CA1299127C - Enzymatic process for preparing sulfur containing l-amino acids - Google Patents
Enzymatic process for preparing sulfur containing l-amino acidsInfo
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- CA1299127C CA1299127C CA000506867A CA506867A CA1299127C CA 1299127 C CA1299127 C CA 1299127C CA 000506867 A CA000506867 A CA 000506867A CA 506867 A CA506867 A CA 506867A CA 1299127 C CA1299127 C CA 1299127C
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- cysteine
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- cystine
- ammonium
- hydrosulfide
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/12—Methionine; Cysteine; Cystine
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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Abstract
Abstract of the Disclosure L-Cysteine and/or L-cystine may be prepared by reacting a .beta.-substituted L-alanine represented by the general formula (I):
Description
12,991~7 Enzymatic Process for Preparing Sulfur Containing L-Amino Acids Field of the Invention The present invention relates to a process for preparing L-cysteine and/or L-cystine by reacting a ~-substituted L-alanine with a metal sulfide, a metal hydrosulfide, a m~tal polysul~ide, ammonium sulfide, ammonium hydrosulfide or an ammonium polysulfide in the presence of tryptophan synthase.
L-Cysteine and L-cystine are widely utilized for cosmetic applications, in the food industry as a food additive, and for other applications, as well as for medical applications, for example, as a constituent of transfusions.
Prior Art Various processes for preparing L-cysteine and L-cystine have hitherto known: typically, (1) extraction from natural materials such as hair, (2) chemical syntheses as disclosed in9 for example, Japanese Patent Application - Laying-open No. SH0 57-200356, (3) an enzymatic synthesis from DL-2-aminothiazoline-4-carboxylic acid as di~closed in Japanese Patent Publication No. SH0 54-2272, and (4) a reaction of a ~-substituted alanine with a metal 6ulfide or hydrosulfide in the presence of cysteine desulfhydrase as :, 7 lZ~
disclosed in Japanese Patent Publication NOD SHO 57-21311 are known. However, these are not necessarily advantageous for an industrial process.
Summary of the Invention Considering such status of the art, the present inventors have studied a new process for preparing L-cysteine and/or L-cystine at low cost and found that L-cysteine and/or L-cystine may be produced by a reaction of a ~-substituted L-alanine with a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, ammonium hydrosulfide or an ammonium polysulfide in the presence of tryptophan synthase. Thus, we have attained this invention on the basis of this discovery.
Description of the Invention Tryptophan synthase is known to exist widely in microorganisms, higher plants and others: refer to, for example, Bacteriological Reviews, Vol. 39, NoO 2, pp. 87-120 (1975).
In the invention, tryptophan synthase derived from microorganisms is usually used but the enzyme source is not limited to this. Strains producing tryptophan synthase include, for example, Escherichia coli MT-10232 (FERM BP-l9), Escherichia coli MT-10242 (FERM BP-20), Neurospora crassa ~ 7 ATCC-14692 and Saccharom~ces cerevisiae ATCC-26787.
Extraction methods of tryptophan synthase from cultured cells are known and described in The Journal of Biological Chemistry, Vol. 249, No. 24, pp. 7756-7763 (1974) for E. coli; ibid., Vol. 250, No. 8, pp. 2941-2946 (1975) for NeurosPora crassa; and European Journal of Biochemistry, Vol. 102, pp. 159-165 (1979) for Saccharom~ces cerevisiae;
respectively.
Tryptophan synthase used in the invention is not necessarily an extracted and purified enzyme. Cultures of a tryptophan synthase producing microorganism, live cells collected from the cultures by centrifugation or a similar method, dried cells thereof, and cell debris obtained by grinding, ultrasonicating or otherwise treating the cells or by autolysis thereof may be utilized as an enzyme source in the invention. Extracts from these cells and crude enzymes obtained from the extracts may also be utilized.
Further, immobilized cells or enzymes may of course be utilized in the invention.
Any synthetic or natural medium may be used for the culture of tryptophan synthase producing cells, provided that it contains a carbon source, a nitrogen source, minerals and, optionally, a small amount of minor nutrient(s).
Addition of a small amount of tryptophan or indole to the 2~ culture medium may 60metimes be effective. Also, the amount .
of tryptophan synthase produced may sometimes be increased by adding a small amount of indoleacrylic acid to the medium.
The culture is aerobically carried out by shaking or aeration agitating culture~ The temperature is in the range of 20 to 40~C, usually 25 to 37C. The pH of the culture medium is 5 to 8~
It is well known that tryptophan synthase is a multifunctional enzyme, that is, the enzyme cataly~es not only the synthesis of L-tryptophan from indole-3-glycero-phosphoric acid and L-serine but also many other reactions:
refer to, for example, Advances in Enzymology and Related Areas of Molecular Biology, Vol~ 49, pp. 127-185 (1979).
