JPH0353914B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to S-carboxylmethyl-L-cysteine (hereinafter simply referred to as carboxymethylcysteine or SMC), which is a derivative of L-cysteine.
Concerning the manufacturing method. Carboxymethylcysteine is a compound that has uses as a therapeutic agent for bronchitis, particularly as an expectorant or antidote.The production method involves reducing cystine separated from human hair hydrolyzate to L-cysteine, -Produced by reacting cysteine with monohalogenoacetic acid. As mentioned above, since L-cysteine is made from human hair or the like, there is a limit to its supply, and there has been a desire to develop an alternative industrial production method. 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 cysteine or cystine using the action of microorganisms was developed (see JP-A-51-70881, JP-A-51-54983 or JP-B-Sho 54-2272). Although this method is suitable for the industrial production of L-cysteine, cysteine cannot be directly obtained because the hydrolysis reaction of ATC to cysteine is strongly inhibited by cysteine produced in the reaction solution. Therefore, in this method, the reaction of hydrolyzing ATC to generate cysteine and the oxidation reaction of cysteine to cystine are carried out simultaneously to generate cystine that does not inhibit the ATC hydrolysis reaction, and this cystine is reduced to form cysteine. We have no choice but to adopt a method to do so. Therefore, this method using ATC as a raw material has long and complicated steps, and furthermore, there has been a desire to improve the low yield. Therefore, the present inventors have conducted intensive research with the aim of developing a method for producing carboxymethylcysteine directly from ATC efficiently and with high yield, and as a result, they have found that they have the ability to hydrolyze ATC to produce cystine. We discovered that carboxymethylcysteine can be directly produced in high yield by allowing microorganisms to act on ATC in the presence of monohalogenoacetic acid. The present invention was completed based on this discovery. The microorganism used in the present invention is a microorganism that has the ability to hydrolyze ATC to produce cysteine or cystine.
and belong to the genera Achromobacter, Alcaligenes, Bacillus, Brevibacterium, Enterobacter, Erwinia, Etschierichia, Flavobacterium, Micrococcus, Mycoplana, Pseudomonas, Sarcina, or Serratia as described in Japanese Patent Publication No. 54-2272. , is a microorganism that has the ability to hydrolyze ATC to produce cysteine or cystine, and more specific examples include the following strains.

[Table] A medium for culturing microorganisms is a medium containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients. For example, as a carbon source, glucose, sucrose, xylose, molasses, etc. Sugars, organic acids such as acetic acid, alcohols such as ethanol, glycerol, methanol, etc. Nitrogen sources such as ammonium sulfate and ammonium chloride; organic nutritional sources such as yeast extract, peptone,
Ions such as magnesium, iron, manganese, potassium, sodium, and phosphoric acid are appropriately used as inorganic ions in meat extracts, corn staple liquor, and the like. The microorganisms may be cultured according to a conventional method, and the pH of the medium may be adjusted to 6 to 9, and the microorganisms may be cultured aerobically at 20 to 40°C. During cultivation, it is desirable to add a small amount of ATC to obtain microbial cells with high ATC water-degrading activity. In the method of the present invention, the microorganism thus obtained is used as an enzyme source. As an enzyme source, the culture solution obtained in this way, isolated bacterial cells separated from the culture solution, washed viable bacterial cells, freeze-dried cells, acetone-dried bacterial cells, and physically, chemically or biochemically destroyed bacterial cells can be used. The enzyme source may be a bacterial cell, an extract, a crude product, a purified product, a purified protein standard, or an immobilized product of the bacterial cell or a purified product. The raw material ATC is D-, which is supplied by a synthetic method.
All of the isomer, L-isomer, and racemic isomer are used. The substrate concentration differs depending on whether it is a batch method or a continuous method, but in a batch method, it is generally 0.1 to 30% in the aqueous medium.
It is preferably about 0.5 to 10%, and in a continuous system, it is preferable to lower the concentration slightly. Monohalogenoacetic acid includes monofluoroacetic acid,
Monochloroacetic acid, monobromoacetic acid, monoiodoacetic acid or their salts are used, and the concentration is 0.1
A range of ~10% is desirable. 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 will be held around 7.0-9.5. The reaction time varies depending on the method of standing, stirring, flowing down, etc., and the form and potency of the enzyme preparation, so it is not uniform, but in the batch method it is usually about 10 minutes to 72 hours. During the reaction, it is desirable to add hydroxylamine or semicarbatide, which can improve the reaction yield. Under such conditions, when microorganisms are allowed to act on ATC in the presence of monohalogenoacetic acid, carboxymethylcysteine is directly produced in the reaction solution. Carboxymethylcysteine can be collected from the reaction solution by following a known method. For example, carboxymethylcysteine easily crystallizes under acidic conditions, so after removing insoluble substances from the reaction solution, it is crystallized by acidification. It is easily collected by this method. Examples will be described below. Example 1 A culture medium having the composition shown in Table 1 was prepared, and 50 ml was dispensed into a 500 ml shaking flask, followed by heating at 120° C. for 10 minutes.

