CA1291159C - Method for production of cystine from cysteine - Google Patents
Method for production of cystine from cysteineInfo
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- CA1291159C CA1291159C CA000536982A CA536982A CA1291159C CA 1291159 C CA1291159 C CA 1291159C CA 000536982 A CA000536982 A CA 000536982A CA 536982 A CA536982 A CA 536982A CA 1291159 C CA1291159 C CA 1291159C
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- cysteine
- cystine
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- oxidation
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Abstract
Abstract This invention relates to a method for the production of cystine from cysteine. Commercially viable prior art methods result in low yields unless a catalyst is used. In this invention, any aqueous solution containing cysteine, obtained by the enzymatic method or fermentation method, is oxidised in the presence of dimethyl sulfoxide to produce cystine in high yields.
Description
~9~59 This invention relates to a method for the produc-tion of cystine by the oxidation of cysteine obtained by an enzymatic method or a fermentation method.
L-cysteine and L-cystine are elementary S-containing amino acids which have found utility as medicines, raw materials for medicines, food additives, and additives for cosmetics. Particularly in recent years, the demand for these compounds as a raw material for cold permanent wave liquids~has grown steadily.
' In the isolation of cysteine from a cysteine-containing reaction solution obtained by a fermentation method, an enæymatic method, a synthetic method, or any other method (herèinafter~ the term "reaction solution"
shall mean the solution which exists after completion of the reaction of the enzymatic, fermentation, or synthetic mèthod),~ the composition of the reaction solution is compIex and the solubility of cysteine in water is very high. Therefore various methods are used to effect isolation; and~; purification of cysteine by preliminarily causing;~ he ~cysteine ~to;react ~with a strong acid such as hydrochloric~ acid~or p-toluene sulfonic acid thereby formlng a~corresponding~ salt.
:
: ~
L-cysteine and L-cystine are elementary S-containing amino acids which have found utility as medicines, raw materials for medicines, food additives, and additives for cosmetics. Particularly in recent years, the demand for these compounds as a raw material for cold permanent wave liquids~has grown steadily.
' In the isolation of cysteine from a cysteine-containing reaction solution obtained by a fermentation method, an enæymatic method, a synthetic method, or any other method (herèinafter~ the term "reaction solution"
shall mean the solution which exists after completion of the reaction of the enzymatic, fermentation, or synthetic mèthod),~ the composition of the reaction solution is compIex and the solubility of cysteine in water is very high. Therefore various methods are used to effect isolation; and~; purification of cysteine by preliminarily causing;~ he ~cysteine ~to;react ~with a strong acid such as hydrochloric~ acid~or p-toluene sulfonic acid thereby formlng a~corresponding~ salt.
:
: ~
- 2 ~
These methods generally are complex in procedure and produce cysteine in very low yields. It i5 also dif-ficult to remove the cysteine in a highly pure form from the reaction solution of the complex composition obtained from the fermentation method. Particularly, isolation and purification of cysteine from a reaction solution obtained by the fermentation or enzymatic method results in heavy losses because the reaction solution contains impurities originating in microorganisms.
Since cysteine is relatively susceptible to oxidation, a method which generally comprises forcibly oxidizing the cysteine in the reaction solution into cystine, separating the cystine in a purified form, and then electrolytically reducing the separated cystine to obtain pure cysteine has been proposed.
Several methods have been disclosed which effect oxidation of the cysteine in the cysteine fermentation broth. The method disclosed in Japanese Patent Publication SHO 57(1982~-7,634 accomplishes the oxidation using air or a peroxide, such as H2O2, with the pH value of the reaction mixture kept in the range of 5 to 10.
Experiment indicates ~that in the method using H22 or air oxidation, the reaction proceeds in the form of radical oxidation and results in drawbacks such as diffi-culty of control of the reaction conditions and occurrenceof decomposition products in a fairly larye pro~ortion.
When the reaction solution is a fermentation broth which contains, in the cysteine reaction system, a metal-lic ion such as Fe~+ or Mn~, the metallic ion catalyzes the radical reaction in the method described above, and cystine is obtained in a relatively high yield. The yield iB poor, howeve~, where the reaction solution contains . , :
virtually no metallic ion. However, if the added metallic ion such as Fe++ or Mn++ is mixed into the separated cystine, it has the possibility of producing adverse effects on the electrolytic reduction of cystine.
For removal of used microorganic cells or used enzyme from the cysteine reaction solution obtained by the fermentation method or enzymatic method, an effective method comprises adjusting the pH value of the reaction solution to a level of not more than 4 by the addition of HCl or H2SO4, adding activated carbon thereto, thermally treating the resultant solution to cause the used enzyme or used microorganic cells to be flocculated and adsorbed -on the activated carbon, and subjecting the resultant com-posite to solid-liquid separation. However, the resulting solution is not suited to the oxidation of cysteine using H2O2 or air, since this requires keeping the reaction solution at a pH of not less than 5 as specifically pointed out in the aforementioned specification.
