JPH03180188A - Production of cysteine - Google Patents

Abstract

PURPOSE:To improve yield by reacting cystine with H2 in the presence of a specific enzymic liquid within an alkaline region. CONSTITUTION:A microorganism (e.g. Clostridium thermoaceticum ATCC 31490) having hydrogenase activity is cultured to provide a culture, which is then centrifuged to collect bacterial cell. The resultant bacterial cells are destroyed and centrifuged to afford an enzymic liquid having alkali-tolerant hydrogenase activity. In a buffer solution containing cystine, are then added 0.01-10wt.% (expressed in terms of proteins) aforementioned enzymic liquid and, as necessary, >=0.005mumol/l coenzyme (e.g. nicotinamide adenine dinucleotide) or methylviologen. H2 is subsequently sealed within an alkaline region at pH8-12 and reacted therewith.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing cysteine, more specifically, using an enzyme solution having alkaline-resistant hydrogenase activity,
This invention relates to a method for producing cystine from cystine in high yield.

[Prior art and problems to be solved by the invention] Cysteine is an amino acid with a mercapto group, and is used in a wide variety of fields such as medicines, pharmaceutical raw materials, food additives, feed additives, and cosmetic additives. In recent years, demand has increased as a raw material for cold perm liquid.

Conventionally, known methods for producing cysteine include extraction methods from natural products, organic synthesis methods, fermentation methods, and enzymatic methods. The extraction method from natural products involves hydrolyzing cystine or natural products with a high cysteine content, such as hair, with hydrochloric acid, and electroreducing the extracted cystine from the resulting amino acid mixture to obtain cystine. However, this extraction method produces other impurities due to the electrode reaction, so it is not particularly suitable for use as a medicine or food additive. In addition, electric reduction requires a large amount of electricity and electrolyte, resulting in high cost. No practical organic synthesis methods have been disclosed, and instead of these methods, the fermentation method (Japanese Unexamined Patent Publication No. 59-2
No. 8485) has been proposed, but it has drawbacks such as expensive raw materials, insufficient production of cysteine, difficulty in purifying impurities produced by bacteria, and difficulty in controlling bacteria. be. Also, the enzymatic method (Unexamined Japanese Patent Publication No. 6)
No. 3-7790) has also been proposed, but the production amount of cysteine is low and it is not an effective manufacturing method.

Therefore, the present inventors first developed a method for producing cystine by reacting cystine with hydrogen in the presence of an enzyme solution having hydrogenase activity (Japanese Patent Application No. 63-247052). According to this production method, cysteine can be produced from cystine with high yield and high purity. However, this method has a drawback in the reaction rate and is not necessarily fully satisfactory as an industrial production method.

Therefore, the development of a more industrially advantageous method for producing cysteine has been eagerly awaited.

[Means to solve the problem]

In view of the above-mentioned circumstances, the present inventors have conducted intensive research without being bound to the idea that it is preferable to conduct a reaction using a conventional enzyme solution having hydrogenase activity at a value near the optimum p)l of the microorganism from which the hydrogenase was obtained. The fact that the enzyme solution obtained from a specific microorganism can perform a good reaction even in an alkaline region that is not near the optimum pH of the microorganism, and furthermore, if hydrogen is reacted with cystine in an alkaline region in the presence of this enzyme solution, it can be used industrially. They discovered that cysteine can be produced advantageously and completed the present invention.

That is, the present invention provides a method for producing cystine, which is characterized by reacting cystine with hydrogen in an alkaline region in the presence of an enzyme solution having alkali-resistant hydrogenase activity.

Cystine, which is a raw material for the method of the present invention, is L-form, D-form or DL-form.
It can be any part of the body.

