KR890004021B1 - Method for producing l-systeine or/and l-systine - Google Patents

Abstract

L-cysteine and L-cystine are prepd. by reacting β-substd. Lalanine of formula (I) with metal sulfide, metal hydrosulfide, metal polysulfide, ammonium sulfide, ammonium hydrosulfide or ammonium polysulfide in the presence of tryptophan synthase. In (I), X is halogen, -OR or -SR; R is hydrogen, alkyl, acetyl, bentyl or sulfonic acid. L-cysteine and L-cystine are useful as cosmetics, food additives and blood components.

Description

Method for preparing L-cysteine, L-cystine and mixtures thereof

The present invention relates to L-cysteine by reacting β-substituted L-alanine in the presence of metal sulfides, metal hydrosulfides, metal polysulfides, ammonium sulfide, ammonium hydrosulfide, or ammonium polysulfide with tryptophan systhase. And / or a method for preparing L-cystine.

L-cysteine and L-cystine are widely used in cosmetic applications, food industry and other uses as food additives, as well as in medical applications such as, for example, transfusion components.

Various conventional methods for producing L-cysteine and L-cystine are known: representative examples include (1) extraction from natural substances such as hair, and (2) Japanese Patent Application Publication No. 57-200356. Chemical synthesis method as disclosed in Japanese Patent Application, (3) enzymatic synthesis method from DL-2-aminothiazoline-4-carboxylic acid as disclosed in Japanese Patent Publication No. 54-2272, and ( 4) The reaction of β-substituted alanine with metal sulfide or hydrosulfide in the presence of cysteine dehydrosulfase enzyme disclosed in Japanese Patent Publication No. 57-21311 is known. However, the above-mentioned methods are not satisfactory enough as an industrial manufacturing method.

In view of the above situation, the present inventors have studied a new method for producing L-cysteine and / or L-cystine at low cost, and found that β-substituted L-alanine in the presence of tryptophan synthase was used as a metal sulfide, metal It was found that L-cysteine and / or L-cystine were prepared by reacting with hydrosulfide, metal polysulfide, ammonium sulfide, ammonium hydrosulfide or ammonium polysulfide. The present invention has been completed based on the above findings.

Tryptophan synthase is known to be present extensively in microorganisms, higher plants, etc., for example, described in Bacteriological Reviews, Vol. 39, No. 2, pages 87-120 (1975).

In the present invention, tryptophan synthase, which is usually obtained from microorganisms, is used, but the enzyme source is not limited thereto. Examples of strains producing tryptophan synthase include Escherichia coli MT-10232 (FERM BP-19), Escherichia coli MT-10242 (FERM BP-20), Neurospora Krasa ( Neurospora crassa, bread mold) ATCC-14692 and Saccharomyces cerevisiae ATCC-26787.

Methods for extracting tryptophan synthase from cultured cells are known and are also described in Journal of Biological Chemistry, Vol. 249, No. 24, pp. 7756-7763 (1974). Extraction from Neurospora Krasa in Journal No. 250 No. 8, pages 2941-2946 (1975) and Saccharomyces cerevisiae in European Journal of Biochemistry 102, pp. 159-165 (1979). Extraction methods from Ae are published respectively.

Tryptophan synthase used in the present invention does not necessarily need to be an extracted and purified enzyme. Cultivate the microorganisms producing tryptophan synthase, harvest the viable cells from the culture using centrifugation or similar methods, dry the cells and grind to obtain cell fragments, and then by ultrasonic disruption or other methods. Cells can be treated or subjected to autolysis to use them as enzyme sources of the present invention. It is also possible to use extracts from the cells and crude enzymes obtained from the extracts, and immobilized cells or enzymes may of course be used in the present invention.

For culturing tryptophan synthase-producing cells, any synthetic or natural medium containing a carbon source, a nitrogen source, minerals and optionally a small amount of eutrophic (s) can be used. Sometimes it is efficient to add small amounts of tryptophan or indole to the culture medium. In addition, when a small amount of indole acrylic acid is added to the medium, the yield of tryptophan synthase sometimes increases.

Cultivation is carried out under aerobic conditions, such as shaking or aeration stirred culture. The temperature is in the range of 20-40 ° C., typically 25-37 ° C., and the pH of the culture medium is 5-8.

