NL8600984A - METHOD FOR THE ENZYMATIC PREPARATION OF L-CYSTEINE AND / OR L-CYSTINE. - Google Patents

Description

1 -1-25508 / Vk / mvl

Short designation: Process for the enzymatic preparation of L-cysteine and / or L-cystine.

The invention relates to a process for the enzymatic preparation of L-cysteine and / or L-cystine.

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

Various methods are known for the preparation of L-cysteine and L-cystine, such as 1) the extraction from natural materials such as hair, 2) chemical synthesis as mentioned for example in Japanese patent application SHO 57-200356, 3) an enzymatic synthesis from 15. DL-2-aminothiazoline-A-carboxylic acid as disclosed in Japanese patent application SHO 54-2272, and 4) a reaction of a 3-substituted alanine with a metal sulfide or hydrosulfide in the presence of cysteine disulf hydrase as disclosed in Japanese patent application SHO 57 -21311. However, these methods are not methods which can be successfully used as industrial processes.

In the context of the studies conducted, these known methods have been studied and a new method has been developed for the preparation of L-cysteine and / or L-cystine, which method is characterized in that a β-substituted L-alanine represented by 25 general formula

X-CHo-CHC00H 2 I

m2 where X has the meaning of a halogen atom or an -OR or -SR group where R is a hydrogen atom or an alkyl, acetyl, benzyl or sulfonic acid group is reacted with a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, ammonium hydrosulfide or an ammonium polysulfide in the presence of tryptophan synthase.

Tryptophan synthase is known to be widespread in microorganisms, higher plant species and other materials as mentioned, for example, in Bacteriological Reviews, Vol. 39, No. 2, biz. 87-120 (1975).

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According to the invention, tryptophan synthase is derived from microorganisms and commonly used, but the enzyme source is not limited thereto. Strains producing tryptophan synthase include, for example, Escherichia coli MT-10232 (FERM BP-19), Escherichia coli MT-.10242 (FERM BP-20), Neurospora crassa ATCC-14692 and Saccharomyces cerevisiae ATCC-26787.

The extraction of tryptophan synthase from cultured cells is known as described in The Journal of Biological Chemistry,

Volume 249, no. 24, biz. 7756-7763 (1974) for E. coli; ibid., Vol. 250, 10 No. 8, biz. 2941-2946 (1975), for Neurospora crassa and European Journal of Biochemistry, Band 102, biz. 159-165 (1979) for Saccharomyces cerevisiae.

Tryptophan synthase used according to the invention is not necessarily an extracted and purified enzyme. Culturing a tryptophan synthase producing microorganism, living cells, collected from cultures by centrifugation or a similar method, dried cells thereof and comminuted cellular material obtained by grinding, ultrasonic comminuting or other treatments of the cells or by autolysis thereof are used as a source of the enzyme material according to the invention. Extracts from these cells and crude enzymes obtained from the extracts can also be used. Furthermore, immobilized cells or enzymes can of course be used in the method of the invention. *

Any synthetic or natural medium can be used to grow tryptophan synthase producing cells provided it contains a carbon source, a nitrogen source, minerals and optionally a small amount of the smaller amounts of nutrients. The addition of a small amount of tryptophan or indole to the culture medium can sometimes be effective. Also, the amount of tryptophan synthase produced can sometimes be increased by the addition of a small amount of indole acrylic acid to the medium.

The cultivation is carried out aerobically by shaking or aerating the culture with stirring. The temperature is in the range of 20 to 40 ° C usually from 25 to 37 ° C. The pH of the culture medium is 5 to 8.

It is well known that tryptophan synthase is a multifunctional enzyme in which the enzyme catalyzes not only the synthesis of L-tryptophan from indole-3-glycerol phosphoric acid and L-serine but -. V ”h -3- 25508 / Vk / mvl /, \ also many other reactions referring, for example, to Advances in Enzymology and Related Areas of Molecular Biology, Band 49, biz. 127-185 (1979).

