JPS6125359B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing dopamine using an enzymatic method, and more specifically, the present invention relates to a method for producing dopamine using an enzymatic method, and more specifically, dopamine is produced by allowing an enzyme produced by a microorganism belonging to the genus Streptococcus that catalyzes the reaction of producing dopamine from L-dopa to act on L-dopamine. It's a method. Dopamine is a very important compound as a raw material for the synthesis of pharmaceuticals such as trimethoquinol. Although many chemical synthesis methods have been known as methods for producing dopamine, a method for enzymatically producing dopamine from L-dopa, such as the method of the present invention, has not yet been reported. The present inventors conducted a wide search to find microorganisms that have the ability to produce dopamine from L-dopa, and found that microorganisms belonging to the genus Streptococcus,
For example, it was discovered that Streptococcus fuecalis has a strong ability to produce dopamine from L-dopa, and the enzyme produced by this microorganism was
It has been found that dopamine can be produced almost quantitatively by acting on dopa. That is, the present invention provides an enzymatic method characterized in that a microorganism belonging to the genus Streptococcus (hereinafter simply referred to as a microorganism of the genus Streptococcus) having the ability to produce dopamine from L-dopa is made to act on L-dopa. This is the method for producing dopamine. In the present invention, any microorganism belonging to the genus Streptococcus that has the ability to produce dopamine from L-dopa can be used as the Streptococcus microorganism, such as Streptococcus, ATCC 8043,
Examples include ATCC11420, ATCC11700, and ATCC12984. As the medium for culturing the above-mentioned microorganisms of the genus Streptococcus, various nutrient sources commonly used in the cultivation of ordinary microorganisms can be used. As carbon sources, glucose, sucrose, lactose, maltose, etc. can be suitably used, and as nitrogen sources and organic micronutrients, peptone, yeast extract, corn steep liquor, etc. can be suitably used. Further, as inorganic salts, in addition to magnesium salts, manganese salts, and iron salts, ammonium chloride and sodium acetate may be added. The amount of L-tyrosine or L-dopa added to the medium as described above is about 0.1 to 1% to the medium, thereby increasing the production amount of the enzyme that catalyzes the reaction of producing dopamine from the L-dopa by about 5 to 1%. This can be dramatically improved by 20 times. For example, when L-tyrosine or L-dopa is not added to the culture medium of Streptococcus fuecalis ATCC 12984, only 0.04 units of the enzyme are produced per ml of medium, but when 0.5% L-tyrosine is added, approximately Production of the enzyme was observed to be 0.8 units, and when 0.5% L-dopa was added, about 0.2 units per m ² of the medium. In addition, in the culture medium of the above-mentioned Streptococcus microorganism, a surfactant such as polyoxyethylene sorbitan monooleate is added to the medium at a rate of about 0.01 to
Particularly good results can be obtained in the production of the enzyme by adding about 0.5%. Cultivation of Streptococcus microorganisms can be carried out by inoculating the microorganisms into a medium such as the one described above, and allowing the medium to stand or shake. Culture is
pH is approximately 5-8, especially pH 7, and culture temperature is 25℃ ~
It can be carried out suitably at 40°C, especially around 30°C to 37°C, and the enzymes in the bacterial cells are produced at the same time as the bacterial cells grow, so the final stage of the logarithmic growth phase, when the amount of bacterial growth is at its maximum. The highest enzyme production is obtained. The enzymatic reaction uses the culture solution of the Streptococcus microorganism thus obtained, and the viable cells of the microorganism isolated from the culture solution or its processed product as an enzyme source;
This is preferably carried out by acting on the L-dopa. Examples of processed bacterial cells include freeze-dried bacterial cells, cell-free extracts obtained by ultrasonic treatment, and the enzyme purified from the extracts, as well as live bacterial cells or enzymes that can be processed using polyacrylamide gel method, etc. Immobilized bacterial cells and immobilized enzymes obtained by immobilization by methods such as carrageenan gel method can also be suitably used. When using a culture solution of a Streptococcus microorganism as an enzyme source in an enzyme reaction, approximately 5% L-dopa is added to the culture solution at an appropriate time from the start to the end of culture of the microorganism. By reacting, the desired dopamine can be produced in the culture solution. Further, when using the cells of a Streptococcus microorganism or a processed product thereof as an enzyme source, it is desirable to add a small amount of pyridoxal phosphoric acid to the reaction solution in an amount of about 0.001 to 0.05%. In this case, the pH of the reaction solution
is preferably between 4 and 6, especially 5.3, and the reaction temperature is preferably between 20°C and 45°C, especially around 30°C and 37°C. Approximately 0.5 glucose during reaction
Addition of ~5% is suitable for producing the target dopamine, and since the reaction solution is maintained within the above pH range by the organic acid produced from the glucose, there is no need to particularly adjust the pH. Adjust the above optimal pH using a mineral acid such as hydrochloric acid or an organic acid such as acetic acid.
Even better results can often be obtained by maintaining the In any of the above cases, by adding approximately 0.01 to 0.1% of an aluminum salt such as aluminum sulfate or aluminum acetate to the enzyme reaction solution,
It is possible to quantitatively produce the desired high concentration of dopamine. In this way, more than 30 mg of dopamine is accumulated per 1 ml of the reaction solution, but dopamine can be obtained by, for example, filtering the reaction solution to remove bacterial cells and other insoluble matter, and then crystallizing it by adding hydrochloric acid to the concentrated solution, or by treating it with an ion exchange resin. It can be easily obtained by known isolation procedures such as. Example 1 Glucose 1%, peptone 1%, yeast extract
0.25% ammonium sulfate, 0.5% ammonium sulfate, 0.2% polyoxysorbitan monooleate, and 0.5% L-dopa, adjusted to pH 7,
Dispense into ml shake flasks and sterilize. In this, Streptococcus fuecalis
Inoculated with ATCC12984, 18 at 37℃, 140C.pm
Incubate with shaking for an hour. Using the bacterial cells obtained by centrifugation, (i) Suspend 0.1 g of bacterial cells, 100 ml of water, and 5 g of L-Dopa, and adjust the pH to 5.3 with sodium hydroxide. (ii) 0.1 g of bacterial cells, 100 ml of water, 2 g of glucose, L-
Suspend 5g of Dopa and adjust pH with sodium hydroxide.
adjusted to 5.3. (iii) 0.1 g of bacterial cells, 100 ml of water, 2 g of glucose, aluminum sulfate, 0.03 g of 16-18 hydrate, and 5 g of L-dopa were suspended and the pH was adjusted to 5.3 with sodium hydroxide. Each of the above was reacted at 37°C for 24 hours, and the amount of dopamine produced was compared. The results are shown in Table 1.