However, the present inventors have for the first time discovered that the reaction according to the invention may be catalyzed by tryptophan synthase.
~-Substituted L-alanines, one of the substrates for the reaction, which may be used in the invention include, for example, ~-halogeno-L-alanine, such as ~-chloro-L-alanine and ~-bromo-L-alanine; 0-alkyl-L-serines, such as 0-methyl-L-sexine and 0-ethyl-L-serine; S-alkyl-L-cysteines, such as S~methyl-L-cysteine and S-ethyl-L-cysteine; 0-acetyl-L-serine; 0-benzyl-L-serine; S~benzyl~L-cysteine; L_serine 0-sulfat~e; L_serine; and the like.
Sulfides, hydrosulfides or poly~ulfides, the other ~%~
substrate, which may be used in the invention include, for e~ample, 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 ~-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 suitably selected from the range of 20 to 60C. The reaction period of time is generally 1 to 100 hours for a batch reaction although it may vary depending on the titer f the enzyme the concentrations of the substrates used and other conditions. The reaction is carried out stationarily or under slow agitation.
The concentrations of the substrates, the ~-substituted L,alanine and the sulfide, hydrosulfide or polysulfide, are not particularly limited but usually in the range of about 0.1-30~ by weight. The substrates may be added wholly at one time when the reaction is started or, alternatively, they may be added partially as the reaction proceeds. Desirably, the sulfide, hydrosulfide or polysulfide i~ present in the reaction medium at an equimolar amount or .
`
. ,~ ., , - .
~Z~3~312 more based on the ~-substituted L,alanine. In the reaction, it is desirable to add a s~all amount of a coenzyme, pyridoxal phosphate, in addition to the substrates.
When cells or culture media of a microorganism capable of producing tryptophan synthase are utilized 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. The alcohols which may be used include ethanol, l-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, l-pentanol, l-octanol and the like. The esters which may be used include ethyl formate, propyl formate, butyl formate, ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate and the like. The ketones which may be used include acetone, methyl ethyl ketone, methyl isobutyl ketone and the like. The surfactants which may be used include anionic surfactants, such as sodium dodecyl sulfate and sodium deoxycholate, nonionic surfactants, such as octyl phenyl ethers, and other surfactants. The amount of these compounds added to the reaction medium is usually in the range of 0.01-5% by weight although it may vary with the type of the compounds or the strain used.
When the reaction is thus carried out, the reaction medium will generally contain a mixture of L-cysteine and L-cystine Rince L-cysteine produced is readily oxidized and _ 7 ~
converted to L-cystine~ The amount of L-cystine gradually increases as the reaction proceeds. However, the ratio of the concentration of L_cysteine to that of L-cystine can be varied by controlling the reaction conditions4 L-Cysteine or L-cystine may be recovered from the reaction medium in any conventional manner. For example, L,cystine9 which is difficultly soluble in water, can readily be isolated by aerating the reaction medium to oxidize a major proportion of I~cysteine to L-cystine after the completion of the reaction~ L-cysteine may be obtained by subjecting the thus obtained L-cystine to electrolytic reduction.
Quantitative measurement of L-cysteine and L-cystine was carried out by liquid chromatography. Further, liquid chromatography using a column for separating optical isomers showed that both the cysteine and cystine produced had the L-configuration.
Description of the Preferred Embodiments The invention will hereinbelow be illustrated by the following examples.
Example l Escherichia coli MT-10242 ~FERM BP-20) was inoculated in a liquid ~edium o~ 1% meat extract, 0.5%
12~
peptone, 0.1~ yeast extract and 0.2% KH2P04, pH 7.0, and cultured with shaking at 30nC for 20 hoursO .4fter the culture, cells were collected by centrifugation and the enzyme was purified according to the method of 0. Adachi et al., The Journal of Biological Chemistry, Vol. 249, No. 24, pp. 7756-7763 (1974). The thus obtained enzyme tryptophan synthase having a specific activity (titer) of 9.2 units per mg was used in the following reaction. The enzyme activity was determined by the method of C Yanofsky et al., lethods in Enzymology, VolO 15, pp. 801-807 (1962).
One unit is represented by the amount OI the enzyme producing 1 ~umol/min of tryptophan from l,serine and indole at 370C, pH 7.8.
Tryptophan synthase, 0.5 mg, was added to 10 ml f a reaction medium, pH 8.5, containing 50 mM L-serine, 100 mM of a sulfide, hydrosulfide or polysulfide indicated in Table 1 and 0.1 mM pyridoxal phosphate, and shaked slowly at 35C for 10 hours. The concentrations of L-cysteine and L-cystine produced and the yields of the total thereof based on L~serine are shown in Table 1.