In the context of the research conducted, the inventors first found, however, that the reaction according to the invention can be catalyzed by tryptophan synthase.

For example, β-substituted L-alanines, one of the substrates for the reaction that can be used according to the invention, include β-halogen-L-alanine such as β-chloro-L-alanine and β-bromo-10-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 and the like.

Sulfides, hydrosulfides or polysulfides, which are the other substrates that can be used according to the invention include, for example, metal sulfides such as sodium sulfide, potassium sulfide 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 and the like.

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 usually selected in the range from 20 to 60 ° C. The reaction time is generally 1 to 10 hours for a batch reaction, although this may vary depending on the purity of the enzyme, the concentrations of the substrates used and the other conditions. The reaction is carried out stationary or with slow stirring.

The concentrations of the substrates, the β-substituted L-alanine and the sulfide, hydrosulfide or polysulfide are not particularly limited, but usually they are in the range of about 0.1 to 30% by weight. The substrates can be added as a whole at one time when the reaction is started, or alternatively they can be partially added as the reaction continues.

Desirably, the sulfide, hydrosulfide or polysulfide is present in the reaction medium in an equimolar amount or more, based on -v * -4- 25508 / Vk / mvl nr »r ψ- ~ the β-substituted L- alanine. In the reaction, it is desirable to add a small amount of a coenzyme, pyridoxal phosphate in addition to the substrates.

When cells or culture media of a microorganism capable of preparing tryptophan synthase are used as a source of the enzyme, the yield can be increased by adding at least one compound selected from alcohols, esters, ketones and surfactants to the reaction medium. The alcohols that can be used include ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, t-butyl alcohol, 1-pentanol, 1-octanol and the like. The esters that can be used include ethyl formate, propyl formate, butyl formate, ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate and the like. The ketones that can be used include acetone, methyl ethyl ketone, methyl isobutyl ketone and the like. The surfactants that can be used include anionic surfactants such as sodium dodecyl sulfate and sodium deoxycholate, nonionic surfactants such as octyl phenyl ethers and other surfactants. The amounts of these compounds added to the reaction medium usually range from 0.01 to 5% by weight, although this amount may vary depending on the type of the compounds used or the strain used.

When the reaction is conducted in this manner, the reaction medium will generally contain a mixture of L-cysteine and L-cystine because L-cysteine, which is thereby prepared, is easily oxidized and converted to L-cystine. The amount of L-cystine gradually increases as the reaction continues. The ratio of the concentration of L-cysteine to that of L-cystine can be varied by controlling the reaction conditions.

L-cysteine or L-cystine can be recovered from the reaction medium in a manner known per se. For example, L-cystine which is sparingly soluble in water can be easily separated by aerating the reaction mixture with most L-cysteine being converted to L-cystine after the completion of the reaction.

L-cysteine can be obtained by subjecting the thus obtained L-cystine to electrolytic reduction.

A quantitative measure of L-cysteine and L-cystine Λ * · r /. 25508 / Vk / mvl was performed by liquid chromatography. Liquid chromatography using a column to separate the optical isomers further showed that both cysteine and cystine prepared in this manner had the L configuration.

The invention is further illustrated by the following examples.

Example I

Escherichia coli MT-10242 (FERM BP-20) was inoculated into a liquid medium of 1% meat extract, 0.5% peptone, 0.1% yeast extract, 0.2% KHgPO 4, at a pH of 7.0 and cultivated with shaking at 30 ° C for 20 hours. After the culture, the cells were collected by centrifugation and the enzyme was purified by the method of 0. Adachi et al., The Journal of Biological Chemistry, Vol. 249, no. 24, biz. 7756-7763 (1974). The enzyme thus obtained, tryptophan synthase, with a specific activity (titer) of 9.2 units per mg was used in the following ______________ reaction. Enzyme activity was determined by the method of C. Yanofsky et al., Methods in Enzymology, Vol. 15, pp 801-807 (1962). One unit is represented by the amount of enzyme that makes 1 pmol / minute of tryptophan from L-serine and indole at 37 ° C and pH 7.8.