[Table] In this medium, Pseudomonas desmolytica
AJ3868 FERM-P2816 and FERM BP-323 were inoculated and cultured with shaking at 30°C for 24 hours. The culture solution was centrifuged to collect bacterial cells, and 5.0 g of wet bacterial cells were added to 100 ml of a substrate solution having the composition shown below, and the reaction was carried out for 8 hours while being kept at 30° C. and stirring gently from time to time.

[Table] After the reaction is complete, the carboxymethyl cysteine produced in the reaction solution is removed using cation exchange resin "ZYPAX".
The amount produced was 0.91 g/dl as determined by high performance liquid chromatography using "SCX". When a similar reaction was carried out using 0.9 g of sodium monofluoroacetate, 1.2 g of sodium monobromoacetate, or 1.6 g of sodium monoiodoacetate instead of monochloroacetic acid, the amounts of carboxymethylcysteine produced were 0.9 g/dl and 0.87 g, respectively. /dl and 0.91g/dl. Example 2 The wet bacterial cells 5.0 obtained by the method of Example 1 were freeze-dried, or the wet bacterial cells 5.0 were suspended in 50 ml of a 0.1 M, PH 7.0 phosphate buffer solution and subjected to ultrasonic treatment ( Tomy Seiko LIR-200P model, 20KC, 5 minutes), and then 5.0g of wet bacterial cells in deionized water.
20 ml of suspension, add 3.8 g of acryl amiva and 225 mg of methylene bisacrylamide, remove oxygen by flowing N ₂ gas, and add 17.5 g of ammonium persulfate.
An immobilized product was prepared by adding 40 μg of N ¹ ,N ¹ -dimethylaminopropionitrile and 40 μg of N 1 ,N 1 -dimethylaminopropionitrile. These preparations were added to 100 ml of a substrate solution having the composition shown in Table 2 and kept at 30°C for 16 hours to carry out an enzyme reaction. After the reaction was completed, insoluble substances were removed from each reaction solution, and the amount of carboxymethylcysteine produced was measured. The results are shown in Table 3.

[Table] Example 3 The microorganisms shown in Table 4 were cultured in the medium shown in Table 1 according to the method of Example 1. Each culture solution was centrifuged, 5.0 g of wet bacterial cells were suspended in 100 ml of the substrate solution shown in Table 2, and the reaction was carried out at 30° C. for 24 hours. After the reaction was completed, carboxymethylcysteine produced in the reaction solution was quantified by high performance liquid chromatography. The results are shown in Table 4.

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Claims (1)

[Claims] 1 A microorganism capable of hydrolyzing 2-amino-thiazoline-4-carboxylic acid to produce L-cysteine or L-cystine is allowed to act on 2-amino-thiazoline-4-carboxylic acid in the presence of monohalogenoacetic acid. 1. A method for producing S-carboxymethyl-S-cysteine, which comprises producing S-carboxymethyl-L-cysteine and collecting it.