The enzyme solution used in the present invention is not particularly limited as long as it has an alkali-resistant hydrogenase activity, and may be a solution in which an alkali-resistant hydrogenase is dissolved or a culture of a microorganism that has an alkali-resistant hydrogenase activity. As a culture of a microorganism having alkaline-resistant hydrogenase activity, for example, Clostridium
ium), Escherichia coli, sulfate-reducing bacteria, methane bacteria, hydrogen bacteria,
Examples include cultures of bacteria belonging to Azotobacter and the like. As such a culture, it is preferable to use, for example, a supernatant obtained by culturing these bacteria by a conventional method, collecting the bacteria using a centrifuge or the like, subjecting them to ultrasonic destruction, and centrifuging them. The amount of the enzyme solution added is about 0.01 wt% or more in terms of protein based on the buffer solution containing cystine.
Particularly about 0.05 wt% or more, and even about 0.05 to t Ow
t% is preferred. The reaction is preferably carried out under a high hydrogen partial pressure in order to improve the reaction rate. In addition, when the microorganism from which the alkali-resistant hydrogenase was obtained is an anaerobic bacterium, it is necessary to make the gas phase anaerobic, that is, an oxygen-free state.

When using a culture of microorganisms, the reaction temperature is preferably within about 8 hours, particularly within about 8 hours, of the optimal growth temperature of the microorganisms. For example, Clostridium thermoaceticum (45-65℃), Methanosarcina parkeri (Methanosarcina
32 when using DSM 804
~42°C is preferred. The reaction time is usually about 8 hours or less when the reaction is carried out by a batch method, but an appropriate reaction time can be selected depending on changes in other conditions. Furthermore, the reaction can be carried out continuously by continuously taking out the produced cysteine.

The present invention is characterized in that the reaction is carried out in an alkaline region, thereby increasing the solubility of cystine and speeding up the reaction. Here, the alkaline region is p
This refers to a pH range of about 8 or higher, preferably about 9 or higher where cystine has sufficient solubility. pH of reaction solution
can be increased as long as the enzyme solution is not deactivated, but it is usually preferably about 12 or less.

In the present invention, the reaction is further promoted by adding a coenzyme or methyl viologen. As a coenzyme, for example, F420 [N-(N-[0-[5-(8-hydroxy-5-deazaiisoalloxazin-10-yl)-2,3
,4-)lihydroxy-4-pentoxyhydroxyphosphinyl]-L-lactyl]-γ-L-g, rutamyl]
-L-glutamic acid], N^0+ [nicotinamide adenine dinucleotide], etc. are used. The amount of coenzyme or methyl viologen added is about 0.005 JJmol/1 or more, especially about 0.0 to the buffer solution containing cystine.
1 pmol/1 or more, furthermore about 0. Ofμmol/
1~20 mmoj! /j! is preferred.

[Action and effect of the invention]

The present invention is an excellent method for producing cysteine that has the following unique effects and is of high industrial value.

(1) Cystine, which is inexpensive and exists in abundance in nature as a natural product such as electricity storage, can be used as a raw material, so cystine can be stably produced at low cost.

(2) Conventional electrical reduction methods require large amounts of electricity and electrolyte, resulting in high costs, but the method of the present invention allows cysteine to be produced at extremely low costs.

(3) It is possible to produce highly pure cysteine that can be used as a pharmaceutical or food additive, which was not possible using conventional electroreduction methods.

(4) Since the reaction can be carried out in an alkaline environment where cystine has a high solubility, the reaction apparatus can be extremely miniaturized and productivity can be dramatically improved.

〔Example〕

EXAMPLES The present invention will be described in more detail with reference to Examples below, but these are merely given for the purpose of illustration and the present invention is not limited thereto.

Example 1 Minimal medium with glycose as the sole carbon source at 60°C and pH
Clostridium thermoaceticum ATCC31490 was batch cultured for 4 days under anaerobic conditions of 7.0. Bacterial cells were collected from the culture by centrifugation, washed with Tris-hydrochloride buffer, destroyed by reacting with a solution containing lysozyme and deoxyribonuclease 1 at 37°C for 90 minutes, and then centrifuged. The clear solution was used as an enzyme solution. The production and storage of this enzyme solution were all carried out anaerobically. This enzyme solution has cystine 3 mmoj! It was added at a ratio of 3 μm in terms of protein to 4 mt' of Tris hydrochloride B solution containing 50 mmof/Il and pH 9 at a ratio of 3 μm/Il.

