CH666695A5 - ENZYMATIC PROCESS FOR THE PREPARATION OF SULFUR-CONTAINING L-AMINO ACIDS. - Google Patents

Description

DESCRIPTION

The present invention relates to a process for preparing L-cysteine and / or L-cystine by reacting an L-alanine substituted in the β position with a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, ammonium hydrosulfide or an ammonium polysulfide, in the presence of tryptophan synthase.

L-cysteine and L-cystine are widely used in cosmetics, in the food industry, as a food additive, and for other applications as well as for medical applications, for example as a liquid ingredient for transfusions.

Various processes for preparing L-cysteine and L-cystine are known up to now. By way of example characteristic of such known processes, the following may be cited: (1) extraction from natural materials, such as hair and body hair, (2) chemical syntheses as described, for example, in the application for Japanese published patent No. SHO 57-200356, (3) enzymatic synthesis from DL-2-aminothiazoline-4-carboxylic acid, as described in Japanese published patent No. SHO 54-2272, and (4) reaction d an alanine substituted in β with a metal sulfide or hydrosulfide, in the presence of cysteine desulfhydrase, as described in the Japanese patent published No. SHO 57-21311. However, such methods do not necessarily have the advantages required for industrial implementation.

In view of the aforementioned state of the art, the present invention results from studies undertaken with a view to the development of a new process for the preparation of L-cysteine and / or L-cystine, of a low cost price, and the discovery that L-cysteine and / or L-cystine can be produced by reacting a ß-substituted L-alanine with a metal sulfide, a metallic hydrosulfide, a metallic polysulfide, ammonium sulfide, ammonium hydrosulfide or an ammonium polysulfide, in the presence of tryptophan synthase. Consequently, the method according to the invention has the characteristics specified in claim 1.

As is known, tryptophan synthase is widely distributed in microorganisms, higher plants and other sources of this substance. On this subject, one can refer, for example, to the article published in "Bacteriological Reviews", vol. 39, No. 2, pages 87-120 (1975).

For the implementation of the method according to the invention, tryptophan synthase produced from microorganisms will usually be used, but this is not the only source of enzyme that can be used. Examples of strains of microorganism producing tryptophan synthase include, for example, the following: Escherichia coli MT-10232 (FERM BP-19), Escherichia coli MT-

10242 (FERM BP-20), Neurospora crassa ATCC-14692 and Saccharo-myces cerevisiae ATCC-26787.

Methods of extracting tryptophan synthase from cell cultures are known and described, for example, in "Journal of Biological Chemistry", vol. 249, No. 24, pages 7756-7763 (1974) for E. coli ', idem, vol. 250, No. 8, pages 2941-2946 (1975) for Neurospora crassa; and "European Journal of Biochemistry", vol. 102, pages 159-165 (1979) for Saccharomyces cerevisiae, respectively.

The tryptophan synthase which can be used for the implementation of the method according to the invention is not necessarily an extracted and purified enzyme. As an enzyme source, for the implementation of the method according to the present invention, it is possible to use, for example, cultures of microorganisms producing tryptophan synthase, living cells collected from cultures by centrifugation or method of the same kind, desiccants from such cells and cell debris obtained by grinding, treating the cells with ultrasound or other suitable ways, or by autolysis thereof. Extracts from such cells and crude enzymes obtained from these extracts can also be used. In addition, immobilized cells or enzymes can of course be used.

Any synthetic or natural medium can be used for the cultivation of cells producing tryptophan synthase, provided that they contain a carbon source, a nitrogen source, minerals and, optionally, a small quantity of secondary nutritive agents. It may sometimes be useful to add small amounts of tryptophan or indole to the culture medium. Also, the amount of tryptophan synthase produced can sometimes be increased by adding small amounts of indole-acrylic acid to the culture medium.

The culture is carried out aerobically, by shaking the culture or by subjecting it to agitation by aeration. Preferably, the culture temperature is in the range of 20 to 40 ° C, usually 25 to 37 ° C. The pH of the culture medium is 5 to 8.

As is known, tryptophan synthase is a multi-functional enzyme, that is to say that the enzyme not only catalyzes the synthesis of L-tryptophan from indole-3-glycerophosphoric acid and L -serine, but also many other reactions, as described, for example, in "Advances in Enzymology and Related Areas of Molecular Biology", vol. 49, pages 127-185 (1979).

However, the present invention results from the discovery that the reaction according to the invention can be catalyzed by tryptophan synthase.