Furthermore, experiment has confirmed that oxi-dation with air effected under s~rongly acidic conditionsin the neighbourhood of pH 1 produces substantially no cystine.
Among other known methods for the oxidation of cysteine are: the method resorting to oxidation with methyl xanthide as described in U.S. Patent 4,039,586;
and, ~he~method relying on~irradiation with y ray as described~in~Chemical Communicationj 1968, 826-827. These me~thods, however, are not commercially feasible.
:
The~inventors made a diligent study in search of a solution for the problema described above.
:
:
.
To be specific, this invention relates to a method for the production of cystine characterized by the steps of oxidizing cysteine in the cysteine-containing a~ueous solution, obtained by the enzymatic method or fermentation method, in the presence of dimethyl sulfoxide (this serves as an oxidizing agent) to convert cysteine into cystine, and separating out the resulting cystine.
The reaction solution subjected to the oxidation need only be a cysteine-containing reaction solution obtained by the fermentation method or enzymatic method. For example, the reaction solution obtained by culturing an L-cysteine-producing microorganism such as oE genus Pseudomonas using 2-amino-thiazolin-4-carboxylic acid (ATC) as disclosed in the aforementioned specification of Japanese Patent Publication SH0 57(1982)-7,634 (published February 12, 1982) can be used.
Desirably, this invention is applied to the reaction solution which has a relatively high inorganic salt content and which originates in a process for the production of cysteine.
The inventors have formerly developed and applied for a patent relating to a method for obtaining L-cysteine by using L-serine as a starting material and sub~ecting it to an enzymatic reaction using a sulfur-containing compound such as Na,S, NaHS, or H2S which functions as a sulfyhdryl group-introducing agent in the presence of tryptophan synthase.
The reaction solution obtained by this enzymatic reaction contains such inorganic salts as NaC1 and (NH4)aS04.
When the method is applied to the enzymatic or fermentation reaction solution, the reaction velocity in the oxidation-step, in the presence of dimethyl sulfoxide (hereinafter referred to briefly as 'IDMSO''), tends to be increased by the catalytlc action of the salt present in ` 1~;2 ~' . ~ :
.
~.:
:
.
- 5 - ~ 59 the reaction solution presumably because the oxidation is an ionic reaction. Therefore, this invention relates to a method of oxidizing cysteine in a reaction solution obtained by the enzymatic method (hereinafter referred, inclusively of the fermentation method, to as "enzymatic method") into cystine. Since the reaction proceeds under mild conditions, it is not affected by the composition of the reaction solution.
The reaction solution, therefore, may contain an insignificant amount of the cystine formed in the course of the cysteine production and may even contain the used enzyme or used microorganic cells originating in the culture. For example, the cysteine contained in a reaction solution freshly resulting from an enzymatic or fermentation reaction, and which still contains the used enzyme or used microorganic cells, may be oxidized into cystine by the addition of DMSO to the solution. Used enæyme or used microorganic cells then may be removed from the solution to isolate the produced cystine.
As already described above, however, the removal of the used microorganic cells from the reaction solution in the present invention can be carrièd out most effec-tively by a method which comprises thermally treating the reaction solution under an acidic condition of not more than pH 4, preferably in the neighbourhood of pH 1 in the presence of an adsorbent thereby flocculating the used microorganic cells, causing the floccules to be adsorbed on the activated carbon, and eluting the adsorbate. DMSO
is added to the acidic a~ueous solution containing cysteine,~ which resulted from removal of the used micro-organic cells from the reaction solution as described above, as an oxidative reagent to convert the cysteine into cystine. In the oxidation reaction of cysteine into cystine, the reaction velocity increases in proportion as ~ . .
- 6 - ~ 9 the pH value of the solution decreases and the yield of cystine increases in proportion as the acidity of the solution increases.
The amount of DMS~ to be used is sufficient on the level of about 0.2 to 2.0 mols, preferably 0.5 to 1.0 mol, per mol of the cysteine contained in the reaction solution.
Though the temperature at which the reaction of this invention is to be carried out is not rigidly de-fined, it is desired to fall in the range of 5C to 100C.
This invention does not require forced supply of oxygen to the reaction system. The reaction can be carried out in any conventional hermetically sealable reaction vessel, and is possible even in an atmosphere of N2 gas.
Oxidation is effected by the use of DMSO, and thus the present invention enjoys the following advantages.
(1) In the oxidation with air or with H2O2, no proper reaction velocity can be obtained unless a metal such as Fe++, Mn++, or Cu++ is added as a catalyst to the reaction system. The method of the present invention does not require a catalyst.