Tryptophan synthase in an amount of 0.5 mg was added to 10 ml of reaction medium at a pH of 8.5 containing 50 mmol of L-serine, 100 mmol of sulfide, hydrosulfide or polysulfide as indicated in Table A and 0.1 mmol of pyridoxal phosphate and shaken slowly at 35 ° C for 10 hours. The concentrations of L-oysteine and L-cystine obtained thereby and the yields of the total are based on L-serine as shown in Table A.

TABLE A

. . L-cysteine L-cystine yield type sulfides (Lol) (Lol) (mol *) 30 ---- sodium sulfide 18 6.5 62 sodium hydrosulfide 16 5 52 potassium sulfide 11 3.5 36 ammonium sulfide 8 1.5 22 35 ammonium hydrosulfide 6 2 20 ammonium trisulfide 6 1.5 18 sodium disulfide 11 3 34 V »* -6- 25508 / Vk / mvl

Example II

According to the method of W.H. Matchett et al., The Journal of Biological Chemistry, Band 250, No. 8, biz. 2941-2946 (1975) Neurospora crassa ATCC-14692 was grown and the enzyme was purified.

The enzyme tryptophan synthase thus obtained had a specific activity of 1.3 unit per mg.

The enzyme liquid was used in the next reaction. A reaction medium (pH: 8.5, 10ml) with 50mmol L-serine, 100mmol sodium hydrosulfide, 0.1mmol pyridoxal phosphate and 1.3 unit trypto-10 fan synthase was shaken slowly at 35 ° C for 10 hours. 12 mmol L-cysteine and 4 mmol L-cystine were prepared in the reaction medium.

Example III

Saccharomyces cerevisiae ATCC-26787 was seeded in a liquid medium of 1% peptone, 0.5% yeast extract, 2% glucose and 0.01% indole acrylic acid at pH 6.0 and shaken with stirring at 30 ° C for 20 hours . The cultured cells were then collected by centrifugation. The enzyme was purified by the method of M. Dettwiler et al., European Journal of Biochemistry, Band 102, 159-165 (1979). Thus, tryptophan synthase having a specific activity of 1.2 unit per mg was obtained.

The enzyme liquid was used to effect the next reaction. A reaction liquid (10ml) containing 50mmol L-serine, 100mmol sodium hydrosulfide, 0.1mmol pyridoxal phosphate and 1.2 unit tryptophan synthase at pH 8.5 was gently shaken at 35 ° C for 10 hours. The resulting reaction liquid contained 11 mmol L-cysteine and 3 mmol L-cystine.

Example IV

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

A reaction medium (100ml) containing 200mmol L-serine, 300mmol sodium sulfide, 0.1mmol pyridoxal phosphate and 5g wet cells and adjusted to pH 8.5 with HCl was shaken at 35 ° C for 24 hours. After the reaction was completed, the amount of -7-25508 / Vk / mvl

S 4, V

L-cystine present in the reaction mixture is reduced to L-cysteine by dithiothreitol. The amount of L-cysteine prepared was 98 mmol.

Example V

Tryptophan synthase obtained in Example I was used in the following reaction.

The tryptophan synthase (500 units) was added to 100 ml of a reaction liquid containing 300 mmol of β-substituted L-alanine listed in Table B, 600 mmol of sodium hydrosulfide, 1 mmol of pyridoxal phosphate and 480 mmol of tris-aminomethane-HCl buffer at pH 10 8.5 and shaken slowly at 35 ° C for 1 hour. After the reaction was completed, L-cystine present in the reaction liquid was reduced to L-cysteine with dithiothreitol and then the amount of L-cysteine was determined. The results are shown in Table B.