Tryptophan synthase is a multifunctional enzyme, ie, it is known that the enzyme is involved in the synthesis of L-tryptoic acid from indole-3-glycerophosphate and L-serine as well as many other reactions; This is described, for example, in Advances in Enzymology and Related Areas of Molecular Biology, Vol. 49, pages 127-185 (1979).

However, the inventors first found out that tryptophan synthase is involved in the reaction according to the present invention.

Examples of β-substituted L-alanine which may be used in the present invention, which are one of the substrates of the present invention reaction, are β-halogeno-L- such as β-chloro-L-alanine and β-bromo-L-alanine. Alanine; O-alkyl-L-serine such as O-methyl-L-serine and O-ethyl-L-serine; S-alkyl-L-cysteine such as S-methyl-L-cysteine and S-ethyl-L-cysteine; O-acetyl-L-serine; O-benzyl-L-serine; S-benzyl-L-cysteine; L-serine-O-sulfate; L-serine; Etc. can be mentioned.

Examples of sulfides, hydrosulfides or polysulfides usable in the present invention, which are another substrate, include metal sulfides such as sodium sulfide, potassium sulfide and lithium sulfide; Metal hydrosulfides such as sodium hydrosulfate, potassium hydrosulfide and lithium hydrosulfide; Metal polysulfides such as sodium polysulfide and potassium polysulfide; Ammonium sulfide; Ammonium hydrosulfide; Ammonium polysulfide, and the like.

According to the present invention, β-substituted L-alanine and sulfides, hydrosulfides or polysulfides are typically reacted in an aqueous medium at pH 6-10 in the presence of tryptophan synthase. The reaction temperature is appropriately selected within the range of 20 to 60 ℃. The reaction time varies depending on the titer of the enzyme, the concentration of the substrate used and the conditions, but is generally 1 to 100 hours in the medium reaction, and the reaction is performed under standing or slow stirring.

The concentration of substrate, β-substituted L-alanine and sulfides, hydrosulfides or polysulfides is not limited to specific values, but is usually in the range of about 0.1 to 30% by weight. Substrates may be added all at once at the beginning of the reaction, or may be added in portions as the reaction proceeds. Sulphides, hydrosulfides or polysulfides are suitably present in equimolar amounts or more, based on β-substituted L-alanine, in the reaction medium. In the reaction, in addition to the substrate, it is preferable to add a small amount of coenzyme phosphate pyridoxal.

When using a cell or culture medium of a microorganism capable of producing tryptophan synthase as an enzyme source, the yield may be increased by adding at least one compound selected from alcohols, esters, ketones and surfactants to the reaction medium. As the alcohol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, t-butyl alcohol, 1-pentanol, 1-octanol and the like can be used. Examples of esters that can be used include ethyl formate, propyl formate, butyl formate, ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate and the like. Ketones that can be used include acetone, methyl ethyl ketone and methyl isobutyl ketone. Surfactants that can be used include anionic surfactants such as sodium dodecyl sulfate and sodium deoxycholate, nonionic surfactants such as octyl phenylether and other surfactants. The amount of the compound added in the reaction medium depends on the type of compound or the strain used, but is usually in the range of 0.01 to 5% by weight.

When the reaction is carried out as described above, since the produced L-cysteine is easily oxidized and converted into L-cystine, the reaction medium generally contains a mixture of L-cysteine and L-cystine, and as the reaction proceeds, The amount gradually increases. However, by controlling the reaction conditions, the concentration ratio of L-cysteine and L-cystine can be changed.

The resulting L-cysteine or L-cystine can be recovered from the reaction medium using any conventional method. For example, L-cystine, which is difficult to dissolve in water, can be easily separated by oxidizing most of L-cysteine to L-cystine after completion of the reaction, through the reaction medium. L-cysteine can also be obtained by performing electrolytic reduction on the L-cystine obtained above.

Quantitative determination of L-cysteine and L-cystine is performed using liquid chromatography. Column liquid chromatography to separate the optical isomers also showed that the produced cysteine and cystine both had L-coordination.

The following examples illustrate the invention in more detail.