.
~2~ 7 Table 1 Kind of Sulfides etc. I,-cysteine L-cystine Yield (mM) (mM) (mole %) 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 According to the method of W. H. Matchett et al., The Journal of Biological Chemistry, Vol. 250, No. 8, pp. 2941-2946 (1975), Neurospora crassa ATCC-14692 was cultured and the enzyme was purified. The thus obtained enzyme tryptophan synthase had a specific activity of 1.3 units per mg.
The enzyme liquid 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 pyridoxal phosphate and 1.3 units tryptophan synthase was gently shaked at 35C ~or 10 hours. There were produced in the reaction medium 12 mM L-cysteine and 4 mM L_cystine.
.
~2~ 27 _ 10 --Example 3 Saccharorn~ces cerev_s_ae ATCC-26787 was inoculated in a liquid medium of 1% peptone, 0.5% yeast extract, 2% glucose and 0.01~ indoleacrylic acid, pH 6.0, and cultured with shaking at 30~C for 20 hours. After the culture cells were collected by centrifugation. The enzyme was purified according to the method of M. Dettwiler et al., European Journal of Biochemistry, Vol. 102, pp. 159-165 tl979). Thus, tryptophan synthase with a specific activitY
of 1.2 units per mg was obtained.
The enzyme liquid was used to effect the following reaction. A reaction liquid (10 ml) containing 50 m~"l L-serine, 100 mM sodium hydrosulfide, 0.1 mr~ pyridoxal phosphate and 1.2 units tryptophan synthase, pH 8.5, was gently shaked at 35C for 10 hours. The resultant reaction liquid contained 11 mM L-cysteine and 3 mM L,cystine produced.
Example 4 Escherichia coli MT-10242 (FERM BP-20) was inoculated in a liquid medium of pH 7.0 containing 1% meat extract, 0.5% peptone, 0.1% yeast extract and 0.2% KH2P04 and 6haked at 30C for 20 hours. After the culture, cells ~ere collected by centrifugation and used as an enzyme source of tryptophan synthaæe. The wet cells had a 6pecific activity of 120 units per g.
~2~t91;~7 A reaction medium (100 ml) containing 200 mM
L-serine, 300 mM-sodium sulfide, 0.1 mM pyridoxal phosphate and 5 g of the wet cells a~d adjusted by HCl to pH Of 8.5 was shaked at 35~C for 24 hours. After the reaction was completed, L-cystine present in the reaction medium was reduced to L-cysteine by dithiothreitolO The amount of L-cysteine produced was 98 mM.
Example 5 Tryptophan synthase obtained in Example 1 was used in the following reaction.
The tryptophan synthase (500 units) was added to 100 ml of a reaction liquid containing 300 m~l of a ~-substituted L-alanine indicated in Table 2, 600 mM sodium hydrosulfide, 1 mM pyridoxal phosphate and 480 mM tris-aminomethane-HCl buffer, pH 8.5, and gently shaked at ~5~C
for 1 hour. After the reaction was completed, L-cystine present in the reaction liquid was reduced to L-cysteine by dithiothreitol and then the amount of L-cysteine was measured.
The results are shown in Table 2.
12~
Table 2 ~-Substituted L-alanineL-cysteine (mM) ~-Chloro-L_alanine 82 0-Methyl-L-serine 116 0-Acetyl-L-serine 95 0-Benzyl-L-serine 24 S-Methyl-L-cy~teine 27 S-Ethyl L-cysteine 18 S-Benzyl-L-cysteine 5 L-Serine 0-sulfate 37 L-Serine 123 Example 6 Escherichia coli ~T-10232 (FERM BP-19) was inoculated in a liquid medium of pH 7.0 containing 1~ met extract, 0O5% peptone, 0.1% yeast extract and 0.2% KH2P04 and shaked to culture at 30~C for 20 hours. After the culture, cells were collected by centrifugation and freezed at -20 7C to store as an enzyme source of tryptophan synthase. The wet cells had a tryptophan synthase activity of 89 units per g- The stored cells were used in the following reaction.
A reaction medium (100 ml) containing 200 mM
L_serine~ 300 mM sodium sulfide, 0.1 mM pyridoxal phosphate, 100 mM trisaminomethane-HCl buffer (pH 8.5) and 5 g of the wet cells was gently ~haked at 35C for 10 hours. After lZ~
the reaction was completed, L,cystine present in the reaction medium was reduced to L,cysteine by dithiothreitol and the amount of L-cysteine produced was then measured. The amount was 114 mM.
Example 7 The wet cells obtained in Example 6 were used in the following reaction.
