisting of and homoserine.