JPH0349555B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to L-sulfur-containing amino acids, specifically L-
The present invention relates to a method for producing cysteine and L-cystine. Conventionally, L-cysteine and L-cystine are
L-cysteine, such as hair, and natural products with a high content of L-cysteine are hydrolyzed with mineral acids, and L-cysteine is isolated from the resulting amino acid mixture. -Produced by reducing cystine. In contrast, the present applicant has already developed 2-amino-thiazoline-4-carboxylic acid (hereinafter referred to as
It is written as ATC. ) was used as a raw material, and a method for producing L-cysteine or L-cystine using the action of microorganisms was developed (see Japanese Patent Application Laid-Open No. 136619/1983). In this method, a microorganism having the ability to convert ATC into a sulfur-containing amino acid is used as an enzyme source, and since this enzyme is an inducible enzyme, the raw material ATC is required as an enzyme inducer. However, ATC is expensive, and it is not economical to use part of the raw material as an enzyme inducer. An inexpensive enzyme inducer has been desired. Therefore, as a result of various research aimed at developing an inexpensive enzyme inducer, the present inventors found that DL-
The present invention was completed by discovering that an inexpensive S-(β-carboxy-β-chloroethyl)-isothiourea (hereinafter referred to as SCCI), which is an intermediate in the chemical synthesis of ATC, can be used. That is, the present invention
A method for producing a sulfur-containing amino acid, which comprises culturing a microorganism capable of converting ATC into a sulfur-containing amino acid in a medium containing SCCI, and allowing the cultured microorganism to act on ATC to convert it into a sulfur-containing amino acid. It is. The microorganisms used in the present invention are wild strains existing in nature, strains stored in public microbial repositories, or microorganisms artificially mutagenized from them, and cystine or (and) cysteine production from ATC is used. For example, Achromobacter, Alcaligenes, Bacillus, Brevibacterium, Enterobacter, Erwinia,
These microorganisms belong to the genera Ethscherichia, Flavobacterium, Micrococcus, Mycoplana, Pseudomonas, Sarcina, or Serratia.
More specific examples include the following strains: Sarcina lutea AJ-1217 (Sarcina lutea)
ATCC272, Achromobacter delmalvaa AJ
−1983 (Achremebaeter delmarvae) FERM−
P3483, Alcaligenes denitrophincus AJ
−2553 (Alcaligenes denitrificans)
ATCC15173, Pacillus brevis AJ-1282
(Bacillus brevis) ATCC8185, Brevibacterium flavum AJ-1516 (Brevibaeterium
flavum) ATCC13826, Enterobacter aerogenes AJ-2643 (Enterobacter aerogenes
IAM1019) FERM-P2764, Erwinia carotovora AJ-2753 (Erwinia carotovora CCM873)
FERM-P2766, Pseudomonas thiazolinophyllum AJ-3854 (Pseudomonas
thiazolinophilum) FERM-P2810, Pseudomonas desmolytica AJ2368 (P.desmolyitca)
FERM−P2816, Etschierhia coli AJ−
2592 (Escherichia coli IAM1132) FERM−
P2763, Micrococcus sodonsis AJ−1753
(Micrococcus sodonensis) ATCC11800, Mycoplana dimorpha AJ-2809 (Mycoplana dimorpha AJ-2809)
dimorpha) ATCC4249, Serratia marcescens AJ-2698 (Serratia marcescens IAM1206)
FERM-P2765, Flavobacterium acidoficum AJ-2494 (Flavobacterium acidoficum)
ATCC8366, Pseudomonas obalis AJ−
2236 (Pseudomenas ovalis IAM1454) FERM−
P2762 The raw material ATC is supplied by a synthetic method and is generally racemic. However, according to the method of the present invention, both the D-form and the L-form are converted to L-form cystine or cysteine. The medium for culturing the microorganisms mentioned above is
A nutrient medium containing 0.01-3% SCCI is used. SCCI is a chemical synthesis intermediate of ATC,
It is cheaper than. As a nutrient medium, carbon source,
The medium contains a nitrogen source, inorganic ions, and, if necessary, organic micronutrients as appropriate. For example, carbon sources include sugars such as glucose, sucrose, xylose, and molasses; organic acids such as acetic acid; and alcohols such as ethanol, glycerol, and methanol; nitrogen sources include ammonium sulfate and ammonium chloride; organic nutritional sources include yeast extract;
Ions such as magnesium, iron, manganese, potassium, sodium, and phosphoric acid are appropriately used as inorganic ions such as peptone, meat extract, and corn staple liquor. Cultivation may be carried out by a conventional method, for example, the pH of the medium is set to 6 to 9, and after inoculation, the culture is carried out aerobically at 20 to 40°C for 1 to 3 days. The microorganisms cultured in this way are used as a substrate for ATC.
It acts on ATC to produce sulfur-containing amino acids. The above-mentioned cultured microorganisms are culture fluid obtained by culturing, isolated bacterial cells, washed living bacterial cells, freeze-dried bacterial cells, acetone-dried bacterial cells, physically, chemically or biochemically destroyed bacterial cells, and extracted bacterial cells. liquid, crudely purified product, purified product,
It refers to a purified protein standard, or an immobilized product of bacterial cells or a purified product.The substrate concentration when acting on ATC differs depending on whether it is a batch method or a continuous method, but in a batch method, it is generally 0.1 to 0.1 in an aqueous medium. 30%,
Preferably, it is about 0.5 to 10%, and in a continuous system, it is preferable to lower the concentration slightly. The reaction is usually carried out in an aqueous medium at 15-60°C, preferably around 30-50°C, and at a pH of 6-10, preferably
It is held around 7.0 to 9.5. Reaction time: standing still;
The batch method usually takes about 10 minutes to 72 hours, although it is not uniform because it depends on the means of stirring, flowing down, etc., and the form and potency of the enzyme preparation. As the reaction progresses, L-cysteine and L-cystine coexist in the reaction solution, but usually L-cysteine coexists with L-cysteine.
Cysteine is oxidized by oxygen in the air and L-
It easily changes to cystine, and over time it changes to L-
The amount of cystine increases. However, it is also possible to change the concentration ratio of L-cysteine and L-cystine by changing reaction conditions and the like. In general, L-cystine could be obtained by converting most of it into L-cystine by aeration oxidation and crystallizing it by taking advantage of its poor solubility. Also, L-
When producing cysteine, it can be collected as L-cysteine by a conventional method of electroreduction. Note that the yield of L-cysteine and/or L-cystine can be improved by adding hydroxylamine or semicarbazide to the reaction solution. Quantification of L-cystine and L-cysteine was carried out by liquid chromatography and microbial assay. The latter is the lactic acid bacterium Leuconostocci titroborum.
This method uses ATCC8081, but since this bacterium depends on both L-cystine and L-cysteine, the sum of both amino acids is quantified, and it is usually expressed as L-cystine. .
Note that this bacterium cannot grow on D-form cystine or cysteine. Example 1 A culture medium having the composition shown in Table 1 was prepared, and 50 ml was dispensed into a 500 ml flask and sterilized by heating at 120° C. for 10 minutes.