A reaction liquid (100 ml) of pH 8.0 containing 200 mM (2.1 g) L_serine, 1000 mM sodium hydrosulfide, 0.1 mM pyridoxal phosphate and 5 g of the wet cells was gently stirred at 35~C for 10 hours. L-serine was fur~her added at the time of 2 hours and 5 hours after the reaction was started,each in an amount of 2.1 g. During the reaction, the pH of the reaction liquid was maintained at 8.0 by adding 6N phosphoric acid. When the reaction was completed, 4.3 g of L_cysteine and 1.lg of L,cystine were accumulated in the reaction liquid.
Example 8 The wet cells obtained in Example 4 were used directly as an enzyme source of tryptophan synthase in the ~ollowing reaction.
A reaction medium tlOO ml, pH 8.5) containing 200 mM L-serine, 300 mM sodium sulfide, 1 mM pyrido~al ~2~31Z7 phosphate, 5 g of the wet cells and a compound indicated in Table 3 was gently shaked at 35C for 5 hours. The pH of the medium was controlled to 8.5 + 003 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 produced was then measured.
The results are shown in Table 3.
12~91~
Table 3 Compound added (Concentration g/l) Amount of L-cysteine (mM) Gontrol (O) 67.2 Ethanol (10) 99.6 l-Propanol (10) 119.4 2-Propanol (10) 105.6 l-Butanol (10) 140.0 2-Butanol (10) 128.7 Isobutyl alcohol (5) 11408 Tert-butyl alcohol (5) 107.2 l-Pentanol (5) 106.5 l-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) ~11.8 Methyl isobutyl ketone(5) 167.0 Sodium deogycholate (1) 139.4 Sodium dodecyl sulfate(1) 160.9 Triton X-100 (1) 160.7 ~ de /~ar~
L-Cysteine and L-cystine are widely utilized for cosmetic applications, in the food industry as a food additive, and for other applications, as well as for medical applications, for example, as a constituent of transfusions.
Prior Art Various processes for preparing L-cysteine and L-cystine have hitherto known: typically, (1) extraction from natural materials such as hair, (2) chemical syntheses as disclosed in9 for example, Japanese Patent Application - Laying-open No. SH0 57-200356, (3) an enzymatic synthesis from DL-2-aminothiazoline-4-carboxylic acid as di~closed in Japanese Patent Publication No. SH0 54-2272, and (4) a reaction of a ~-substituted alanine with a metal 6ulfide or hydrosulfide in the presence of cysteine desulfhydrase as :, 7 lZ~
disclosed in Japanese Patent Publication NOD SHO 57-21311 are known. However, these are not necessarily advantageous for an industrial process.
Summary of the Invention Considering such status of the art, the present inventors have studied a new process for preparing L-cysteine and/or L-cystine at low cost and found that L-cysteine and/or L-cystine may be produced by a reaction of a ~-substituted L-alanine with a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, ammonium hydrosulfide or an ammonium polysulfide in the presence of tryptophan synthase. Thus, we have attained this invention on the basis of this discovery.
Description of the Invention Tryptophan synthase is known to exist widely in microorganisms, higher plants and others: refer to, for example, Bacteriological Reviews, Vol. 39, NoO 2, pp. 87-120 (1975).
In the invention, tryptophan synthase derived from microorganisms is usually used but the enzyme source is not limited to this. Strains producing tryptophan synthase include, for example, Escherichia coli MT-10232 (FERM BP-l9), Escherichia coli MT-10242 (FERM BP-20), Neurospora crassa ~ 7 ATCC-14692 and Saccharom~ces cerevisiae ATCC-26787.
Extraction methods of tryptophan synthase from cultured cells are known and described in The Journal of Biological Chemistry, Vol. 249, No. 24, pp. 7756-7763 (1974) for E. coli; ibid., Vol. 250, No. 8, pp. 2941-2946 (1975) for NeurosPora crassa; and European Journal of Biochemistry, Vol. 102, pp. 159-165 (1979) for Saccharom~ces cerevisiae;
respectively.
Tryptophan synthase used in the invention is not necessarily an extracted and purified enzyme. Cultures of a tryptophan synthase producing microorganism, live cells collected from the cultures by centrifugation or a similar method, dried cells thereof, and cell debris obtained by grinding, ultrasonicating or otherwise treating the cells or by autolysis thereof may be utilized as an enzyme source in the invention. Extracts from these cells and crude enzymes obtained from the extracts may also be utilized.
Further, immobilized cells or enzymes may of course be utilized in the invention.
Any synthetic or natural medium may be used for the culture of tryptophan synthase producing cells, provided that it contains a carbon source, a nitrogen source, minerals and, optionally, a small amount of minor nutrient(s).
