JPS60172292A - Method of racemization of homocysteine - Google Patents

Abstract

PURPOSE:To racemize the titled compound useful for synthesizing L-cysteine, etc. under mild conditions without racemizing other amino acids, by cultivating a strain belonging to the genus Pseudomonas, treating homocysteine in the presence of the culture or cultivated mold. CONSTITUTION:A strain belonging to the genus Pseudomonas (e.g., Pseudomonas putida IFO 12996, etc.) or to the genus Aeromonas (e.g., Aeromonas punctata subspecies caviae MT-10243, etc.) is cultivated, and in the presence of the culture or a bacterial cell separated from the culture or an extract obtained from the bacterial cell, homocysteine is treated with an aqueous solution containing pyridoxal phosphate, 1,4-dithiothreitol, and sodium pyrophosphate, to racemize homocysteine.

Description

[Detailed Description of the Invention] For more details, please refer to Pseudomonas (Pseud
-omonas) genus or Aeromonas (Aero
The present invention relates to a method for culturing a strain belonging to the genus Monas) and racemizing homocysteine in the presence of the culture, cultured cells isolated from the culture, or extracts from the cultured cells.

Homocysteine is used as a raw material for L-7 stain or L-methionine synthesis.

Homocystine is an amino acid that has one asymmetric carbon in its molecule, and exists in D-type and D-type.

Homocysteine obtained by the fermentation method and enzymatic method is usually L
Homocystine obtained by synthetic methods is usually DL
It is a type. If necessary, homocystine is separated from D-homocystine by repeating optical resolution and racemization.
- Homocystine or L-homocystine can be converted to D-homocystine.

Conventionally, as a method for racemizing homocystine, a method in which an aqueous solution of homocystine is treated at high temperature and high pressure is known, but this method has the following drawbacks. That is, (1)
Racemization requires a large amount of energy because of the high-pressure heating operation. (2) Other coexisting amino acids also racemize. For example, in the case of a mixture of D-homocystine and L-cysti, not only p-homocystine but also
L-cysteine is also racemized. (3) Also inactivates coexisting enzymes. For example, in the process of producing L-cysteine from DL-homocysteine in the presence of cystathionine β-cy/tase and r-cystathionase, if unreacted D-homocysteine is racemized by this method, the produced L Not only does it racemize the -7 stain, but it also deactivates the enzyme that catalyzes the reaction.

The present inventors have investigated various racemization methods that do not have the above-mentioned drawbacks, and have found that a culture of a certain bacterial strain of the genus Pseudomonas or Aeromonas, or a cultured bacterial cell isolated from the culture, or a cultured bacterial cell isolated from the cultured bacterial cell, or discovered that homocysteine racemizes in the presence of an extract of

Many strains belonging to the genus Sheetmonas or Aeromonas may be used in the practice of the present invention, such as Pseudomonas putida (pse), as shown in the Examples below.
udomonas putida) I F 012
996, Aeromonas punctata Φsubspecies Φcavier
caviae) MT-1
0243 (FERM BP-21) and the like are used.

Cultivation of these microorganisms is usually carried out under aerobic conditions such as shaking culture or submerged culture with aeration.

The culture temperature is 20-50℃, and the pH of the medium during culture is
It is desirable to maintain it near 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 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, fat sea meal or its digested products. Natural organic nitrogen sources can be used, such as Many natural organic nitrogen sources can serve as both a nitrogen source and a carbon source. Furthermore, it is convenient to use potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, and the like as inorganic substances, if necessary.

Enzyme sources used in the present invention include microbial cultures as they are, live microbial cells collected from the culture solution by methods such as centrifugation, dried microbial cells, or crushed microbial cells, autolysis, ultrasonication, etc. Processed bacterial cells obtained by treatments such as treatment, extracts from these bacterial cells, and crude enzymes obtained from the extracts can be used. Of course, these immobilized enzymes or immobilized bacterial cells may also be used.

The racemization reaction of homocystine is carried out in an aqueous solution, but the concentration of homocystine is not particularly limited.

The reaction temperature is preferably 20 to 50°C, and the pH of the reaction solution is preferably 5 to 1°.

Next, the present invention will be explained with reference to examples. In the examples, the degree of racemization of homocystine was determined by separating and quantifying the D-isomer and the D-isomer by optical rotation and liquid chromatography. Note that all values are expressed in weight%.

Example 1 A loopful of Pseudomonas spotted mite FO12996 was inoculated into a Sakaro flask containing 50 rnl of a medium having the following composition, and cultured with shaking at 30°C for 24 hours.

Medium composition Meat extract i, o% Peptone 0.5% Yeast extract 01% Initial pH 7.0 1 ton of culture solution was centrifuged to collect bacterial cells and subjected to the next racemization reaction. L-homocystine 62, pyridoxal phosphate 10m9.1.4-diteothreitol 100mg
, 1100 m of aqueous solution containing 1.62 sodium pyrophosphate
The centrifuged microbial cells obtained from culture solution 1 were added to /', and the reaction was carried out at 37° C. for 48 hours with gentle stirring under a nitrogen seal. Analysis after the reaction revealed that the reaction solution contained 3.2r of L-homocystine and 2.71r of D-homocystine.

Example 2 A similar test was conducted using D-homocystine in place of L-homocystine in Example 1. Results of analyzing the reaction solution 1. It contained 3.31 D-homocysteine and 2.52 L-homocysteine.

Example 3 In place of Pseudomonas putida IFO12996 of Example 1, Aeromonas punctata subsp. cavier MT-10243 (Feikoken Joyori No. 21)
A similar test was conducted using Results of analyzing the reaction solution 1. L
-Homocystine 3.15', D-homocystine 28
2 were included.

Example 4 A similar test was conducted using D-homocystine instead of L-homocystine in Example 3. As a result of analyzing the reaction solution, D-homocystine was 6.01, L-homocystine was 2.
．． 91 were included.

patent applicant Mitsui Toatsu Chemical Co., Ltd.

Claims (1)

[Claims] It is characterized by culturing a strain belonging to the genus Sheetomonas or the genus Aeromonas, and racemizing homocysteine in the presence of the culture, cultured cells isolated from the culture, or extracts from the cultured cells. Homocystine racemization method.