Also, as oxidation with air or with H22 is a radical reaction, it does not terminate with the occu~rrence of cystine but proceeds to the occurrence of decomposition products such as cysteic acid. As a result, ~the recovery of cystine inevitably entails heavy loss and the oxidation reaction is not easily controlled. With oxidation carried out in the presence of DMSO as contem-plated in th~e present invention, the oxidation is an ionic .
LS~
reaction and thus it proceeds moderately and producescystine substantially quantitatively from cysteine.
Furthermore, when the reaction is carried out in the pre-sence of an electrolytic inoryanic salt, the reaction ve-locity of the oxidation is increased. The effect of thiselectrolytic inorganic salt is particularly prominent when the method is applied to the L-cysteine reaction solution which is obtained by using L-serine as a substrate and effecting the enæymatic reaction of a sulfur-containing compound in the presence of tryptophane synthase.
(2) Preferably, the aqueous cysteine solution to be subjected to the oxidation of this invention has as low a pH value as possible. Where the aqueous cysteine solution resulting from the removal of the used micro-organic cells, the removal being carried out usingactivated carbon under an acidic condition (pH 4 or less), is subjected to the oxidation reaction, the aqueous solution can be put to use directly in the oxidation reaction without requiring a treatment for pH adjustment.
As compared with the conventional method which resorts to oxidation with air or with H2O2, the method of the present invention which relies on the oxidation with DMSO has noticeable advantages such as lowered ratio of decomposition of cysteine into cysteic acid, particularly under a strongly acidic condition. Data supporting this fact are shown in the following table.
In the test oxidation, an aqueous 10~ L-cysteine solution was used as a model solution. DMSO and H22 were each used in an amount of 0.75 mol per mol of L-cysteine.
In the case of the oxidation with air, air was passed through the solution at a rate of three times the volume of the solution per minute. In all the tests, the oxidation was carried out at room temperature for eight ,, ~ -- 8 - ~9~59 hours. In the table, the first of each pair of figures represents the conversions (oxidation ratios) of cysteine and the second the formation ratios of cystine (selectivities to cystine).
I DMSO ¦ Air ¦ H22 . ._ , _ pH198/96 5/4 99/68 pH787/85 85/79 99/76 1 0 . . _ .
Note) In the tests using air and H2O2, FeCl2 was added in a minute amount to permit the presence of Fe++ ion in the reaction system.
Now, the present invention will be described more specifically below with reference to working examples. In these working examples, cysteine and cystine were analyzed as follows. The analysis of cysteine was performed by the known Gaitonde method.
, About 0.5 g of a given solution was taken as a sample, weighed accurately, and diluted with 2N HCl to 10 to 20 times the original volume. The diluted solution was further diluted~with distilled water to about 100 times.
The solution finally diluted ~to 1,000 to 2,000 times the original volume was coloured with an acidic ninhydrin reagent ~and tested~ for absorbancy at 560 nm with an absorptiometer. Separately, standard samples of known concentrations were prepared; in advance to obtain a calibration~curve of the absorbancy at 560 nm. Then the cysteine ; concentration of the given~ solution was calc~ulated~from the callbration curve.
- ~ , As ~regards the cystine concentration, a given solu~ion~diluted to ~1~,000 to 2,000 times the original volyme as~described; above~ was mixed with a roughly equal :
:
- 9 ~
volume of a 5-uM 1,4-dithiothreitol (reducing agent), adjusted to pH 8.0 to 8.5 by the addition of 2N NaO~, and left standing at room temperature for one hour for thorough reducing of cystine into cys-teine to permit calculation of the cysteine concentration by the method described above. The cystine concentration was found by deducting the cysteine concentration before the reduction from the concentration so calculated.
Example 1:
In a separable flask having an inner volume of 300 ml and fitted with a stirrer, 91 g of an aqueous L-serine solution containing 22.0 wt% of L-serine (0~19 mol as L-serine), 28.1 g of sodium hydrosulfide dihydrate (NaSH 2H2O) (0.38 mol), and 10 g of a culture broth of tryptophan synthase originating from Escherichia coli MT-10242 (FERM BP-20) and still containing the used cells (dry content 2.2 g), were combined, adjusted to pH 7.5 by the addition of an aqueous 5% sodium hydroxide solution, and diluted with water to a total volume of 200 ml. The resultant mixture was placed in a constant temperature bath at 35C and left reacting therein for 24 hours.
After completion of the reaction, the reaction mixture was adjusted to pH 1.0 with hydrochloric acid, then combined with 5 g of activated carbon (produced by Takeda Chemical Industries, Ltd. and marketed under trademark designation of "Tokusei Shirasagi"), and heated at 80C for 30 minutes. After this heat treatment, the reaction mixture kept at 80C was hot filtered through a Buchner ~funnel to remove used microorganic cells and obtain 188 g of a réaction filtrate in a treated form.