Addition of a small amount of tryptophan or indole to the 2~ culture medium may 60metimes be effective. Also, the amount .
of tryptophan synthase produced may sometimes be increased by adding a small amount of indoleacrylic acid to the medium.
The culture is aerobically carried out by shaking or aeration agitating culture~ The temperature is in the range of 20 to 40~C, usually 25 to 37C. The pH of the culture medium is 5 to 8~
It is well known that tryptophan synthase is a multifunctional enzyme, that is, the enzyme cataly~es not only the synthesis of L-tryptophan from indole-3-glycero-phosphoric acid and L-serine but also many other reactions:
refer to, for example, Advances in Enzymology and Related Areas of Molecular Biology, Vol~ 49, pp. 127-185 (1979).
However, the present inventors have for the first time discovered that the reaction according to the invention may be catalyzed by tryptophan synthase.
~-Substituted L-alanines, one of the substrates for the reaction, which may be used in the invention include, for example, ~-halogeno-L-alanine, such as ~-chloro-L-alanine and ~-bromo-L-alanine; 0-alkyl-L-serines, such as 0-methyl-L-sexine and 0-ethyl-L-serine; S-alkyl-L-cysteines, such as S~methyl-L-cysteine and S-ethyl-L-cysteine; 0-acetyl-L-serine; 0-benzyl-L-serine; S~benzyl~L-cysteine; L_serine 0-sulfat~e; L_serine; and the like.
Sulfides, hydrosulfides or poly~ulfides, the other ~%~
substrate, which may be used in the invention include, for e~ample, 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 ~-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 suitably selected from the range of 20 to 60C. The reaction period of time is generally 1 to 100 hours for a batch reaction although it may vary depending on the titer f the enzyme the concentrations of the substrates used and other conditions. The reaction is carried out stationarily or under slow agitation.
The concentrations of the substrates, the ~-substituted L,alanine and the sulfide, hydrosulfide or polysulfide, are not particularly limited but usually in the range of about 0.1-30~ by weight. The substrates may be added wholly at one time when the reaction is started or, alternatively, they may be added partially as the reaction proceeds. Desirably, the sulfide, hydrosulfide or polysulfide i~ present in the reaction medium at an equimolar amount or .
`
. ,~ ., , - .
~Z~3~312 more based on the ~-substituted L,alanine. In the reaction, it is desirable to add a s~all amount of a coenzyme, pyridoxal phosphate, in addition to the substrates.
When cells or culture media of a microorganism capable of producing tryptophan synthase are utilized 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. The alcohols which may be used include ethanol, l-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, l-pentanol, l-octanol and the like. The esters which may be used include ethyl formate, propyl formate, butyl formate, ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate and the like. The ketones which may be used include acetone, methyl ethyl ketone, methyl isobutyl ketone and the like. The surfactants which may be used include anionic surfactants, such as sodium dodecyl sulfate and sodium deoxycholate, nonionic surfactants, such as octyl phenyl ethers, and other surfactants. The amount of these compounds added to the reaction medium is usually in the range of 0.01-5% by weight although it may vary with the type of the compounds or the strain used.
When the reaction is thus carried out, the reaction medium will generally contain a mixture of L-cysteine and L-cystine Rince L-cysteine produced is readily oxidized and _ 7 ~
converted to L-cystine~ The amount of L-cystine gradually increases as the reaction proceeds. However, the ratio of the concentration of L_cysteine to that of L-cystine can be varied by controlling the reaction conditions4 L-Cysteine or L-cystine may be recovered from the reaction medium in any conventional manner. For example, L,cystine9 which is difficultly soluble in water, can readily be isolated by aerating the reaction medium to oxidize a major proportion of I~cysteine to L-cystine after the completion of the reaction~ L-cysteine may be obtained by subjecting the thus obtained L-cystine to electrolytic reduction.
Quantitative measurement of L-cysteine and L-cystine was carried out by liquid chromatography. Further, liquid chromatography using a column for separating optical isomers showed that both the cysteine and cystine produced had the L-configuration.
Description of the Preferred Embodiments The invention will hereinbelow be illustrated by the following examples.
Example l Escherichia coli MT-10242 ~FERM BP-20) was inoculated in a liquid ~edium o~ 1% meat extract, 0.5%
12~
peptone, 0.1~ yeast extract and 0.2% KH2P04, pH 7.0, and cultured with shaking at 30nC for 20 hoursO .4fter the culture, cells were collected by centrifugation and the enzyme was purified according to the method of 0. Adachi et al., The Journal of Biological Chemistry, Vol. 249, No. 24, pp. 7756-7763 (1974). The thus obtained enzyme tryptophan synthase having a specific activity (titer) of 9.2 units per mg was used in the following reaction. The enzyme activity was determined by the method of C Yanofsky et al., lethods in Enzymology, VolO 15, pp. 801-807 (1962).
