JPS589678B2 - L- - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-tartaric acid, and more specifically, in a hydrolysis reaction of cis-epoxysuccinic acid using an enzyme capable of ring-opening hydrolysis of an epoxy ring, L-tartaric acid is By adding a surfactant of L
- The objective is to provide an economically superior method for producing L-tartaric acid by reducing the rate of hydrolysis to tartaric acid.

Conventionally, L-tartaric acid has been produced exclusively by an extraction method using raw tartar as a by-product during wine production.

However, in such a production system that relies on natural resources, the raw material situation is always unstable, and in recent years, the supply of L-tartaric acid has been particularly tight due to a shortage of raw tartar.

Therefore, there has been a strong desire to develop a new method for producing tartaric acid using inexpensive petrochemical products as raw materials without worrying about supply sources.

In this sense, the already known method for producing tartaric acid by a synthetic method using maleic acid as a raw material partially meets the above expectations, but because the tartaric acid produced is DL-integrated (racemic), , has drawbacks such as significantly lower solubility than L-tartaric acid, and is difficult from a practical standpoint.

Alternatively, a method of obtaining L-tartaric acid by optical resolution and racemization from the DL of tartaric acid can be considered, but this method increases the number of steps and is inevitably economically disadvantageous.

On the other hand, there have been reports on the production of L-tartaric acid by fermentation using glucose, etc. as a raw material (for example, Japanese Patent Publication No. 33154/1983).
There are some, but the yield is low.

The present inventors have already succeeded in producing L-tartaric acid, which is the same as natural tartaric acid, by combining a synthesis method and a biochemical hydrolysis reaction using maleic acid, which is a petrochemical product that is reliable and inexpensive, as a raw material. We proposed an industrial method for biochemically converting cis-epoxysuccinic acid, which is easily obtained from maleic acid by epoxidation with hydrogen peroxide, into L-tartaric acid (Japanese Patent Application No. 17973/1983).
issue).

The present invention uses an enzyme capable of ring-opening hydrolysis of the epoxy ring of cis-epoxy succinic acid to hydrolyze a cis-epoxy succinic acid derivative under appropriate conditions.
The present invention was completed based on the knowledge that the hydrolysis rate is significantly accelerated by adding a specific surfactant to the reaction solution.

That is, the present invention involves the reaction production of L-tartaric acid from cis-epoxysuccinic acid or its derivatives in the presence of an enzyme capable of ring-opening hydrolysis of an epoxy ring, in which (1) a higher fatty acid salt, an alkyl At least one anionic surfactant selected from the group consisting of phosphate esters and alkyl polyoxyethylene phosphate esters, or (2) polyoxyethylene alkyl ethers, polyoxyethylene sorbicun fatty acid esters, and polyoxyethylene esters. This is a method for producing L-tartaric acid, which comprises adding at least one nonionic surfactant selected from the group consisting of polyoxypropylene block polymers.

The cis-epoxysuccinic acid or derivative thereof used in the present invention can be easily obtained by epoxidizing maleic anhydride, maleic acid, or a derivative thereof using a known method, for example, with hydrogen peroxide.

As derivatives of cis-epoxysuccinic acid, its inorganic salts such as sodium, potassium, calcium and ammonium salts, and water-soluble ones such as organic salts, esters and amides are used.

The concentration of cis-epoxysuccinic acid or its derivative used is 0.1-3 mol/l, preferably 0.1-1.5 m
ol/l is good.

For example, as cis-epoxy succinic acid is hydrolyzed to L-tartaric acid, solid or concentrated cis-epoxy succinic acid solution may be added sequentially to maintain the concentration of cis-epoxy succinic acid at about 1 mol/l. That's a good method.

Reaction temperature is 20-60℃, preferably 25-45℃, PH
is preferably 5 to 10, preferably 6 to 9.

In addition, the enzyme used in the present invention is a microorganism that has the ability to generate an enzyme that cleaves and hydrolyzes the epoxy ring of cis-epoxysuccinic acid, such as Nocardia tatricans, a species previously proposed by the present inventors.
rtaric ans NOV. SP) (Feikoken Bibori No. 2882), etc., the usual methods include the crushing method in which auxiliaries such as glass powder or alumina powder are added and ground in a mortar, the high-pressure method using a French press, Cells may be disrupted by sonication, ultrasonication, or other methods, and the cell wall fraction may be separated by centrifugation, and the supernatant may be used as a crude enzyme solution.