This reaction filtrate was found by analysis to contain 20.1 g (0.166 mol) of L-cysteine and 2.0 g (0.008 mol) of L-cystine bo~h reduced as L-cysteine. The conversion of .
.. . . ~ . ,. . .= . .. .
- 1 o - ~ i9 L-serine to L-cysteine and L-cystine was found by calcu-latlon to be 95.8%.
The reaction solution thus obtained by the heat treatment was transferred into a separable flask having an inner volume of 300 ml and fitted with a stirrer and stirred therein with 9.7 9 of DMSO at room temperature for eight hours to obtain 196.2 g of a solution having L-cystine crystals precipitated therein (the solution had still pH 1). In this solution, 0.2 g of L-cysteine remained.
The solution resulting from the oxidation was neutralized to about pH 5 by the addition of an aqueous 5%
sodium hydroxide solution to induce precipitation of crystals. The crystals were separated by filtration and dried to obtain 19.89 (0.165 mol as 100%) of white powdery crystals. These crystals were found to possess a specific rotation~[a]D = -218 (C = 2, 2N HCl), an Fe content of not more than 10 ppm, and a purity of not less than 98.5~. As regards the ratio of recovery, 95.0% of all the L-cysteine and L-cystine present in the reaction solution were recovered. The ratio of decomposition was not more than 2%.
Comparative Experiment:
The` same reaction solution obtained by following the procedure of Example 1 was heat treated and then neutralized with an aqueous 5~ sodium hydroxide solution to about pH 5. The reaction solution resulting from this heat treatment was transferred into the same separable flask having an inner volume of 300 ml as used in Example 1, mixed therein with 0.2 g of FeCl2 and blown with air introduced~at a flow rate of about 200 ml/min. at room ~g~s~
temperature, to produce 200.8 g of a solution having L-crystine crystals precipitated therein. In the solution, 4.2 g of L-cysteine remained.
By separating the crystals from the reaction solu-tion through filtration and drying the separated crystals,there was obtained 14.8 g of white powdery crystals. The crystals were found to possess a specific rotation, [a]D = -215 (C = 2, 2N HCl), a Fe content of 30 ppm, and a purity of not less than 98O5%~ As regards the ratio of recovery, 70.3% of all L-cysteine and L-cystine present in the reaction solu~ion were recovered. The ratio of decomposition was 9.9%.
Example 2:
In a separable flask having an inner volume of 300 ml and fitted with a stirrer, 91 g of an aqueous L-serine solution containing 22.0 wt% of L-serine (0.19 mol of L-serine), 28.1 g (0.38 mol) of sodium hydrosulfide dihydrate (NaSH-2H20), and 10 g of a culture broth of tryptophan synthase originating from Escherlchia coli MT-10242 (FERM BP-20) and still containing the used cells (dry content 2.2 g), were combined, adjusted to pH 7.5 by the addition of an aqueous 5~ sodium hydroxide solution and then diluted with water to a total volume of 200 ml.
The diluted solution was placed in a constant temperature bath at 35C and left reacting therein for 24 hours.
The reaction solution obtained after completion of the reaction was found by analysis to contain 18.5 g (0.1~53 mol) o~ L-cysteine and 3.2 g (0.013 mol) of L-cystin~ both reduced as L-cysteine. The conversion of L-seeine to L-cysteine and L-cystine was calculated to be 94.2%.
.
91~L5~3 The reaction solution consequently obtained was adjusted to pH 0.5 by the addition of abaut 30 ml of concentrated hydrochloric acid and combined with 0.97 g of DMSO and stirred at 35C for eight hours for oxidation.
At this stage, the oxidized solution contained 0.7 g (0.006 mol) of L-cysteine and 20.7 g (0.086 mol) of L-cystine. The conversion of L-serine to L-cystine was calculated to be 90.6~.
The oxidized solution and 5 g of activated carbon (product of Takeda Chemical Industries, Ltd.) added thereto were heated at 80C for 30 minutes. m e resultant reaction mixture kept at 80C was hot filtered through a Buchner funnel to remove used microorganic cells and obtain 2.30 g of a homogeneous filtrate (having still pH
0.5) in a treated form. This filtrate was found by analysis to contain 0 3 g (0.002 mol) of L-cysteine and 20.8 g (0.087 mol) of L-cystine. The conversion from L-serine to L-cystine was found to be 91.3%.
Tbe solution resulting from the separation of the used mircoorganic cells was neutralized to pH 2 by dropwise addition of about 5 ml of 30% NaOH solution to induce precipitation of L-cystine crystals. The crystals were separated by vacuum filtration with a Buchner funnel~
washed thoroughly with 50 ml of water, and dried to afford 20.2 g (0.084 mol as 100%)- of white powdery cFystals.
