JPH0254075B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for racemizing methionine, alanine or homoserine. More specifically, a strain belonging to the genus Aeromonas that has the ability to racemize methionine, alanine, or homoserine is cultured, and methionine, This invention 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.
The amino acid obtained by organic synthesis is the 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. Some DL-amino acids, such as methionine, are used in large quantities as feed additives. Furthermore, L-type and D-type amino acids become DL-type by racemization. DL amino acids can be separated into D-type and L-type by known optical resolution methods. Therefore, L type, D
By racemization and optical resolution, the desired type of amino acid can be obtained, regardless of whether it is the amino acid type or the DL type. As understood from the above description, racemization of amino acids is one of the important steps in 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 a large amount of 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 various studies on racemization methods that do not have these drawbacks, the present inventors have found that a culture of a strain belonging to the genus Aeromonas, a cultured bacterial cell isolated from the culture, or an extract from the cultured bacterial cell, methionine, It was discovered that it catalyzes the racemization reaction of 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 that the present inventors isolated from nature.
Aeromonas punctata subspicis
Aeromonas punctata
subspecies caviae ) MT―10243 (FERM BP―
21) and MT-10244 (FERM 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 ATCC15468, the type strain of Aeromonas punctata subspice caviae, but taxonomically. are considered to belong to the same taxonomic group. Next, the taxonomic properties of these strains will be described. Morphological properties: Colonies of both MT-10243 and MT-10244 after 24 hours on nutrient agar and plain heart infusion agar are smooth and opaque with entire edges. On sheep blood agar, it is smooth and shiny all around, with gray pigment present in areas of active growth. MT after 24 hours on rabbit and sheep blood agar
-10243 shows very weak hemolysis around the village, but MT-10244 does not show any hemolysis.
Both strains are highly motile, Gram-negative, short rods that also have polar or purely polar uniflagellates. Biochemical properties:

[Table] Generation

【table】

[Table] Le culture medium)
The above mycological properties were described in the Bergeys-Manual of
Determinative Bacteriology 8th edition (1974)
Judging from the classification criteria, strains MT-10243 and MT-10244 are Aeromonas punctata subspice caviae.
punctata subspecies caviae). The strains of the genus Aeromonas used in the present invention are commonly called anaerobic bacteria, but their growth usually progresses more smoothly when cultured under aerobic conditions rather than anaerobic conditions.
The culture temperature is 20-40°C. It is desirable to maintain the pH of the medium during culture at neutral or slightly alkaline. The culture period is usually 1 to 3 days. As the carbon source and nitrogen source used in the culture medium, any type of carbon source and nitrogen source may be used as long as they are available to the bacteria used. That is, various carbohydrates such as glucose, glycerol, fructose, sucrose, starch hydrolyzate, and molasses can be used as carbon sources. 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,
Natural nitrogen sources such as fish meal or its digested product, defatted soybean meal or its digested product 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 crude enzymes obtained from the extracts can be used. It is. Of course, these immobilized enzymes or immobilized bacterial 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 limitations on the concentration of these amino acids. The reaction temperature is 20
~50°C, and the pH of the reaction solution is preferably within 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 light intensity and bioassay. Note that all percentages are expressed in weight%. Example 1 A loopful of Aeromonas punctata subspice caviae MT-10243 was inoculated into a Sakaguchi flask containing 50 ml of the following medium, and incubated at 30°C for 24 hours.
Cultured with shaking for hours. Medium composition Meat extract 1.0% Peptone 0.5% Yeast extract 0.1% Initial pH 7.0 Culture solution 1 was centrifuged to collect the bacterial strains and subjected to the next racemization reaction. D-methinion 50g, pyridoxal phosphate 100g
Add the bacterial cells obtained from 1 minute of phosphate buffer solution containing 35 mg of
The reaction was carried out at ℃ for 24 hours. After the reaction, analysis of the reaction solution revealed that the reaction had progressed to almost 100% and the reaction solution contained approximately equal amounts of D-methionine and L-methionine. D- instead of D-methionine
In experiments using alanine and D-homoserine, D-form and L-form were similarly obtained in approximately equal amounts. Example 2 MT instead of strain MT-10243 in Example 1
A similar experiment was conducted using 10244. Analysis of the reaction solution revealed that methionine, alanine, and homoserine contained approximately the same amount of D and L forms. Example 3 Using L-methionine, L-alanine and L-homoserine in place of D-methionine, D-alanine and D-homoserine in Example 1, respectively,
As a result of conducting the same experiment as in Example 1, it was found that the L-form and D-form of each amino acid were contained in approximately equal amounts.

Claims (1)

[Claims] 1. Cultivate a strain belonging to the genus Aeromonas and having the ability to ceramize methionine, alanine, or homoserine, and in the presence of the culture, cultured bacteria isolated from the culture, or extract from the cultured bacteria, methionine, A method for racemizing amino acids, which comprises racemizing amino acids selected from the group consisting of alanine and homoserine.