JPS63258850A - Production of l-cystine - Google Patents

Abstract

PURPOSE:To obtain l-cystine useful as a drug, a drug raw material, food additive, cosmetic additive or a raw material for cold permanent wave solution, by subjecting l-serine to enzymatic reaction to give l-cysteine and oxidizing the l-cysteine in the presence of dimethyl sulfoxide. CONSTITUTION:l-Serine is reacted with hydrogen sulfide, an alkali metallic sulfide or an alkali metallic hydroxide in the presence of tryptophan synthase to give l-cysteine. l-Cysteine in an aqueous solution containing l-cysteine is subjected to enzymatic reaction in the presence of dimethyl sulfoxide at 20-60 deg.C at reaction pH 5-10 and formed cysteine is converted into cystine to give the aimed substance. The amount of dimethyl sulfoxide used is 0.2-5.0mol., preferably 0.5-2.0mol. based on l-serine used.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention uses an enzyme method! - Concerning a method for obtaining cystine.

Cystine can be easily converted to cysteine by reduction, and 2-cystine and 2-cystine are used as medicines or raw materials for medicines, food additives, cosmetic additives, etc., and especially in recent years, they have been used as cold perm liquids. It is an amino acid containing S element, which is in increasing demand as a raw material for.

(Prior art and problems to be solved by the invention) Conventional! - Known methods for producing cysteine include (1) extraction from natural products, (2) organic synthesis, (3) fermentation, and (4) enzymatic methods. However, the supply of raw materials is unstable and other unnecessary amino acids are mixed in. In addition, in organic synthesis methods,
Requires division of L. Furthermore, the fermentation method has drawbacks such as a low accumulation amount, so it is difficult to say that it is an industrially advantageous production method.

Enzymatic methods for synthesizing 2-cysteine using enzymes include (1) using cysteine synthase and cysteine desulfhydrase! - Synthesis from serine and hydrogen sulfide, (2) Synthesis from β-substituted alanine and metal sulfide using cysteine desulfhydrase, (3) 2-amino-thiazoline-4-carboxylic acid ( ATC), ff1-ATC-hydrolase, ATC
- Synthesis methods using racemase, S-carbamyl-l-cysteine hydrolase, etc. are known. The present applicant has previously applied for a method of reacting 2-serine with a compound having a sulfhydryl group such as a metal hydrosulfide in the presence of tryptophan synthase (Japanese Patent Application No. 84545/1982).

Regardless of these fermentation methods, in both fermentation and synthesis methods, when it comes to separating cysteine from a cysteine-containing reaction solution, it is difficult to separate cysteine from a cysteine-containing reaction solution because the composition of the reaction solution is complex and cysteine has a very high solubility in water. It is extremely difficult to isolate and purify cystine directly from the reaction solution containing cysteine; the usual method is to forcibly oxidize cysteine, purify and isolate it as cysteine, and if necessary recover it as refined cysteine by electrolytic reduction. It is being

In addition, some methods for oxidizing cysteine to cystine are known. For example, in Japanese Patent Publication No. 7634/1983, cysteine in a cysteine fermentation aqueous solution is maintained at pH 5 to 10, and air oxidation or H! 0. Oxidation methods using peroxides such as

However, the method, for example! - When obtaining cystine from serine, it cannot be applied until after the enzymatic reaction has been completed and cysteine has been produced, and the oxidation method using ntog or air has difficulty controlling the cystine oxidation reaction conditions; If not strictly controlled, there were problems such as a considerable proportion of decomposition products other than cystine being produced.

In addition, in the enzymatic reaction of l-serine, it can be expected that the direct use of hydrogen sulfide (Hls) as the sulfhydryl group compound will have higher reactivity, and although it is cheaper, H and S gases are Since it has little solubility in water, water-soluble salts such as sodium bisulfide have usually been used as HzS sources.

(Means for Solving the Problems) The present inventors have arrived at the method of the present invention as a result of intensive studies in consideration of the above problems.