These crystals were found to posse9s a specific rotation, [a]D = -218.6 (C = 2, 2N HCl), a Fe contént of not more than 10 ppm, and purity of not le86 than 98.5%~
As regards the ratio of recovery, nearly 100% of all L-cysteine and ~L-cystine present in the reaction solution were recovered.
These methods generally are complex in procedure and produce cysteine in very low yields. It i5 also dif-ficult to remove the cysteine in a highly pure form from the reaction solution of the complex composition obtained from the fermentation method. Particularly, isolation and purification of cysteine from a reaction solution obtained by the fermentation or enzymatic method results in heavy losses because the reaction solution contains impurities originating in microorganisms.
Since cysteine is relatively susceptible to oxidation, a method which generally comprises forcibly oxidizing the cysteine in the reaction solution into cystine, separating the cystine in a purified form, and then electrolytically reducing the separated cystine to obtain pure cysteine has been proposed.
Several methods have been disclosed which effect oxidation of the cysteine in the cysteine fermentation broth. The method disclosed in Japanese Patent Publication SHO 57(1982~-7,634 accomplishes the oxidation using air or a peroxide, such as H2O2, with the pH value of the reaction mixture kept in the range of 5 to 10.
Experiment indicates ~that in the method using H22 or air oxidation, the reaction proceeds in the form of radical oxidation and results in drawbacks such as diffi-culty of control of the reaction conditions and occurrenceof decomposition products in a fairly larye pro~ortion.
When the reaction solution is a fermentation broth which contains, in the cysteine reaction system, a metal-lic ion such as Fe~+ or Mn~, the metallic ion catalyzes the radical reaction in the method described above, and cystine is obtained in a relatively high yield. The yield iB poor, howeve~, where the reaction solution contains . , :
virtually no metallic ion. However, if the added metallic ion such as Fe++ or Mn++ is mixed into the separated cystine, it has the possibility of producing adverse effects on the electrolytic reduction of cystine.
For removal of used microorganic cells or used enzyme from the cysteine reaction solution obtained by the fermentation method or enzymatic method, an effective method comprises adjusting the pH value of the reaction solution to a level of not more than 4 by the addition of HCl or H2SO4, adding activated carbon thereto, thermally treating the resultant solution to cause the used enzyme or used microorganic cells to be flocculated and adsorbed -on the activated carbon, and subjecting the resultant com-posite to solid-liquid separation. However, the resulting solution is not suited to the oxidation of cysteine using H2O2 or air, since this requires keeping the reaction solution at a pH of not less than 5 as specifically pointed out in the aforementioned specification.
Furthermore, experiment has confirmed that oxi-dation with air effected under s~rongly acidic conditionsin the neighbourhood of pH 1 produces substantially no cystine.
Among other known methods for the oxidation of cysteine are: the method resorting to oxidation with methyl xanthide as described in U.S. Patent 4,039,586;
and, ~he~method relying on~irradiation with y ray as described~in~Chemical Communicationj 1968, 826-827. These me~thods, however, are not commercially feasible.
:
The~inventors made a diligent study in search of a solution for the problema described above.
:
:
.
To be specific, this invention relates to a method for the production of cystine characterized by the steps of oxidizing cysteine in the cysteine-containing a~ueous solution, obtained by the enzymatic method or fermentation method, in the presence of dimethyl sulfoxide (this serves as an oxidizing agent) to convert cysteine into cystine, and separating out the resulting cystine.
The reaction solution subjected to the oxidation need only be a cysteine-containing reaction solution obtained by the fermentation method or enzymatic method. For example, the reaction solution obtained by culturing an L-cysteine-producing microorganism such as oE genus Pseudomonas using 2-amino-thiazolin-4-carboxylic acid (ATC) as disclosed in the aforementioned specification of Japanese Patent Publication SH0 57(1982)-7,634 (published February 12, 1982) can be used.
Desirably, this invention is applied to the reaction solution which has a relatively high inorganic salt content and which originates in a process for the production of cysteine.
The inventors have formerly developed and applied for a patent relating to a method for obtaining L-cysteine by using L-serine as a starting material and sub~ecting it to an enzymatic reaction using a sulfur-containing compound such as Na,S, NaHS, or H2S which functions as a sulfyhdryl group-introducing agent in the presence of tryptophan synthase.
The reaction solution obtained by this enzymatic reaction contains such inorganic salts as NaC1 and (NH4)aS04.
When the method is applied to the enzymatic or fermentation reaction solution, the reaction velocity in the oxidation-step, in the presence of dimethyl sulfoxide (hereinafter referred to briefly as 'IDMSO''), tends to be increased by the catalytlc action of the salt present in ` 1~;2 ~' . ~ :
.
~.:
:
.