Example 1

Inoculate Escherichia coli MT-10242 (FERM BP-20) in a liquid medium (pH 7.0) containing 1% broth, 0.5% peptone, 0.1% yeast extract and 0.2% KH ₂ PO ₄ and shake at 30 ° C. Incubate for 20 hours. After incubation, the cells are harvested by centrifugation and the enzyme is purified according to O. Adachi et al., O. Adachi et al., Biochemical Journal, Vol. 249, pp. 7756-7763 (1974). The enzyme obtained, tryptophan synthase having a specific activity (titer) of 9.2 units per mg mg, is used in the following reaction. Enzyme activity is measured using C. Yanofsky et al. (C.Yanoflsy et al, Methods in Enzymology, Vol. 15, pp. 801-807 (1962)). One unit represents the amount of enzyme that produces 1 μmol / minute tryptophan from L-serine and indole at 37 ° C. and pH 7.8.

0.5 mg of tryptophan synthase was added to 10 ml of a reaction medium of pH 8.5 containing 50 mM L-serine, 100 mM sulfide, hydrosulfide or polysulfide and 0.1 mM pyrophosphate, and 10 hours at 35 ° C. Shake slowly while. The concentrations of L-cysteine and L-cystine produced and their total yield based on L-serine are shown in Table 1 below.

TABLE 1

Example 2

Purify the enzyme by culturing Neurospora Krasa ATCC-14692 according to WHMatchett et al., Biochemical Journal, Vol. 250, No. 8, pages 2941-2946 (1975). do. The enzyme obtained, tryptophan synthase, has an activity of 1.3 units / mg.

The following reaction is carried out using the enzyme solution. The reaction medium (pH 8.5, 10 ml) containing 50 mM L-serine, 100 mM sodium hydrosulfide, 0.1 mM pyrophosphate and 1.3 units of tryptophan synthase was gently shaken at 35 ° C. for 10 hours. As a result, 12 mM L-cysteine and 4 mM L-cystine are produced in the reaction medium.

Example 3

Saccharomyces cerevisiae ATCC-26787 is inoculated in a liquid medium (pH 6.0) containing 1% peptone, 0.5% yeast extract, 2% glucose and 0.01% indoleacrylic acid and incubated at 30 ° C. for 20 hours. The cultured cells are harvested by centrifugation and the enzyme is purified according to the method of M. Detwiler et al., European Biochemistry Journal, Vol. 102, pages 159-165 (1979). Tryptophan synthase with a specific activity of 1.2 units / mg is obtained.

The following reaction is carried out using the enzyme solution. The reaction solution (10 ml) at pH 8.5 containing 50 mM L-serine, 100 mM sodium hydrosulfide, 0.1 mM ancinpyridoxal, and 1.2 units of tryptophan synthase was gently shaken at 35 ° C. for 10 hours. The resulting reaction solution contains 11 mM L-cysteine and 3 mM L-cystine prepared.

Example 4

Inoculate Escherichia coli MT-10242 (RERM BP-20) in a liquid medium (pH 7.0) containing 1% broth, 0.5% peptone, 0.1% yeast extract and 0.2% KH ₂ PO ₄ , and at 20 ° C. Incubate for time. After incubation, cells are harvested by centrifugation and used as an enzyme source of tryptophan synthase. The wet cells have a specific activity of 120 units / g.

The reaction medium (100 ml) containing 200 mM L-serine, 300 mM sodium sulfide, 0.1 mM phosphate pyridoxal and 5 g of wet cells and adjusted to pH 8.5 using HCl is shaken at 35 ° C. for 24 hours. After the reaction is completed, dithiothreitol is used to reduce L-cystine present in the reaction medium to L-cysteine. The amount of L-cysteine produced is 98 mM.

Example 5

The following reaction was carried out using the tryptophan synthase obtained in Example 1.

Tryptophan synthase (500 units) in 100 ml of the reaction solution containing 300 mM β-substituted L-alanine, 600 mM sodium hydrosulfide, 1 mM pyrophosphate, and 480 mM tris aminomethane-HCl buffer (pH 8.5) shown in Table 2 below. Is added and gently shaken at 35 ° C. for 1 hour. After the reaction is completed, dithiothreitol is used to reduce L-cystine present in the reaction solution to L-cysteine, and then the amount of L-cysteine is measured. The results are shown in Table 2 below.