[Table] The amount of dopamine is measured using n-butanol: acetic acid:
After performing thin layer chromatography with a solvent of water (4:1:1), quantitative determination was performed using a densitometer. Example 1 Glucose 1%, peptone 1%, yeast extract
0.25%, ammonium sulfate 0.5%, polyoxysorbitan, monooleate 0.2% and L-tyrosine 0.5% and adjusted to pH 7.
Dispense into 500ml shake flasks and sterilize. In this, Streptococcus fuecalis
Inoculate ATCC12984 and culture with shaking at 37℃, 140C.p, m. for 18 hours. Add 7 g of glucose, 1 mg of pyridoxal phosphate, and aluminum sulfate 16-18 hydrate to 350 ml of a suspension of bacterial cells in water obtained by centrifugation.
Add 100 mg and 12 g of L-dopa and react at 30°C for 48 hours. After the reaction is completed, the pH is adjusted to 2 with hydrochloric acid, the bacterial cells are removed by filtration, the filtrate is concentrated, concentrated hydrochloric acid is added thereto, the mixture is cooled, and the precipitated crystals are collected by filtration. Obtain 10.3g of dopamine hydrochloride. mp241℃ (decomp) Example 2 Glucose 1%, peptone 1%, yeast extract
0.25% ammonium sulfate, 0.5% ammonium sulfate, 0.2% polyoxysorbitan monooleate, and 0.5% L-dopa, adjusted to pH 7.
Dispense into 500ml shake flasks and sterilize. In this, Streptococcus fuecalis
Inoculated with ATCC12984, 18 at 37℃, 140C.p, m.
Incubate with shaking for an hour. Add 7 g of glucose, 1 mg of pyridoxal phosphate, and aluminum sulfate 16-18 hydrate to 350 ml of a suspension of bacterial cells in water obtained by centrifugation.
Add 100 mg and 4 g of L-dopa, and react at 30°C for 48 hours. Thereafter, 3.4 g of dopamine hydrochloride was obtained by processing in the same manner as in Example 1. Example 3 Glucose 0.5%, peptone 1%, yeast extract 1%, ammonium chloride 0.5%, L-tyrosine 0.5
Adjust 350 ml of medium containing % to pH 7, dispense into 500 ml shake flasks and sterilize. This is inoculated with Streptococcus fuecalis ATCC12984, and cultured with shaking at 37°C for 16 hours. The cells obtained by centrifugation were suspended in 350 ml of water, and 3.5 g of glucose, 1 mg of pyridoxal phosphate, 175 mg of aluminum sulfate 16-18 hydrate and L-dopa were added.
Add 17.5g, adjust the pH to 5.3 with hydrochloric acid, and raise to 48% at 37℃.
Allow time to react. Thereafter, the same treatment as in Example 1 is carried out to obtain 15.1 g of dopamine hydrochloride.

Claims (1)

[Claims] 1. A microorganism belonging to the genus Streptococcus that has the ability to produce dopamine from L-dopa is
- A method for producing dopamine, which is characterized by producing dopamine by acting on dopa. 2. The production method according to claim 2, wherein the microorganism belonging to the genus Streptococcus is Streptococcus fuecalis. 3 Microorganisms belonging to the genus Streptococcus are
Claim 1: When acting on dopa, glucose is added in an amount of about 0.5 to about 5% after the reaction.
Manufacturing method described in section. 4 Microorganisms belonging to the genus Streptococcus are
4. The method according to claim 3, wherein about 0.01 to about 0.1% of aluminum salt is added after the reaction when acting on DOPA. 5 Microorganisms belonging to the genus Streptococcus are
The manufacturing method according to claims 3 and 4, wherein DOPA is allowed to act at a pH of about 4.5 to 7 and a temperature of about 20°C to about 45°C. 6 Microorganisms belonging to the genus Streptococcus are
3. The production method according to claim 1 or 2, wherein the microorganism is cultured in the presence of L-tyrosine or L-dopa when producing vapamine by acting on dopa. 7 Microorganisms belonging to the genus Streptococcus are
7. The production method according to claim 6, wherein the microorganism is cultured by adding about 0.01 to about 0.5% polyoxyethylene sorbitan monooleate to the medium when producing dopamine by acting on dopa.