JPH0329395B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for efficiently producing L-cysteine using the action of microorganisms. Conventionally, L-cysteine (hereinafter abbreviated as cysteine) and L-cystine (hereinafter abbreviated as cystine) have been produced by hydrolyzing cysteine such as hair or natural products with a high cystine content with mineral acids, and using the resulting amino acid mixture to obtain cystine. Cysteine was produced by reducing the cystine thus obtained. In contrast, the present applicant has already developed 2-amino-thiazoline-4-carboxylic acid (hereinafter referred to as
It is written as ATC. ) as a raw material and a method for producing cysteine or cystine using the action of microorganisms (see JP-A-51-70881, JP-A-51-54983 and 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 performed simultaneously to generate cystine that has no inhibitory effect, and then the generated cystine is reduced to obtain cysteine. I have no choice but to adopt it. Therefore, this production method requires long and complicated steps and is not necessarily satisfactory in terms of yield, so further improvements have been desired in these respects. Therefore, the present inventors conducted intensive research to develop a method for efficiently producing cysteine from ATC, and found that microorganisms capable of producing cysteine or cystine from ATC were used in the presence of acetone.
When reacted with ATC, 2,2-dimethylthiazoline-4-carboxylic acid (hereinafter abbreviated as DTC) is produced in good yield, and by hydrolyzing this DTC under acidic conditions, cysteine can be produced in a short time and in high yield. It was discovered that it is generated by The present invention was completed based on this discovery. That is, in the present invention, a microorganism capable of hydrolyzing 2-amino-thiazoline-4-carboxylic acid to produce cysteine or cystine is allowed to act on ATC in the presence of acetone to produce DTC, and the DTC is then subjected to acidic conditions. The present invention relates to a method for producing cysteine, which is characterized in that cysteine is produced by hydrolysis below. The microorganism used in the present invention is a microorganism that has the ability to hydrolyze ATC to produce cysteine or L-cystine, and is disclosed in, for example, JP-A-51-54983, JP-A-51-70881, and JP-B-Sho 54-2272. It belongs to the genera Achromobacter, Allicaligenes, Bacillus, Brevibacterium, Enterobacter, Erwinia, Etschierichia, Flavobacterium, Micrococcus, Mycoplana, Pseudomonas, Sarcina, or Serratia, which are listed in the official publication, and hydrolyzes ATC to produce cysteine. or microorganisms that have the ability to produce cystine, and more specific examples include the following strains: Sarcina lutea ATCC 272 Achromobacter delmarvae FERM-P3483 Alcaligenes・Alcaligenes denitrificans ATCC 15173 Bacillus brevis
ATCC 8185 Brevibacterium flavum ATCC 13826 Enterobacter aerogenes
FERM P-2764, FERM BP-321 Erwimia carotovora
carotovora) FERM−P2766 Pseudomonas thiazolinophilum
FERM P-2810, FERM BP-322 Pseudomonas desmolytica
desmolytica)
FERM P-2816, FERM BP-323 Escherichia coli
FERM−P2763 Micrococcus sodonensis
sodonensis ATCC 11880 Mycoplana dimorpha
dimorpha) ATCC 4249 Serratia marcetuscens (Serratia
marcescens) FERM−P2765 Flavobacterium acidoficum ATCC 8366 Pseudomonas
ovlis) FERM P-2762, FERM BP-1328, etc. 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, sugars such as glucose, sucrose, xylose, and molasses, acetic acid, etc. organic acids, alcohols such as ethanol, glycerol, and methanol; 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 the enzyme source, the culture solution obtained in this way, isolated bacterial cells isolated from the culture solution, washed viable bacterial cells, Freeze-dried product, acetone-dried bacterial cells, physically, chemically or biochemically destroyed bacterial cells, extracts, crudely purified products, purified products, purified protein standards, or immobilized bacterial cells or purified products etc. are used as enzyme sources. ATC, which is a raw material for cysteine, can be used in any of the D-form, L-form, and racemic form, which are supplied by a synthetic method. 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.