TABLE 2

Example 6

Inoculate Escherichia coli MT-10232 (FERM BP-19) in a liquid medium (pH 7.0) containing 1% broth, 0.5% peptone, 0.1% yeast extract and 0.2% KH ₂ PO ₄ and 20 at 30 ° C. Shake culture for time. After incubation, cells are harvested by centrifugation, frozen at -20 ° C and stored as an enzyme source of tryptophan synthase. The wet cells have a tryptophan synthase activity of 89 units / g. The following reactions are carried out using storage cells.

The reaction medium (100 ml) containing 200 mM L-serine, 300 mM sodium sulfide, 0.1 mM pyrophosphate, 100 mM trisaminomethane-HCl buffer (pH 8.5) and 5 g of wet cells (100 ml) was shaken gently at 35 ° C. for 10 hours. After the reaction was completed, L-cystine present in the reaction medium was reduced to L-cysteine using dithiothreitol, and the amount of L-cysteine prepared was 114 mM.

Example 7

The following reaction was carried out using the wet cells obtained in Example 6.

The reaction solution (100 ml, pH 8.0) containing 200 mM (2.1 g) L-serine, 1000 mM sodium hydrosulfide, 0.1 mM pyrophosphate and 5 g of wet cells is gently stirred at 35 ° C. for 10 hours. Two and five hours after the start of the reaction, 2.1 g of L-serine is added again. During the reaction, 6N phosphoric acid is added to maintain the pH of the reaction solution at 8.0. When the reaction was completed, 4.3 g of L-cysteine and 1.1 g of L-cystine were accumulated in the reaction solution.

Example 8

In the following reaction, the wet cells obtained in Example 4 are used directly as an enzyme source of tryptophan synthase.

The reaction medium (100 ml, pH 8.5) containing 200 mM L-serine, 300 mM sodium sulfide, 1 mM pyrophosphate, 5 g of wet cells and the compounds shown in Table 3 below is gently shaken at 35 ° C. for 5 hours. During the reaction, the pH of the medium is adjusted to 8.5 ± 0.3. After the reaction is completed, dithiothreitol is used to reduce the L-cystine present in the reaction medium to L-cysteine and then measure the amount of L-cysteine produced. The results are shown in Table 3 below.

TABLE 3

Claims (6)

L-characterized by reacting β-substituted L-alanine of formula (I) with metal sulfide, metal hydrosulfide, metal polysulfide, ammonium sulfide, ammonium sulfide or ammonium polysulfide in the presence of tryptophan synthase Method for preparing cysteine and L-cystine. X-CH ₂ -CHCOOH (I) │ NH ₂ [Wherein X is a halogen atom or an -OR or -SR group, R is a hydrogen atom or an alkyl, acetyl, benzyl or sulfonic acid group.] The process for preparing L-cysteine and L-cystine according to claim 1, wherein the reaction is carried out in the presence of 0.01 to 5% by weight of at least one compound selected from alcohols, esters, ketones and surfactants. Β-substituted L-alanine of the following general formula (I) is reacted in the presence of a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, ammonium sulfide or ammonium polysulfide with tryptophan synthase, and vented to the reaction medium. Method for producing L-cystine characterized by the sikim. X-CH ₂ -CHCOOH (I) │ NH ₂ [Wherein X is a halogen atom or an -OR or -SR group, R is a hydrogen atom or an alkyl, acetyl, benzyl or sulfonic acid group.] The process for preparing L-cystine according to claim 3, wherein the reaction is carried out in the presence of 0.01 to 5% by weight of at least one compound selected from alcohols, esters, ketones and surfactants. Β-substituted L-alanine of formula (I) below is reacted with metal sulfide, metal hydrosulfide, metal polysulfide, ammonium sulfide, ammonium sulfide or ammonium polysulfide in the presence of tryptophan synthase, and is characterized by reducing Method for producing L-cysteine X-CH ₂ -CHCOOH (I) │ NH ₂ [Wherein X is a halogen atom or an -OR or -SR group, R is a hydrogen atom or an alkyl, acetyl, benzyl or sulfonic acid group.] A process for preparing L-cysteine according to claim 5, wherein the reaction is carried out in the presence of 0.01 to 5% by weight of at least one compound selected from alcohols, esters, ketones and surfactants.