- 5 - ~ 59 the reaction solution presumably because the oxidation is an ionic reaction. Therefore, this invention relates to a method of oxidizing cysteine in a reaction solution obtained by the enzymatic method (hereinafter referred, inclusively of the fermentation method, to as "enzymatic method") into cystine. Since the reaction proceeds under mild conditions, it is not affected by the composition of the reaction solution.
The reaction solution, therefore, may contain an insignificant amount of the cystine formed in the course of the cysteine production and may even contain the used enzyme or used microorganic cells originating in the culture. For example, the cysteine contained in a reaction solution freshly resulting from an enzymatic or fermentation reaction, and which still contains the used enzyme or used microorganic cells, may be oxidized into cystine by the addition of DMSO to the solution. Used enæyme or used microorganic cells then may be removed from the solution to isolate the produced cystine.
As already described above, however, the removal of the used microorganic cells from the reaction solution in the present invention can be carrièd out most effec-tively by a method which comprises thermally treating the reaction solution under an acidic condition of not more than pH 4, preferably in the neighbourhood of pH 1 in the presence of an adsorbent thereby flocculating the used microorganic cells, causing the floccules to be adsorbed on the activated carbon, and eluting the adsorbate. DMSO
is added to the acidic a~ueous solution containing cysteine,~ which resulted from removal of the used micro-organic cells from the reaction solution as described above, as an oxidative reagent to convert the cysteine into cystine. In the oxidation reaction of cysteine into cystine, the reaction velocity increases in proportion as ~ . .
- 6 - ~ 9 the pH value of the solution decreases and the yield of cystine increases in proportion as the acidity of the solution increases.
The amount of DMS~ to be used is sufficient on the level of about 0.2 to 2.0 mols, preferably 0.5 to 1.0 mol, per mol of the cysteine contained in the reaction solution.
Though the temperature at which the reaction of this invention is to be carried out is not rigidly de-fined, it is desired to fall in the range of 5C to 100C.
This invention does not require forced supply of oxygen to the reaction system. The reaction can be carried out in any conventional hermetically sealable reaction vessel, and is possible even in an atmosphere of N2 gas.
Oxidation is effected by the use of DMSO, and thus the present invention enjoys the following advantages.
(1) In the oxidation with air or with H2O2, no proper reaction velocity can be obtained unless a metal such as Fe++, Mn++, or Cu++ is added as a catalyst to the reaction system. The method of the present invention does not require a catalyst.
Also, as oxidation with air or with H22 is a radical reaction, it does not terminate with the occu~rrence of cystine but proceeds to the occurrence of decomposition products such as cysteic acid. As a result, ~the recovery of cystine inevitably entails heavy loss and the oxidation reaction is not easily controlled. With oxidation carried out in the presence of DMSO as contem-plated in th~e present invention, the oxidation is an ionic .
LS~
reaction and thus it proceeds moderately and producescystine substantially quantitatively from cysteine.
Furthermore, when the reaction is carried out in the pre-sence of an electrolytic inoryanic salt, the reaction ve-locity of the oxidation is increased. The effect of thiselectrolytic inorganic salt is particularly prominent when the method is applied to the L-cysteine reaction solution which is obtained by using L-serine as a substrate and effecting the enæymatic reaction of a sulfur-containing compound in the presence of tryptophane synthase.
(2) Preferably, the aqueous cysteine solution to be subjected to the oxidation of this invention has as low a pH value as possible. Where the aqueous cysteine solution resulting from the removal of the used micro-organic cells, the removal being carried out usingactivated carbon under an acidic condition (pH 4 or less), is subjected to the oxidation reaction, the aqueous solution can be put to use directly in the oxidation reaction without requiring a treatment for pH adjustment.
As compared with the conventional method which resorts to oxidation with air or with H2O2, the method of the present invention which relies on the oxidation with DMSO has noticeable advantages such as lowered ratio of decomposition of cysteine into cysteic acid, particularly under a strongly acidic condition. Data supporting this fact are shown in the following table.
In the test oxidation, an aqueous 10~ L-cysteine solution was used as a model solution. DMSO and H22 were each used in an amount of 0.75 mol per mol of L-cysteine.
In the case of the oxidation with air, air was passed through the solution at a rate of three times the volume of the solution per minute. In all the tests, the oxidation was carried out at room temperature for eight ,, ~ -- 8 - ~9~59 hours. In the table, the first of each pair of figures represents the conversions (oxidation ratios) of cysteine and the second the formation ratios of cystine (selectivities to cystine).
I DMSO ¦ Air ¦ H22 . ._ , _ pH198/96 5/4 99/68 pH787/85 85/79 99/76 1 0 . . _ .
Note) In the tests using air and H2O2, FeCl2 was added in a minute amount to permit the presence of Fe++ ion in the reaction system.