Among the β-substituted L-alanines which constitute one of the substrates for the reaction, it is possible to use, for example: β-halogenated L-alanines, such as ß-chloro-L-alanine and ß-bromo- L-alanine; O-alkyl-L-serines, such as O-methyl-L-serine and O-ethyl-L-serine; S-alkyl-L-cysteines, 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.

As an example of a sulfide, hydrosulfide or polysulfide, which constitute the other substrate which can be used for carrying out the process according to the invention, the following compounds may be mentioned: metal sulfides such as sodium sulfide, potassium and lithium sulfide; metal hydrosulfides such as sodium hydrosulfide, potassium hydrosulfide and lithium hydrosulfide; metal polysulfides such as sodium polysulfides and potassium polysulfides; ammonium sulfide; ammonium hydrosulfide; ammonium polysulfides, etc.

According to the invention, the β-substituted L-alanine and the sulfide, hydrosulfide or polysulfide are usually reacted in an aqueous medium, at a pH of 6 to 10, in the presence of tryptophan synthase. The reaction temperature is chosen in the range from 20 to 60 ° C. The reaction time is generally between 1 and 100 hours, for each batch subjected to the reaction,

although it may vary, depending on the title of the enzyme, the concentra5

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of the substrates used and the other parameters. The reaction can be carried out in a stationary medium or with gentle agitation.

There is no particular limit to the concentrations of the substrates, β-substituted L-alanine and sulfide, hydrosulfide or polysulfide, but these are usually in the range of about 0.1 to 30% by weight. All of the substrates can be added all at once, at the start of the reaction or, alternatively, they can be added in fractions as the reaction progresses. Advantageously, the sulphide, hydrosulphide or polysulphide is present in the reaction medium in an equimolar amount or in excess relative to the L-alanine substituted in ß. It is advantageous to add a small amount of a coenzyme, pyridoxal phosphate, in addition to the substrates.

When cells or a culture medium of a microorganism capable of producing tryptophan synthase are used as the source of the enzyme, the yield can be increased by adding at least one selected compound to the reaction medium. among alcohols, esters, ketones and surfactants. As the alcohol, it is possible to use, for example, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 1-octanol, etc. As the ester, there can be used, for example, ethyl formate, propyl formate, butyl formate, ethyl acetate, propyl acetate, butyl acetate, acetate isobutyle, etc. As the ketone, there can be used, for example, acetone, methyl ethyl ketone, methyl isobutyl ketone, etc. As surfactant, anionic surfactants, such as sodium dodecyl sulfate and sodium deoxycholate, nonionic surfactants, such as ethylphenyl ethers and other surfactants can be used, for example. The amount of these compounds added to the reaction medium is usually in the range of 0.01 to 5%, by weight, although it may vary depending on the type of compound or the strain used.

When the reaction is carried out in this way, the reaction medium generally contains a mixture of L-cysteine and L-cystine, due to the fact that the L-cysteine produced is easily oxidized and transformed into L-cystine. The amount of L-eystine gradually increases as the reaction progresses. However, the ratio of the concentration of L-cysteine relative to that of L-cystine can be varied by acting on the reaction parameters.

The separation of L-cysteine or L-cystine from the reaction medium can be carried out in any suitable known manner. For example, it is easy to isolate L-cystine, which is hardly soluble in water, by aerating the reaction medium, so as to oxidize a large proportion of L-cysteine to L-cystine, after the end of the reaction. L-cysteine can be obtained by subjecting the L-cystine thus obtained to electrolytic reduction.

The quantitative determination of L-cysteine and L-cystine contents was carried out by liquid chromatography. In addition, liquid chromatography, performed using a column for separation of the optical isomers, showed that the cysteine and cystine produced both had the L configuration.

We will now give examples of implementation of the method according to the invention.

Example 1:

Escherichia coli MT-10242 (FERM BP-20) was inoculated

in a liquid medium containing 1% meat extract, 0.5% peptone, 0.1% yeast extract and 0.2% KH2PO4, pH 7.0, and was cultivated for 20 hours, with stirring, at 30 ° C. After the culture, the cells were isolated by centrifugation and the enzyme purified according to the method of O. Adachi et al., Described in "The Journal of Biological Chemistry", vol. 249, No. 24, pages 7756-7763 (1974). The enzyme tryptophan synthase thus obtained, which had a specific activity (titer) of 9.2 units per mg, was used in the following reaction. The activity of the enzyme was determined by the method of

C. Yanofsky et al., Described in "Methods in Enzymology", vol. 15, pages 801-807 (1962). One unit is represented by the quantity of enzymes producing 1 μmol / min of tryptophan from L-serine and indole, at 37 ° C., and at pH 7.8.