[Table] In this medium, Pseudomonas desmolytica
FERM-P2816 was inoculated and cultured with shaking at 30°C for 24 hours. 1.0 g/dl DL-ATC and 1.0 g/dl in each 5.0 ml culture solution
Add 5.0 ml (PH 8.0) containing g/dl KH ₂ PO ₄ ,
The reaction was carried out at 30°C for 5 hours. The amounts of cysteine and cystine produced in the reaction solution were determined using high performance liquid chromatography. The results are shown in Table 2.

[Table] Culture solution 50 obtained by adding SCCI 0.1g/dl above
After centrifuging ml to collect bacterial cells and washing with water,
This bacterial cell was added to DL-ATC・3H ₂ O 3.0g and KH ₂ PO ₄
Add to 100ml of PH8.0 aqueous solution containing 1.0g and heat at 30℃.
The mixture was reacted for 15 hours with gentle stirring. After the reaction was completed, the reaction solution was vigorously stirred to convert cysteine to cystine and precipitate crystals. Concentrated hydrochloric acid was then added to dissolve the crystals, followed by centrifugation to remove insoluble substances. A caustic soda solution was added to the obtained supernatant to neutralize it and precipitate crystals.
Recrystallization was performed again to obtain 1.7 g of white crystals. This crystal completely matched authentic L-cystine in its NMR spectrum, X-ray diffraction, and liquid chromatography, and was identified as L-cystine. Example 2 Medium with the composition shown in Table 1 (SCCI: 0.1g/dl)
The test bacteria shown in Table 3 were cultured in the same manner as in Example 1. Transfer 5.0ml of culture solution to 5.0ml DL−
ATC aqueous solution (DLATC2.0g/dl, KH ₂ PO ₄ 1.0
g/dl, pH 8.0) and reacted at 30°C for 5 hours. Cysteine and cystine in the reaction solution were quantified using high performance liquid chromatography. The results are shown in Table 3.

【table】

Claims (1)

[Claims] 1 A microorganism having the ability to convert 2-amino-thiazoline-4-carboxylic acid to L-cysteine or L-cystine was cultured in a medium containing S-(β-carboxy-β-chloroethyl)-isothiourea. Microorganisms are treated with 2-amino-thiazoline-
L-cysteine or L-cystine is produced by acting on 4-carboxylic acid.
- A method for producing a sulfur-containing amino acid.