TABLE B

15 --- j ^ -substituted L-alanine L-cysteine (mmol) β-chloro-L-alanine 82 O-methyl-L-serine 116 O-acetyl-L-serine 95 20 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 25 L-serine 123

Example VI

Escherichia coli MT-10232 (FERM BP-19) was inoculated in a liquid medium at a pH of 7.0 containing 1% meat extract, 0.5% peptone, 0.1% yeast extract and 0.2% and shaken to cultivating it at 30 ° C for 20 hours. After cultivation, the cells were collected by centrifugation and lyophilized at -20 ° C to store as an enzyme source for tryptophan synthase. The wet cells had a tryptophan synthase activity of 89 units per g. The stored cells were used in the next reaction.

A reaction medium (100ml) containing 200mmol L-serine, 300mmol sodium sulfide, 0.1mmol pyridoxal phosphate, 100mmol trisamino -8- 25508 / Vk / mvl

r .. -V

methane HCl buffer (pH = 8.5) and containing 5 g of wet cells was shaken slowly at 35 ° C for 10 hours. After the reaction was terminated, L-cystine present in the reaction mixture was reduced to L-cysteine by dithiothreitol and the amount of L-cysteine prepared thereby was then measured. The amount was 114 mmol.

Example VII

The wet cells obtained in Example VI were used in the next reaction.

A reaction liquid (100 ml) with a pH of 8.0, containing 10 200 mmol (2.1 g) of L-serine, 1000 mmol of sodium hydrosulfide, 0.1 mmol of pyridixal phosphate and 5 g of wet cells, was stirred together at 35 ° C for 10 hours. L-serine was further added at a time of 2 hours and 5 hours after the reaction started, each in an amount of 2.1 g. During the reaction, the pH of the reaction liquid was kept at 8.0 by adding 6 N phosphoric acid. When the reaction was completed, 4.3 g of L-cysteine and 1.1 g of L-cystine were collected in the reaction liquid.

Example VIII

The wet cells obtained in Example IV were immediately used as an enzyme source of tryptophan synthase in the next reaction.

A reaction medium (100ml) at a pH of 8.5 containing 200mmol L-serine, 300mmol sodium sulfide, 1mmol pyridoxal phosphate, 5g wet cells and a compound indicated in Table C was gently shaken at 35 ° G for 5 hours. The pH of the medium was adjusted to 8.5 to 0.3 during the reaction. After the reaction was completed, L-cystine present in the reaction medium was reduced to L-cysteine by dithiothreitol and the amount of L-cysteine obtained thereby was measured. The results are shown in Table C.

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-9- 25508 / Vk / mvl

TABLE C

added compound concentration (g / l) amount of _ '_ L-cysteine (mmol) blank 0 67.2 5 ethanol 10 99> 6' 1-propanol 10 119.4 2-propanol 10 105.6 1-butanol 10 140, 0 2-butanol 10 128.7 10 isobutyl alcohol 5 114.8 t-butyl alcohol 5 107.2 1-pentanol 5 106.5 1-octanol 10 128.7 ethyl formate 5 96.7 15 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 20 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 25 sodium radodecyl sulfate 1 160.9

Triton X-100 1 160.7 - - ·

Claims (2)

0 * '% e vv. -¾ -10- 25508 / Vk / ravl 1. Process for the preparation of L-cysteine and / or L-cystine, characterized in that a β-substituted L-alaine represented by general formula X-CH.-CHC00H 2 I NH2, wherein X has the meaning of a halogen atom or an -OR or -SR group, wherein R is a hydrogen atom or an alkyl, acetyl, benzyl or sulfonic acid group is reacted with a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, ammonium hydrosulfide or an ammonium polysulfide in the presence of tryptophan synthase. Process for the preparation of L-cysteine and / or L-cystine according to claim 1, characterized in that the reaction is carried out in the presence of at least one substance in an amount of 0.01 to 5% by weight selected from the group consisting of alcohols, esters, ketones and surfactants. Eindhoven, April 1986. 'N * i Ui · f i ... i Af ·. · «/ J