Now, the present invention will be described more specifically below with reference to working examples. In these working examples, cysteine and cystine were analyzed as follows. The analysis of cysteine was performed by the known Gaitonde method.
, About 0.5 g of a given solution was taken as a sample, weighed accurately, and diluted with 2N HCl to 10 to 20 times the original volume. The diluted solution was further diluted~with distilled water to about 100 times.
The solution finally diluted ~to 1,000 to 2,000 times the original volume was coloured with an acidic ninhydrin reagent ~and tested~ for absorbancy at 560 nm with an absorptiometer. Separately, standard samples of known concentrations were prepared; in advance to obtain a calibration~curve of the absorbancy at 560 nm. Then the cysteine ; concentration of the given~ solution was calc~ulated~from the callbration curve.
- ~ , As ~regards the cystine concentration, a given solu~ion~diluted to ~1~,000 to 2,000 times the original volyme as~described; above~ was mixed with a roughly equal :
:
- 9 ~
volume of a 5-uM 1,4-dithiothreitol (reducing agent), adjusted to pH 8.0 to 8.5 by the addition of 2N NaO~, and left standing at room temperature for one hour for thorough reducing of cystine into cys-teine to permit calculation of the cysteine concentration by the method described above. The cystine concentration was found by deducting the cysteine concentration before the reduction from the concentration so calculated.
Example 1:
In a separable flask having an inner volume of 300 ml and fitted with a stirrer, 91 g of an aqueous L-serine solution containing 22.0 wt% of L-serine (0~19 mol as L-serine), 28.1 g of sodium hydrosulfide dihydrate (NaSH 2H2O) (0.38 mol), and 10 g of a culture broth of tryptophan synthase originating from Escherichia coli MT-10242 (FERM BP-20) and still containing the used cells (dry content 2.2 g), were combined, adjusted to pH 7.5 by the addition of an aqueous 5% sodium hydroxide solution, and diluted with water to a total volume of 200 ml. The resultant mixture was placed in a constant temperature bath at 35C and left reacting therein for 24 hours.
After completion of the reaction, the reaction mixture was adjusted to pH 1.0 with hydrochloric acid, then combined with 5 g of activated carbon (produced by Takeda Chemical Industries, Ltd. and marketed under trademark designation of "Tokusei Shirasagi"), and heated at 80C for 30 minutes. After this heat treatment, the reaction mixture kept at 80C was hot filtered through a Buchner ~funnel to remove used microorganic cells and obtain 188 g of a réaction filtrate in a treated form.
This reaction filtrate was found by analysis to contain 20.1 g (0.166 mol) of L-cysteine and 2.0 g (0.008 mol) of L-cystine bo~h reduced as L-cysteine. The conversion of .
.. . . ~ . ,. . .= . .. .
- 1 o - ~ i9 L-serine to L-cysteine and L-cystine was found by calcu-latlon to be 95.8%.
The reaction solution thus obtained by the heat treatment was transferred into a separable flask having an inner volume of 300 ml and fitted with a stirrer and stirred therein with 9.7 9 of DMSO at room temperature for eight hours to obtain 196.2 g of a solution having L-cystine crystals precipitated therein (the solution had still pH 1). In this solution, 0.2 g of L-cysteine remained.
The solution resulting from the oxidation was neutralized to about pH 5 by the addition of an aqueous 5%
sodium hydroxide solution to induce precipitation of crystals. The crystals were separated by filtration and dried to obtain 19.89 (0.165 mol as 100%) of white powdery crystals. These crystals were found to possess a specific rotation~[a]D = -218 (C = 2, 2N HCl), an Fe content of not more than 10 ppm, and a purity of not less than 98.5~. As regards the ratio of recovery, 95.0% of all the L-cysteine and L-cystine present in the reaction solution were recovered. The ratio of decomposition was not more than 2%.
Comparative Experiment:
The` same reaction solution obtained by following the procedure of Example 1 was heat treated and then neutralized with an aqueous 5~ sodium hydroxide solution to about pH 5. The reaction solution resulting from this heat treatment was transferred into the same separable flask having an inner volume of 300 ml as used in Example 1, mixed therein with 0.2 g of FeCl2 and blown with air introduced~at a flow rate of about 200 ml/min. at room ~g~s~
temperature, to produce 200.8 g of a solution having L-crystine crystals precipitated therein. In the solution, 4.2 g of L-cysteine remained.
By separating the crystals from the reaction solu-tion through filtration and drying the separated crystals,there was obtained 14.8 g of white powdery crystals. The crystals were found to possess a specific rotation, [a]D = -215 (C = 2, 2N HCl), a Fe content of 30 ppm, and a purity of not less than 98O5%~ As regards the ratio of recovery, 70.3% of all L-cysteine and L-cystine present in the reaction solu~ion were recovered. The ratio of decomposition was 9.9%.