The gas phase is H, :N, = 3 : 1 (molten ratio) gas 3
It was sealed under atmospheric pressure and reacted for 5 hours with shaking at a temperature of 55°C. When cysteine was quantified using the Gaitonde method, the reduction rate of cysteine to cysteine was 50.2%.

Comparative Example 1 A test was carried out in the same manner as in Example 1, except that the enzyme solution was heated at 121° C. for 15 minutes to lose enzyme activity and used instead of the enzyme solution. In this case, the reduction rate of cystine to cysteine was 0.4%.

As a result, compared with Comparative Example 1, it can be seen that in Example 1, reduction of cystine to cysteine was carried out by an enzymatic reaction.

Example 2 The same procedure as Example 1 was carried out except that methyl viologen was added at a ratio of 0.1 mmol 1/1 and the reaction time was varied. The results are shown in Table 1.

Table 1 Compared to Example 1, it can be seen that the reaction was accelerated by adding methyl viologen.

Reference Example 1 A minimal medium with methanol as the sole carbon source, 37°C, p
Methanosarcina Balke IJ under anaerobic conditions of H6,7
DSM 804 was cultured for 3 days. Collect bacterial cells from the culture by centrifugation, wash with Tris-HCl buffer,
The bacterial cells were destroyed by ultrasonic treatment under cooling, and the supernatant obtained by centrifugation was used as an enzyme solution. The production and storage of this enzyme solution were all carried out anaerobically.

Contains this enzyme solution at a ratio of cystine 3fflIII01/1 at a concentration of 50 mmoj! / 1. 4ml of pH 7 salt hydrochloride buffer! Added 3■ in terms of protein and added 0.1 mmoj of methyl viologen! /1.

The gas phase was sealed at 3 atm with a gas of H,:N2=3:1 (volume ratio), and reacted for 2 hours with shaking at a temperature of 37°C. When cysteine was quantified using the Gaitonde method, the amount of cysteine produced was 1. Ommo 1/Il, reduction rate is 16
．． It was 7%.

Compared to Example 2, this method has a reduction of 1.0 mmoj! It took 40 times as long to obtain cysteine.

Furthermore, in this method, the solubility of cystine was not sufficient, and cystine particles were suspended in the reaction solution.

Reference example 2 Cystine concentration is 1 mmoj! The same procedure as in Reference Example 1 was carried out except that the reaction time was 6 hours.
The amount of cysteine produced was 0.96 mmoj! /
1. The return rate was 48%.

Example 3 Minimal medium with methanol as sole carbon source, 30t, pH
Methanosarcina basilce under anaerobic conditions of 6, 7) D
SM 804 was cultured for 3 days. Bacteria were collected from the culture by centrifugation, washed with Tris-hydrochloride buffer, treated with ultrasonic waves under cooling to destroy the bacterial cells, and the centrifuged supernatant was used as an enzyme solution. This enzyme solution was manufactured and stored in an anaerobic manner. p) 19 Tris-hydrochloride buffer 4- and cystine l containing this enzyme solution at a ratio of 1 mmol cystine @
moj! / 4 mj of pH 10 glycine hydroxide sodium salt buffer containing at a ratio of 1! Each was added at a ratio of 3 μm in terms of protein. Gas phase Hs:N*=3
: 1 (volume ratio) gas was sealed at 3 atm, and reacted at a temperature of 37° C. for 2 hours with shaking. When the amount of cysteine produced was quantified using the Gaitonde method,
Both cysteine amount 2. Ommo 1/j!
, the return rate was 100%. It can be seen that the reaction was accelerated by reacting in the alkaline region compared to Reference Example 2.

Below Up

Claims (1)

[Claims] 1. A method for producing cysteine, which comprises reacting cystine with hydrogen in an alkaline region in the presence of an enzyme solution having alkali-resistant hydrogenase activity. 2. An enzyme solution having alkali-resistant hydrogenase activity,
The method for producing cysteine according to claim 1, which is a culture of a microorganism having hydrogenase activity. 3. The method for producing cysteine according to claim 1 or 2, characterized in that the reaction is carried out by further adding a coenzyme or methyl viologen.