Preferably, it is about 0.5 to 10%, and in a continuous system, it is preferable to lower the concentration slightly. The reaction is carried out in an aqueous solution containing 2 to 40% acetone, and the yield can be improved by adding hydroxylamine or semicarbatide to the reaction solution. Reaction temperature is 15-60℃, preferably 30-50℃, PH
is preferably in the range of 6 to 10, preferably 7.0 to 9.5. When microorganisms act on ATC under these conditions, ATC is hydrolyzed to cysteine and condensed with acetone in the reaction solution to produce DTC. DTC is rapidly and quantitatively hydrolyzed to cysteine under acidic conditions. . Unlike cysteine, DTC produced from ATC does not inhibit the hydrolysis reaction of ATC to cysteine at all, so the reaction yield of DTC, that is, the yield of cysteine, is high, reaching over 90%, and the DTC is converted from ATC to cysteine.
The reaction time is short, and cysteine can be produced in 1 to 25 hours. This time is reduced to about 1/2 compared to the 2 to 50 hours required when producing cystine from ATC, which is known in the art. Furthermore, in the conventional method, there was a risk that the produced cysteine would be decomposed by harmful enzymes, resulting in the generation of hydrogen sulfide, but in the method of the present invention, there is no risk of generation of hydrogen sulfide. It can be said to be advantageous for industrial production. Cysteine is collected from the aqueous solution obtained by hydrolyzing DTC under acidic conditions by removing acetone and then using a known cysteine separation method such as a method using a cation exchange resin or a crystallization method. Cysteine is quantified by high performance liquid chromatography using a cation exchange resin or by a microbial assay using lactic acid bacteria (Leuconostoc titroborum ATCC 8081). 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 1 Medium composition (PH7.0) Component content Glycerol 1.0% Yeast extract 0.5% Peptone 0.5% Meat extract 0.5% NaCl 0.5% DL-ATC 0.2% Pseudomonas desmolytica AJ
3868 FERM P-2816 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, which were added to a substrate solution with the following composition, kept at 30℃, and stirred occasionally.
A time reaction was performed. Table 2 Composition of substrate solution (PH8.0) Component content DL -ATC 2.0g/dl KH ₂ PO ₄ 1.0g/dl Hydroxylamine hydrochloride 0.15g/dl Acetone 0-50% After the reaction is completed, the reaction solution is Hydrochloric acid was added to adjust the pH to 3.0 to generate cysteine, and the amount of cysteine in the reaction solution was quantified. The results are shown in Table 3.

[Table] Example 2 Freeze-dry the wet bacterial cells 5.0 obtained by the method of Example 1, or suspend the wet bacterial cells 5.0 in 50 ml of 0.1 M, pH 7.0 phosphate buffer, and add super Sonication treatment (Tomy Seiko LIR-200P model, 20KC, 5 minutes)
In addition, add 5.0 g of wet bacterial cells to 20 ml of deionized water.
3.8 g of acrylamide and 225 mg of methylenebisacrylamide were added to this, oxygen was removed by flowing N ² gas, and 17.5 mg of ammonium persulfate and 40 μ of N′,N′-dimethylaminopropionitrile were added.
Prepare the immobilized product by adding 5.0 g of
%) was added to 100 ml and kept at 30°C for 16 hours to perform an enzyme reaction. After the reaction was completed, insoluble substances were removed from each reaction solution, the pH was adjusted to 3.0 to generate cysteine, and cysteine was quantified. The results are shown in Table 4. Table 4 Production amount of cysteine ( g/dl) Freeze-dried sample 1.3 Ultrasonication sample 1.4 Fixed sample 1.2 Example 3 The microorganisms shown in Table 5 were used in the culture medium shown in Table 1. Cultivation was performed 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 (acetone amount: 5.0%), and the reaction was carried out by keeping at 30°C for 24 hours. . After the reaction was completed, the pH of the reaction solution was adjusted to 3.0 to generate cysteine, and the amount of cysteine produced was determined. The results are shown in Table 5.

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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 acetone. 2,2-dimethylthiazolidine-4-carboxylic acid is produced, and the 2,2-dimethylthiazoline-4-carboxylic acid is hydrolyzed under acidic conditions to produce L.
- L characterized by producing cysteine
- Method for producing cysteine.