- That is, the present invention provides for oxidizing l-cysteine in a reaction aqueous solution containing l-cysteine obtained by reacting 2-serine with hydrogen sulfide, an alkali metal hydrosulfide, or an alkali metal sulfide through an enzymatic reaction. - When obtaining cystine, an oxidation reaction is also carried out in the reaction system by carrying out an enzymatic reaction in the presence of dimethyl sulfoxide, so the reaction is carried out while sequentially converting the produced cystine into cystine. From l-Serine! - A method for producing cystine.

In this way, in the present invention, dimethyl sulfoxide (hereinafter abbreviated as DMSO) used as an oxidizing agent for cysteine is
By using it in the enzymatic reaction system during the production of l-cysteine, the produced cysteine can be completely converted to cystine in the reaction process, and even if hydrogen sulfide gas is used in the cysteine reaction, DMSO can convert hydrogen sulfide in the reaction system. Since it acts as a solubilizing agent, the enzyme reaction can be carried out while maintaining a sufficient concentration in the aqueous medium.

In addition, DMSO added in the system does not inhibit the enzymatic reaction at all (it is produced during the enzymatic reaction!-cystine is sequentially oxidized to stable!-cystine and precipitated from the enzyme reaction system, so l- It was also found that the enzyme reaction rate during cysteine production could be significantly accelerated.

Therefore, the present invention has produced! -It is now possible to prevent the decomposition of cysteine into by-products! -L-cystine can be obtained from serine in high yield.

The enzyme used in the present invention is not limited in any way as long as it is an enzyme used in the enzymatic reaction between 2-serine and hydrogen sulfide, alkali metal hydrosulfide, and alkali metal sulfide. Enzymes such as rufuhydrase may be used, and tryptophan synthase proposed by the applicant described above may also be used for the reaction.

In particular, in the present invention, it is more effective to use tryptophan synthase (the preferred method,
As the tryptophan synthase enzyme producing bacteria, Escherichia coli MT-10232 (F E RM
B P-19), Escherichia coli MT-1
Microorganisms such as 0242 (FERM BP-20),
Although microorganisms such as Neurospora kratte (ATCC 14692) can be used, it is especially advantageous to use tryptophan synthase obtained from Escherichia coli.

The enzyme does not necessarily have to be extracted and pure; it may be a culture of the above-mentioned production strain, live cells collected from the culture by methods such as centrifugation, frozen cells, freeze-dried cells, or A treated bacterial cell product obtained by sonication or the like is used.

In the method of the present invention, there is no particular restriction on the substrate concentration of 2-serine, but it is usually preferable to use the concentration in the solution in the range of 1 to 25%, and the amount of enzyme used in the reaction solution is as follows:
It varies depending on the usage form of the enzyme and is not particularly limited, and may be changed depending on various conditions such as substrate concentration and enzyme activity. When tryptophan synthase is used, it is desirable to add a trace amount of pyridoxal I) as a coenzyme in addition to the substrate, for example at a concentration in the solution of 0.1 to 50 ppm.

In the present invention, the sulfhydryl group compound to be supplied during the enzyme reaction is an alkali metal hydrosulfide such as NaHS or Its, and hydrogen sulfide gas may be directly introduced into the reaction system. It is preferable that the amount used is at least 1 times the molar amount of i-serine.

Further, the amount of DMSO used, which is a feature of the present invention, is about 0.2 to 5 times the mole of l-serine, and more preferably 0.5 to 2.0 times the mole of l-serine is added to the reaction system. . Generated when the amount used is 0.2 times the mole or less! - Full amount of cysteine! - It is difficult to convert to cystine, and its solubility also decreases when hydrogen sulfide is used. Moreover, if the amount is 5 times or more by mole, the enzyme reaction tends to be significantly inhibited.

The reaction temperature is usually in the range of 20 to 60°C, and the reaction pH
is 5-10. The reaction time is usually 2 to 50 hours, preferably 2 to 10 hours in the case of hydrogen sulfide.

