JPS58146287A - Preparation of l-cysteine derivative - Google Patents

Abstract

PURPOSE:To prepare S-substituted L-cysteine industrially inexpensively, by reacting L-serine with a mercaptan in an aqueous solution by the action of tryptophan synthetase. CONSTITUTION:One mercaptan selected from the group consisting of ethylmercaptan, propylmercaptan, phenylmercaptan, and benzylmercaptan is reacted with L-serine in an aqueous solution in the presence of tryptophan synthetase produced by Escherichia coil MT-10238 (FERM-P 6045), etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-cysteine derivatives. More specifically, it relates to a method for producing corresponding S-substituted cysteine derivatives by reacting L-serine with ethyl mercaptan, propyl mercaptan, butyl mercaptan, phenyl mercaptan, or hensyl mercaptan in an aqueous solution by the action of tryptophan synthetase. The aim is to produce inexpensive cysteine derivatives.

Cysteine derivatives have various physiological activities, and methods of synthesizing them are being investigated, but they are limited to laboratory scale, and there are currently no examples of them being industrialized, and an inexpensive industrial method is desired. It was rare.

As a result of various studies on methods for producing L-cysteine derivatives using enzymatic methods, the present inventors found that cysteine derivatives can be easily produced by reacting L-serine with various mercaptans in an aqueous solution through the action of tryptophan synthetase. The present invention was completed by discovering that derivatives can be produced.

As the tryptophan synthetase producing strain used in the present invention, for example, Escherichia coli MT-10
238 (FEBM P-6145), Neurospora Clap ATC014692, etc. Escherichia
The method for extracting tryptophan ncetase from cultured cells of E. coli is described in The Journal ofBio
Logical chemistry, Vol. 2
52. AS, pp. 19.6594-6599 (1977), vol.

250, Notification 8, pages 2941-2946 (1975). However, the tryptophan/ncetase used in the present invention does not necessarily have to be pure. In other words, cultures of tryptophan cicisetase-producing strains, live bacterial cells collected from the culture by methods such as centrifugal separation, dried bacterial cells, or bacterial cells obtained by grinding, autolysis, sonication, etc. Processed products, furthermore, extracts from these flash bodies and crude enzymes obtained from the extracts can also be used. Of course, these immobilized enzymes or immobilized bacterial cells may also be used.

As a medium for culturing tryptophan synthetase-producing bacteria, any synthetic medium or natural medium can be used as long as it contains a carbon source, a nitrogen source, inorganic substances, and small amounts of micronutrients if necessary. However, it is generally necessary to add trace amounts of tryptophan, anthranilic acid, or indole to the medium. The carbon source and nitrogen source used in the culture medium may be of a type that can be used by the bacteria used. That is, various carbohydrates such as glucose, glycerol, fructose, sucrose, maltose, mannose, MS, m powder hydrolyzate, and molasses can be used as carbon sources. Nitrogen sources include ammonia, various inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, or meat extract, yeast extract,
Natural organic nitrogen sources such as corn steep liquor, casein hydrolyzate, phytosea meal or its digestate, defatted soybean meal or its digestate can be used. Many natural organic nitrogen sources can be both nitrogen and carbon sources. Further, as inorganic substances, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, magnesium sulfate, sulfur chloride, ferrous sulfate, and the like can be used as necessary.

Cultivation is performed under aerobic conditions such as shaking culture or submerged culture with aeration. Culture flow rate is 20-50℃, usually 30-50℃
It is in the range of 67°C. It is desirable to maintain the pH of the medium during culture near neutrality. The culture period is usually 1 to 6 days.

Tryptophan synthetase synthesizes L-tryptophan from indole glycerophosphate and L-serine.
-It is a multifunctional pyridoxal element that catalyzes reactions such as α-, β-elimination, and amine transfer in addition to substitution reactions. The enzymatic chemical properties and catalytic mechanism of this enzyme were detailed by Miles et al., and published in Advancesin Enzymology.
and Re1ated Areas of Mo1
-ecular Biology, Vol. 49.
127-185 (1979). However, the reaction of the present invention using tryptophan synthetase is the first reaction discovered by the present inventors.

The serine that can be used in the present invention is L-type serine. ＼-Serine cannot be used because it is not affected by tryptophan synthetase.

The amount of L-serine and various mercaptan substrates in the reaction solution is not particularly limited, but the concentration in the solution can usually be in the range of 001 to 10 parts by weight. During the reaction, it is desirable to add a trace amount of pyridoxal phosphate, which is a coenzyme, in addition to the substrate, for example, at a concentration in the solution in the range of 01 to 10 ppm.

The amount of tryptophan synthetase in the reaction solution varies depending on the separation and purification or processing method of the enzyme as described above, but is not particularly limited and can be changed as appropriate depending on the ratio to substrate, enzyme activity, and other conditions.

The reaction temperature in this reaction is usually in the range of 20 to 60°C.

The reaction is carried out by a batch method or, in the case of immobilized enzymes, by a continuous method using a column, either standing still or under gentle stirring. Successive additions of enzymes and mercaptans during the reaction increase the product yield. The reaction time varies depending on the reaction conditions, but is usually 5 to 40 hours in the case of a batch method.

The cysteine derivative of the reaction product can be easily isolated by a conventional method such as adsorption/desorption treatment using an ion exchange resin, activated carbon, or the like.

The reaction products thus obtained were fixed by TLC, amino acid analysis, NMR, and other instrumental analyses, and it was confirmed that the reaction products of the present invention are definitely cysteine derivatives corresponding to the respective mercaptans. Confirmed.

Example 1 Enerichia coli MT-10238 (FEBM P
The following reaction was carried out using tryptophantase obtained from cultured bacterial cells of -6145).

L-Serif 30mJ each mercaptan 50mJ separately Pyridoxal phosphate 0.01rnMl
including degree I) H s. A powder of reaction solution K and l-'J butophane synthetase adjusted to 0 (KOH) was added in a total volume of 10 ml and 97 dl. Reaction solution 10+〃
1 was shaken at 60°C for 24 hours. The molar yields of the produced/stin derivatives are shown in Table 1.

Table-1 Example 2 1r-'7 I Ruhi7 Urn-1ri MT-1026
8 (FERM P, 6145) to medium 5 with medium composition 1.
One platinum loopful was inoculated to 0 mg, and cultured with shaking at 30°C for 20 hours.

Medium composition I Meat extract 10 Peptide 7 05% Yeast extract 01 KH2PO4 0.2% Initial pH 7.0 1 ton of culture solution was centrifuged to collect bacterial cells, which were used as an enzyme source for tryptophan synthetase.

L-Serif 30mJ each mercaptan separately 50mJ Pyridoxal phosphate 0.01mM
The reaction solution containing [)H io was adjusted to 8.0 (KOH).

The above-mentioned centrifuged bacterial cells corresponding to 1 minute of culture solution joom were added to ynl, and the reaction was carried out at 60° C. for 48 hours with shaking.

The molar yields of the cysteine derivatives produced were as shown in Table 2.

Patent applicant: Mitsui Toatsu Chemical Co., Ltd.

Claims (1)

[Claims] A type of mercaptan consisting of ethyl mercaptan, propyl mercaptan, butyl mercaptan, phenyl mercaptan and hensyl mercaptan is reacted with L-serine in an aqueous solution by the action of tryptophan synthetase, resulting in S-ethyl-L-cysteine, s- Propyl-L-cysteine, S-butyl-L-cysteine,
S-phenyl-L-cystine Taha S-pencil-
1. A method for producing a cysteine derivative, which comprises producing L-cysteine.