Example 2:
In a separable flask having an inner volume of 300 ml and fitted with a stirrer, 91 g of an aqueous L-serine solution containing 22.0 wt% of L-serine (0.19 mol of L-serine), 28.1 g (0.38 mol) of sodium hydrosulfide dihydrate (NaSH-2H20), and 10 g of a culture broth of tryptophan synthase originating from Escherlchia coli MT-10242 (FERM BP-20) and still containing the used cells (dry content 2.2 g), were combined, adjusted to pH 7.5 by the addition of an aqueous 5~ sodium hydroxide solution and then diluted with water to a total volume of 200 ml.
The diluted solution was placed in a constant temperature bath at 35C and left reacting therein for 24 hours.
The reaction solution obtained after completion of the reaction was found by analysis to contain 18.5 g (0.1~53 mol) o~ L-cysteine and 3.2 g (0.013 mol) of L-cystin~ both reduced as L-cysteine. The conversion of L-seeine to L-cysteine and L-cystine was calculated to be 94.2%.
.
91~L5~3 The reaction solution consequently obtained was adjusted to pH 0.5 by the addition of abaut 30 ml of concentrated hydrochloric acid and combined with 0.97 g of DMSO and stirred at 35C for eight hours for oxidation.
At this stage, the oxidized solution contained 0.7 g (0.006 mol) of L-cysteine and 20.7 g (0.086 mol) of L-cystine. The conversion of L-serine to L-cystine was calculated to be 90.6~.
The oxidized solution and 5 g of activated carbon (product of Takeda Chemical Industries, Ltd.) added thereto were heated at 80C for 30 minutes. m e resultant reaction mixture kept at 80C was hot filtered through a Buchner funnel to remove used microorganic cells and obtain 2.30 g of a homogeneous filtrate (having still pH
0.5) in a treated form. This filtrate was found by analysis to contain 0 3 g (0.002 mol) of L-cysteine and 20.8 g (0.087 mol) of L-cystine. The conversion from L-serine to L-cystine was found to be 91.3%.
Tbe solution resulting from the separation of the used mircoorganic cells was neutralized to pH 2 by dropwise addition of about 5 ml of 30% NaOH solution to induce precipitation of L-cystine crystals. The crystals were separated by vacuum filtration with a Buchner funnel~
washed thoroughly with 50 ml of water, and dried to afford 20.2 g (0.084 mol as 100%)- of white powdery cFystals.
These crystals were found to posse9s a specific rotation, [a]D = -218.6 (C = 2, 2N HCl), a Fe contént of not more than 10 ppm, and purity of not le86 than 98.5%~
As regards the ratio of recovery, nearly 100% of all L-cysteine and ~L-cystine present in the reaction solution were recovered.
Claims (2)
1. A method for the production of cystine, which comprises oxidizing cysteine in a cysteine-containing aqueous solution having a pH value of not more than 4, said solution having been obtained by an enzymatic method of fermentative method, in the presence of dimethyl sulfoxide employed as an oxidizing agent, the dimethyl sulfoxide being present in an amount of about 0.2 to about 2.0 mols per mol of cysteine, thereby converting said cysteine into cystine; and separating the cystine.
2. A method according to claim 1, wherein said cysteine-containing aqueous solution is an aqueous L-cysteine solution obtained by the reaction of L-serine with a sulfhydryl group-containing compound in the presence of tryptophan synthase.
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60258602A JPH0660157B2 (en) | 1985-11-20 | 1985-11-20 | Method for producing cystine from cysteine |
| JP61060853A JPS62221664A (en) | 1985-11-20 | 1986-03-20 | Production of crystine from cysteine |
| US07/049,807 US4769491A (en) | 1985-11-20 | 1987-05-12 | Method for production of cystine from cysteine |
| EP87106852A EP0290643B1 (en) | 1985-11-20 | 1987-05-12 | Method for production of cystine from cysteine |
| CA000536982A CA1291159C (en) | 1987-05-12 | 1987-05-13 | Method for production of cystine from cysteine |
| AU72935/87A AU569677B1 (en) | 1985-11-20 | 1987-05-14 | Production of cystine from cysteine obtained by the enzymatic or fermentative method |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP87106852A EP0290643B1 (en) | 1985-11-20 | 1987-05-12 | Method for production of cystine from cysteine |
| CA000536982A CA1291159C (en) | 1987-05-12 | 1987-05-13 | Method for production of cystine from cysteine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1291159C true CA1291159C (en) | 1991-10-22 |
Family
ID=25671342
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000536982A Expired - Lifetime CA1291159C (en) | 1985-11-20 | 1987-05-13 | Method for production of cystine from cysteine |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1291159C (en) |
-
1987
- 1987-05-13 CA CA000536982A patent/CA1291159C/en not_active Expired - Lifetime
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