One unit is represented by the amount OI the enzyme producing 1 ~umol/min of tryptophan from l,serine and indole at 370C, pH 7.8.
Tryptophan synthase, 0.5 mg, was added to 10 ml f a reaction medium, pH 8.5, containing 50 mM L-serine, 100 mM of a sulfide, hydrosulfide or polysulfide indicated in Table 1 and 0.1 mM pyridoxal phosphate, and shaked slowly at 35C for 10 hours. The concentrations of L-cysteine and L-cystine produced and the yields of the total thereof based on L~serine are shown in Table 1.
.
~2~ 7 Table 1 Kind of Sulfides etc. I,-cysteine L-cystine Yield (mM) (mM) (mole %) 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 According to the method of W. H. Matchett et al., The Journal of Biological Chemistry, Vol. 250, No. 8, pp. 2941-2946 (1975), Neurospora crassa ATCC-14692 was cultured and the enzyme was purified. The thus obtained enzyme tryptophan synthase had a specific activity of 1.3 units per mg.
The enzyme liquid 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 pyridoxal phosphate and 1.3 units tryptophan synthase was gently shaked at 35C ~or 10 hours. There were produced in the reaction medium 12 mM L-cysteine and 4 mM L_cystine.
.
~2~ 27 _ 10 --Example 3 Saccharorn~ces cerev_s_ae ATCC-26787 was inoculated in a liquid medium of 1% peptone, 0.5% yeast extract, 2% glucose and 0.01~ indoleacrylic acid, pH 6.0, and cultured with shaking at 30~C for 20 hours. After the culture cells were collected by centrifugation. The enzyme was purified according to the method of M. Dettwiler et al., European Journal of Biochemistry, Vol. 102, pp. 159-165 tl979). Thus, tryptophan synthase with a specific activitY
of 1.2 units per mg was obtained.
The enzyme liquid was used to effect the following reaction. A reaction liquid (10 ml) containing 50 m~"l L-serine, 100 mM sodium hydrosulfide, 0.1 mr~ pyridoxal phosphate and 1.2 units tryptophan synthase, pH 8.5, was gently shaked at 35C for 10 hours. The resultant reaction liquid contained 11 mM L-cysteine and 3 mM L,cystine produced.
Example 4 Escherichia coli MT-10242 (FERM BP-20) was inoculated in a liquid medium of pH 7.0 containing 1% meat extract, 0.5% peptone, 0.1% yeast extract and 0.2% KH2P04 and 6haked at 30C for 20 hours. After the culture, cells ~ere collected by centrifugation and used as an enzyme source of tryptophan synthaæe. The wet cells had a 6pecific activity of 120 units per g.
~2~t91;~7 A reaction medium (100 ml) containing 200 mM
L-serine, 300 mM-sodium sulfide, 0.1 mM pyridoxal phosphate and 5 g of the wet cells a~d adjusted by HCl to pH Of 8.5 was shaked at 35~C for 24 hours. After the reaction was completed, L-cystine present in the reaction medium was reduced to L-cysteine by dithiothreitolO The amount of L-cysteine produced was 98 mM.
Example 5 Tryptophan synthase obtained in Example 1 was used in the following reaction.
The tryptophan synthase (500 units) was added to 100 ml of a reaction liquid containing 300 m~l of a ~-substituted L-alanine indicated in Table 2, 600 mM sodium hydrosulfide, 1 mM pyridoxal phosphate and 480 mM tris-aminomethane-HCl buffer, pH 8.5, and gently shaked at ~5~C
for 1 hour. After the reaction was completed, L-cystine present in the reaction liquid was reduced to L-cysteine by dithiothreitol and then the amount of L-cysteine was measured.
The results are shown in Table 2.
12~
Table 2 ~-Substituted L-alanineL-cysteine (mM) ~-Chloro-L_alanine 82 0-Methyl-L-serine 116 0-Acetyl-L-serine 95 0-Benzyl-L-serine 24 S-Methyl-L-cy~teine 27 S-Ethyl L-cysteine 18 S-Benzyl-L-cysteine 5 L-Serine 0-sulfate 37 L-Serine 123 Example 6 Escherichia coli ~T-10232 (FERM BP-19) was inoculated in a liquid medium of pH 7.0 containing 1~ met extract, 0O5% peptone, 0.1% yeast extract and 0.2% KH2P04 and shaked to culture at 30~C for 20 hours. After the culture, cells were collected by centrifugation and freezed at -20 7C to store as an enzyme source of tryptophan synthase. The wet cells had a tryptophan synthase activity of 89 units per g- The stored cells were used in the following reaction.