The reaction solution turned white as time passed, indicating that l-cystine crystals were successively precipitated in the reaction solution.

After the reaction is complete, the reaction solution contains unreacted phosphorus and l-
Cystine exists in a precipitated state, but in order to isolate cystine from this reaction solution, enzyme cells are removed, for example, by adding activated carbon to the reaction solution under strong acidity of hydrochloric acid, heat-treating it, and subjecting it to hot filtration. By returning the pu of the reaction solution to around 5 again,! - Highly pure l-cystine can be obtained by crystallizing cystine and performing normal solid-liquid separation. Moreover, if the 2-cystine obtained in this way is electrolytically reduced,
2-cysteine can be obtained.

The present invention will be explained in detail with reference to Examples below, and the cystine analysis method used in the Examples is based on the well-known Gaitonde method.
It was calculated in terms of cysteine by the method of Tonde.

That is, to the test solution diluted 1,000 to 2,000 times, approximately the same amount of 5 NM 1,4-dithiothreitol (on the reducing side) was added, and then the pH was adjusted to 8.0 to 8 with 2 N NaOH.
．． 5, all cystine contained is reduced to cysteine by standing at room temperature for 1 hour, color is developed using an acidic ninhydrin reagent, and absorbance at 560n* is measured using an absorbance meter.

On the other hand, prepare a standard sample with a known concentration, and
A calibration curve of absorbance was prepared in advance, and the concentration of cysteine in the test liquid was calculated based on this calibration curve.

Example 1 In a 200 d separable flask equipped with a stirrer, blowing tube, and exhaust tube, 10 g of 2-serine was added to DMSO5,6
g, pyridoxal phosphate 25id were added, and the total volume was made up to 100 ad with ion-exchanged water. Adjust the pH of the reaction solution to 8.5 with 10% NaOH solution and keep the reaction solution constant at 35°C.
Charge 2.0g of 0) in terms of dry bacterial cells.

Blow-in of hydrogen sulfide gas from a cylinder was started at a rate of about 14 d/min, and the blowing was completed in about 4 hours.

(The amount of hydrogen sulfide used is approximately 1.5 times the mole of β-serine.) Meanwhile, when the blowing was started, the inside of the reaction system gradually turned white! -Cystine was precipitated.

After the reaction is complete, sample the reaction solution as uniformly as possible.
Dissolve in 2N hydrochloric acid and sterilize by centrifugation! - When cystine is analyzed in terms of l-cystine, it is 9.3%, that is! - The conversion rate of serine to l-cystine was about 81%.

Next, add about 25 d of 35% hydrochloric acid to the reaction solution to
Set H to 0.5, charge 1.0 g of activated carbon, and heat at 95°C.
Heat treatment was applied for about 1 hour. Immediately after the heat treatment, hot filtration was performed to eliminate bacteria.

After sterilization, add about 5 d of 40% NaOH to the transparent yellow liquid.
H5, and l-cystine crystals were precipitated by cooling.

The precipitated l-cystine crystals were filtered through a Nutsche filter, washed with 20Id of ion-exchanged water, and then dried to obtain 8.2 g of white l-cystine crystals.

(Yield of 72 mol % based on the charged l-serine) This crystal had good [α)'' = -218.7'', purity of 99.1%, and ash content of 0.2%.

Example 2 In a 200 ml separable flask with a stirrer, 2-
10 g of phosphorus, 5.6 g of DMSO, 25 g of pyridoxal phosphoric acid, 9.5 g of sodium hydrogen sulfide (70% pure reagent)
Add and make the total volume to 200g with ion-exchanged water. 10
% NaOH of the reaction solution was adjusted to 8.0, the reaction solution was maintained at a constant temperature of 35°C, and 2.0 g of the tryptophan synthase-containing microbial cells used in Example 1 was charged in terms of dry microbial cells. The pH during the reaction is maintained at 8.0 with 4 mol/l phosphoric acid.