0.5 mg of tryptophan synthase was added to 10 ml of a reaction medium, pH 8.5, containing 50 mM of L-serine, 100 mM of a sulfide, hydrosulfide or polysulfide, as indicated in Table 1, and 0.1 mM pyridoxal phosphate and stirred gently at 35 ° C for 10 hours. The concentrations of L-cysteine and L-cystine produced as well as the yields in total amount of these compounds, relative to L-serine, are shown in Table 1.

Table 1

Nature of sulfides, etc.

L-cysteine (mM)

L-cystine (mM)

Yield (moles%)

Sodium sulfide

18

6.5

62

Sodium hydrosulfide

16

5

52

Potassium sulfide

11

3.5

36

Ammonium sulfide

8

1.5

22

Ammonium hydrosulfide

6

2

20

Ammonium trisulfide

6

1.5

18

Sodium disulfide

11

3

34

Example 2;

We proceed to the culture of Neurospora crassa ATCC-14692,

according to the method of W.H. Matchett et al., described in "The Journal of Biological Chemistry", vol. 250, No. 8, pages 2941-2946 (1975) and the enzyme is purified. The enzyme tryptophan synthase thus obtained had a specific activity of 1.3 units per mg.

The liquid enzyme was used in the following reaction. A reaction medium (pH 8.5, 10 ml) containing 50 mM L-serine, 100 mM sodium hydrosulfide, 0.1 mM was subjected to moderate stirring at 35 ° C for 10 hours. pyridoxal phosphate and 1.3 unit of tryptophan synthase. There was thus obtained the production of 12 mM L-cysteine and 4 mM L-cystine in the reaction medium.

Example 3:

Saccharomyces cerevisiae ATCC-26787 was inoculated into a liquid medium containing 1% peptone, 0.5% yeast extract, 2% glucose and 0.01% indole-acrylic acid, this medium having a pH 6.0, and cultured with stirring at 30 ° C for 20 hours. After culture, the cells were collected by centrifugation. The enzyme was purified according to the method of M. Dettwiler et al., Described in "European Journal of Biochemistry", vol. 102, pages 159-165 (1979). There was thus obtained tryptophan synthase having a specific activity of 1.2 units per mg.

The liquid enzyme was used to carry out the following reaction. A liquid reaction medium (10 ml) containing 50 mM L-serine, 100 mM sodium hydrosulfide, 0.1 mM pyridoxal phosphate and 1.2 was gently stirred at 35 ° C. for 10 hours. tryptophan synthase unit. The resulting liquid reaction medium contained 11 mM L-cysteine and 3 mM L-cystine produced by the reaction.

Example 4:

Escherichia coli MT-10242 (FERM BP-20) was inoculated into a liquid reaction medium at pH 7.0, containing 1% meat extract, 0.5% peptone, 0.1% extract of yeast and 0.2% KH2PO4, then stirred at 30 ° C for 20 hours. After culture, the cells were collected by centrifugation and used as a source of the tryptophan synthase enzyme. The wet cells had a specific activity of 120 units per g.

A reaction medium (100 ml) containing 200 mM L-serine, 300 mM was kept stirring at 35 ° C. for 24 hours.

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sodium sulfide, 0.1 mM pyridoxal phosphate and 5 g of wet cells, this medium having been adjusted to a pH of 8.5, using HCl. After the end of the reaction, the L-cystine present in the reaction medium was reduced to L-cysteine by means of dithiothreitol. The amount of L-cysteine produced was 98 mM.

fie empie 1:

Tryptophan synthase obtained according to Example 1 was used in the following reaction.

Tryptophan synthase (500 units) was added to 100 ml of a liquid reaction medium containing 300 mM of a β-substituted L-alanine, shown in Table 2, 600 mM of sodium hydrosulfide, 1 mM of pyridoxal phosphate and 480 mM of tris-aminomé-thane-HCl buffer, pH 8.5, then maintained under moderate stirring at 35 ° C, for 1 hour. After the end of the reaction, the L-cysteine present in the liquid reaction medium was reduced to L-cysteine by means of dithiothreitol, then the amount of L-cysteine was measured. The results are shown in Table 2.