A reaction medium (100 ml) containing 200 mM
L_serine~ 300 mM sodium sulfide, 0.1 mM pyridoxal phosphate, 100 mM trisaminomethane-HCl buffer (pH 8.5) and 5 g of the wet cells was gently ~haked at 35C for 10 hours. After lZ~
the reaction was completed, L,cystine present in the reaction medium was reduced to L,cysteine by dithiothreitol and the amount of L-cysteine produced was then measured. The amount was 114 mM.
Example 7 The wet cells obtained in Example 6 were used in the following reaction.
A reaction liquid (100 ml) of pH 8.0 containing 200 mM (2.1 g) L_serine, 1000 mM sodium hydrosulfide, 0.1 mM pyridoxal phosphate and 5 g of the wet cells was gently stirred at 35~C for 10 hours. L-serine was fur~her added at the time of 2 hours and 5 hours after the reaction was started,each in an amount of 2.1 g. During the reaction, the pH of the reaction liquid was maintained at 8.0 by adding 6N phosphoric acid. When the reaction was completed, 4.3 g of L_cysteine and 1.lg of L,cystine were accumulated in the reaction liquid.
Example 8 The wet cells obtained in Example 4 were used directly as an enzyme source of tryptophan synthase in the ~ollowing reaction.
A reaction medium tlOO ml, pH 8.5) containing 200 mM L-serine, 300 mM sodium sulfide, 1 mM pyrido~al ~2~31Z7 phosphate, 5 g of the wet cells and a compound indicated in Table 3 was gently shaked at 35C for 5 hours. The pH of the medium was controlled to 8.5 + 003 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 produced was then measured.
The results are shown in Table 3.
12~91~
Table 3 Compound added (Concentration g/l) Amount of L-cysteine (mM) Gontrol (O) 67.2 Ethanol (10) 99.6 l-Propanol (10) 119.4 2-Propanol (10) 105.6 l-Butanol (10) 140.0 2-Butanol (10) 128.7 Isobutyl alcohol (5) 11408 Tert-butyl alcohol (5) 107.2 l-Pentanol (5) 106.5 l-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) ~11.8 Methyl isobutyl ketone(5) 167.0 Sodium deogycholate (1) 139.4 Sodium dodecyl sulfate(1) 160.9 Triton X-100 (1) 160.7 ~ de /~ar~
Claims (4)
1. A process for preparing L-cysteine or a mixture of L-cysteine and L-cystine comprising reacting a .beta.-substituted L-alanine represented by the general formula (I):
(I) wherein X denotes a halogen atom or an -OR or -SR group where R is a hydrogen atom or an alkyl, acetyl, benzyl or sulfonic acid group, with a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, ammonium hydrosulfide or an ammonium polysulfide in the presence of tryptophan synthase without adding dimethyl sulfoxide.
(I) wherein X denotes a halogen atom or an -OR or -SR group where R is a hydrogen atom or an alkyl, acetyl, benzyl or sulfonic acid group, with a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, ammonium hydrosulfide or an ammonium polysulfide in the presence of tryptophan synthase without adding dimethyl sulfoxide.
2. A process for preparing L-cysteine or a mixture of L-cysteine and L-cystine according to claim 1, wherein the reaction is carried out in the presence of at least one of 0.01 to 5% by weight selected from the group consisting of alcohols, esters, ketones, and surfactants.
3. A process for preparing L-cystine, comprising oxidizing the L-cysteine or the L-cysteine of the mixture of L-cysteine and L-cystine prepared by the reaction as set forth in claim 1, after the reaction.