The reaction was carried out at pH 8 and 0/35°C for about 16 hours, but around 2 hours after the start of the reaction, cystine precipitated in the reaction system and it started to become cloudy.

After the reaction is complete, sample the reaction solution as uniformly as possible.
After dissolving in 2N hydrochloric acid, sterilize by centrifugation to obtain 2-cystine! -When analyzed in terms of cysteine, it is 9.1%! -Serin's! - Conversion rate to cystine was approximately 79%.

Thereafter, treatment was carried out in the same manner as in Example 1 to obtain 7.6 g of t-cystine crystals. (66 for l-serine in the preparation)
．． (Yield: 7 mol %) This crystal had good [α) ”=−220°, purity of 99.3%, and acche content of 0.1%.

Example 3 A 200 d separable flask with a stirrer! −
10g of serine, 5.6g of DMSO, 25d of pyridoxal phosphate, 9.3g of sodium sulfide (reagent purity 96% or more)
Add and make the total volume to 200g with ion-exchanged water.

The reaction solution was diluted with 10% HCl to adjust the reaction po to 8.0.
The tryptophan used in Example 1 was kept at a constant temperature of 5°C.
Charge 2.0g of synthase-containing bacterial cells in terms of dry bacterial cells,
The pH during the reaction is maintained at 8.0 with 10% HCffi.

The reaction was carried out at pt18.0/35°C for about 16 hours,
Around 2 hours after the start of the reaction, cystine precipitated in the reaction system and the mixture began to become cloudy.

After the reaction is complete, sample the reaction solution as uniformly as possible.
After dissolving in 2N hydrochloric acid, sterilize by centrifugation to obtain l-cystine! -8.7% or l when analyzed in terms of cysteine
- The conversion rate of serine to l-cystine was about 74%.

Hereafter, the process is carried out in the same manner as in Example 1. - Obtained 7.4 g of cystine crystals. (64 for l-serine in the preparation)
．． 9 mol% yield) [α] of this crystal? --221°, purity 98.9%, and ash content 0.3%.

Example 4 In a 200 d capacity separable flask with a stirrer, 2-
The total volume was made up to 50 g with 10 g of serine, 5.6 g of DMSO, and ion exchange water.

Cysteine desulfhydrase-containing enzyme solution (Bacillus subtilis AT CC6051 was collected after culturing and disrupted by ultrasonic waves in a buffer) was added, and N
Make ptts and s with aOH solution, and bring the total volume of the reaction solution to 100g.
shall be.

Hydrogen sulfide gas was started to be blown in from the cylinder at a rate of about 14rI11 minutes, and the blowing was completed in about 4 hours.

(The amount of hydrogen sulfide used is approximately 1.5 times the mole of serine)
Meanwhile, when the blowing was started, the inside of the reaction system gradually turned white and l-cystine was precipitated.

After the reaction is complete, sample the reaction solution as uniformly as possible.
Sterilize by dissolving in 2N hydrochloric acid and centrifuging! - Cystine was analyzed in terms of 2-cystine and was found to be 5.8%, that is, the conversion rate of l-serine to l-cystine was approximately 53%. Furthermore, when the concentration of cysteine alone in the reaction system was measured, it was found to be 0.1% or less.

Claims (1)

[Claims] 1) l-cysteine in a reaction aqueous solution containing l-cysteine obtained by reacting l-serine with hydrogen sulfide, an alkali metal sulfide, or an alkali metal hydrosulfide through an enzymatic reaction. , Production of l-cystine from l-serine, characterized by carrying out an enzymatic reaction in the presence of dimethyl sulfoxide when oxidizing to obtain l-cystine, and carrying out the reaction while sequentially converting the produced cystine to cystine. Method. 2) The method according to claim 1, wherein the enzymatic reaction of l-serine is carried out in the presence of tryptophan synthase.