Table 2

L-alanine substituted in

L-cysteine (mM)

ß-chloro-L-alanine

82

O-methyl-L-serine

116

O-acetyl-L-serine

95

O-benzyl-L-serine

24

S-methyl-L-cysteine

27

S-ethyl-L-cysteine

18

S-benzyl-L-cysteine

5

L-serine O-sulfate

37

L-serine

123

Example 6:

Escherichia coli MT-10232 (FERM BP-19) was inoculated in a liquid medium of pH 7.0, containing 1% meat extract, 0.5% peptone, 0.1% yeast extract and 0.2% KH2PO4 then maintained the culture medium with stirring, at 30 ° C, for 20 hours. After culture, the cells were collected by centrifugation and then frozen at -20 ° C to keep them as a source of enzyme tryptophan synthase. The wet cells had a tryptophan synthase activity of 89 units per g. The cells thus preserved were used to carry out the following reaction.

A reaction medium (100 ml) containing 200 mM L-serine, 300 mM sodium sulfide, 0.1 mM pyridoxal phosphate, 100 mM buffer was subjected to moderate stirring at 35 ° C for 10 hours. tris-aminomethane-HCl (pH 8.5) and 5 g of wet cells. After the end of the reaction, the L-cystine present in the reaction medium was reduced to L-cysteine, by dithiothreitol, and the quantity of L-cysteine produced was measured. The amount thus determined was 114 mM.

Example 7:

The wet cells obtained according to Example 6 were used in the following reaction.

A liquid reaction medium (100 ml) at pH 8.0, containing 200 mM (2.1 g) of L-serine, 1000 mM hydrosulfide, was subjected to moderate stirring at 35 ° C for 10 hours. sodium, 0.1 mM pyridoxal phosphate and 5 g of wet cells. L-serine was then added after 2 hours and 5 hours after the start of the reaction, each in an amount of 2.1 g. During the reaction, the pH of the liquid reaction medium was kept at 8.0 by adding 6N phosphoric acid. After the end of the reaction, it was found that 4.3 g of L-cysteine and 1.1 g of L-cystine were accumulated in the liquid reaction medium.

Example 8:

The wet cells obtained in Example 4 were used directly as the source of the tryptophan synthase enzyme in the following reaction.

A reaction medium (100 ml, pH 8.5) containing 200 mM L-serine, 300 mM sodium sulfide, 1 mM pyridoxal phophate, was subjected to moderate stirring at 35 ° C. for 5 hours. 5 g of wet cells and the compound indicated in table 3. The pH of the reaction medium was maintained at 8.5 + 0.3 during the reaction. After the end of the reaction, the L-cystine present in the reaction medium was reduced to L-cysteine, using dithiothreitol, and the quantity of L-cysteine produced was then measured. The results of these measurements are shown in Table 3.

Table 3

Compound added (concentration g / 1)

Amount of L-cysteine (mM)

Witness

(0)

67.2

Ethanol

(10)

99.6

1-Propanol

(10)

119.4

2-Propanol

(10)

105.6

1-Butanol

(10)

140.0

2-Butanol

(10)

128.7

Isobutyl alcohol

(5)

114.8

Tertiary butyl alcohol

(5)

107.2

1-Pentanol

(5)

106.5

1-Octanol

(10)

128.7

Ethyl formate

(5)

96.7

Propyl formate

(5)

99.8

Butyl formate

(5)

104.3

Methyl acetate

(5)

98.4

Ethyl acetate

(5)

108.9

Butyl acetate

(5)

162.5

Isobutyl acetate

(5)

129.1

Acetone

(5)

96.5

Methyl ethyl ketone

(5)

111.8

Methyl isobutyl ketone

(5)

167.0

Sodium deoxycholate

(1)

139.4

Sodium dodecyl sulfate

(1)

160.9

Triton X-100

(1)

160.7

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Claims (2)

666 695 2 CLAIMS 1. Process for the preparation of L-cysteine and / or L-cystine, characterized in that one reacts a L-alanine substituted in ß, represented by the general formula (I): X-CH2-CHCOOH (I) I nh2 in which X represents a halogen atom or a group —OR or —SR in which R is a hydrogen atom or an alkyl, acetyl or benzyl group or a sulphonic acid group, with a metal sulphide, a metal hydrosulphide, a metallic polysulfide, ammonium sulfide, ammonium hydrosulfide or an ammonium polysulfide, in the presence of tryptophan synthase. 2. Method according to claim 1, characterized in that the reaction is carried out in the presence of 0.01 to 5% by weight of at least one compound chosen from alcohols, esters, ketones and surfactants.