4. A process for preparing L-cysteine, comprising reducing the L-cystine of the mixture of L-cysteine and L-cystine prepared by the reaction as set forth in claim 1, after the reaction.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60084545A JPS61242589A (en) | 1985-04-22 | 1985-04-22 | Production of l-sulfur-containing amino acid |
| JP60-84545 | 1985-04-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1299127C true CA1299127C (en) | 1992-04-21 |
Family
ID=13833616
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000506867A Expired - Lifetime CA1299127C (en) | 1985-04-22 | 1986-04-16 | Enzymatic process for preparing sulfur containing l-amino acids |
Country Status (10)
| Country | Link |
|---|---|
| JP (1) | JPS61242589A (en) |
| KR (1) | KR890004021B1 (en) |
| AU (1) | AU562772B2 (en) |
| CA (1) | CA1299127C (en) |
| CH (1) | CH666695A5 (en) |
| DE (1) | DE3613388A1 (en) |
| FR (1) | FR2580667B1 (en) |
| GB (1) | GB2174390B (en) |
| IT (1) | IT1190275B (en) |
| NL (1) | NL8600984A (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0660157B2 (en) * | 1985-11-20 | 1994-08-10 | 三井東圧化学株式会社 | Method for producing cystine from cysteine |
| JPH0623182B2 (en) * | 1986-06-19 | 1994-03-30 | 三井東圧化学株式会社 | Method for separating L-cysteine hydrochloride monohydrate |
| CA1299128C (en) * | 1986-11-19 | 1992-04-21 | Tooru Miyahara | Method of producing l-cystine |
| US5756319A (en) * | 1995-07-18 | 1998-05-26 | Mitsui Toatsu Chemicals, Inc. | Production process of S-phenyl-L-cysteine |
| US6579705B2 (en) * | 2001-04-04 | 2003-06-17 | Consortium Fur Elektrochemische Industrie Gmbh | Process for preparing non-proteinogenic L-amino acids |
| BRPI0407600A (en) | 2003-02-18 | 2006-02-14 | Metabolic Explorer Sa | processes for the preparation of evolved microorganisms and an evolved protein and biotransformation, an evolved gene, an evolved protein, and the use of an evolved microorganism or an evolved protein |
| FR2854902B1 (en) * | 2003-05-14 | 2006-06-09 | Metabolic Explorer Sa | MICROORGANISM WITH MODIFIED CYSTEINE SYNTHASE ACTIVITY AND PROCESS FOR PREPARING CYSTEINE |
| EP1604656A1 (en) | 2004-06-09 | 2005-12-14 | Schwarz Pharma Ag | Novel use of peptide compounds for treating amyotrophic lateral sclerosis (ALS) |
| KR101959061B1 (en) | 2017-03-22 | 2019-03-18 | 대상 주식회사 | Method for Preparing L-Cysteine hydrochloride |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1443280A1 (en) * | 1961-05-31 | 1969-10-23 | Von Holt Dr Med Claus | Process for the preparation of 35S-L-cysteine hydrochloride |
| IT1012509B (en) * | 1971-05-26 | 1977-03-10 | Snam Progetti | PROCEDURE FOR THE PREPARATION OF ORGANIC COMPOUNDS CONTAINING MARKED ATOMS |
| US3974031A (en) * | 1974-05-09 | 1976-08-10 | Mitsubishi Chemical Industries Ltd. | Process for producing L-cysteine or its derivatives |
| JPS5838154B2 (en) * | 1976-05-10 | 1983-08-20 | 三菱化学株式会社 | Method for producing L-cysteines |
| JPS58146287A (en) * | 1982-02-25 | 1983-08-31 | Mitsui Toatsu Chem Inc | Preparation of l-cysteine derivative |
| JPS58187198A (en) * | 1982-04-27 | 1983-11-01 | Showa Denko Kk | Preparation of s-carboxymethyl-l-cysteine |
-
1985
- 1985-04-22 JP JP60084545A patent/JPS61242589A/en active Granted
-
1986
- 1986-04-14 GB GB08609015A patent/GB2174390B/en not_active Expired
- 1986-04-16 CA CA000506867A patent/CA1299127C/en not_active Expired - Lifetime
- 1986-04-17 AU AU56348/86A patent/AU562772B2/en not_active Ceased
- 1986-04-18 NL NL8600984A patent/NL8600984A/en not_active Application Discontinuation
- 1986-04-21 IT IT47912/86A patent/IT1190275B/en active
- 1986-04-21 DE DE19863613388 patent/DE3613388A1/en active Granted
- 1986-04-22 KR KR1019860003103A patent/KR890004021B1/en not_active Expired
- 1986-04-22 CH CH1632/86A patent/CH666695A5/en not_active IP Right Cessation
- 1986-04-22 FR FR868605758A patent/FR2580667B1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| IT8647912A0 (en) | 1986-04-21 |
| IT1190275B (en) | 1988-02-16 |
| KR890004021B1 (en) | 1989-10-16 |
| FR2580667A1 (en) | 1986-10-24 |
| GB2174390A (en) | 1986-11-05 |
| NL8600984A (en) | 1986-11-17 |
| GB2174390B (en) | 1988-10-12 |
| GB8609015D0 (en) | 1986-05-21 |
| CH666695A5 (en) | 1988-08-15 |
| DE3613388C2 (en) | 1987-07-23 |
| DE3613388A1 (en) | 1986-10-30 |
| AU562772B2 (en) | 1987-06-18 |
| FR2580667B1 (en) | 1989-11-03 |
| KR860008273A (en) | 1986-11-14 |
| AU5634886A (en) | 1986-10-30 |
| JPH0527389B2 (en) | 1993-04-21 |
| JPS61242589A (en) | 1986-10-28 |
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