The above-mentioned microorganisms are usually cultured by liquid culture, and also by solid surface culture.

As a culture medium, for example, a natural medium such as meat juice can be used, but an appropriate carbon source (for example, glucose, sucrose, glycerin, ethanol, improbanol, propylene glycol, n-paraffin, etc.), nitrogen source (for example, ammonium salt) can be used. Synthetic media consisting of other inorganic salts (such as nitrates, nitrates, urea, etc.) are also used.

Furthermore, during culture, it is desirable to adaptively enhance necessary enzymes by adding cis-epoxysuccinic acid, which serves as a substrate, to the medium.

The pH of the medium is in the range of 5.5-11, preferably 6-9.

The culture is carried out aerobically, and the culture temperature is 10-45°C, preferably 25-40°C.

Batch culture is usually carried out for half a day to 10 days.

After completion of the culture, centrifugation is performed, and the resulting bacterial cells are dissolved in water or a medium from which the carbon source has been removed from the synthetic medium described above, in which cis-epoxysuccinic acid is dissolved at a concentration of 1 mol/l or less (PH 6 to 9). It is preferable to carry out enzyme induction at 20 to 40°C for 5 to 30 hours.

The hydrolysis reaction in the present invention can be carried out in any manner such as a batch method, a semi-batch method, or a continuous method.

The surfactant to be added to the reaction system, which is a feature of the present invention, is limited to specified anionic surfactants and nonionic surfactants, including cationic surfactants such as cetyltrimethylammonium bromide and laurylpyridinium chloride. The surfactant has an inhibitory effect rather than a promoting effect on the ring-opening hydrolysis of the epoxy moiety.

On the other hand, among anionic surfactants, sulfonates such as alkyl sulfate ester salts and alkylbenzene sulfonates act as inhibitors, while higher fatty acid salts such as sodium laurate. Alkyl (polyoxyethylene) phosphate ester such as DDP-8 (manufactured by Nikko Chemicals). Anionic materials such as alkyl phosphate esters such as SLP (manufactured by Nikko Chemicals) exhibit a remarkable promoting effect.

Also, among nonionic surfactants, Tween-80
Polyoxyethylene sorbicun fatty acid esters such as (manufactured by Kao Atlas Co., Ltd.), polyoxyethylene-polyoxypropylene block polymers such as Pluronic L-61 (manufactured by Nikko Chemicals Co., Ltd.), BL-4.2 (manufactured by Nikko Chemicals Co., Ltd.) Polyoxyethylene alkyl ethers such as are effective.

The concentration of the surfactant used in the reaction system is 1 to 0.0.
01 wt% is preferable, and the optimum concentration varies depending on the type of surfactant.

In this way, in the present invention, the hydrolysis rate of cis-epoxysuccinic acid to L-tartaric acid is improved by adding a specific surfactant to the reaction solution, and as a result, the rate of hydrolysis of cis-epoxysuccinic acid to L-tartaric acid is improved.
-It promotes the production of tartaric acid, but there is no knowledge of the mechanism that promotes its hydrolysis rate, and it is unclear.

Therefore, both the anionic and nonionic surfactants specified in the present invention were discovered through statistical experiments.

In addition, a cationic surfactant consisting of a quaternary ammonium salt such as cetyltrimethylammonium bromide is used in the reaction production of L-tartaric acid from cis-epoxysuccinic acid using a microorganism that has the ability to hydrolyze cis-epoxysuccinic acid. significantly accelerates its production rate, but when using an enzyme capable of hydrolyzing cis-epoxysuccinic acid isolated from cells as in the present invention, it acts as an inhibitor; We believe that surfactants have a completely different mechanism of action in this reaction system.

In the present invention, after the reaction is completed, L-tartaric acid can be easily obtained from the reaction solution by a known method such as precipitation as calcium tartrate.

The obtained monotartaric acid was confirmed to be natural L-tartaric acid from the Rf value of paper chromatography, mobility of filter paper electrophoresis, IR, NMR optical rotation, elemental analysis, etc.

EXAMPLES The present invention will be specifically explained below with reference to Examples, but the present invention is by no means limited to these examples.

Example 1 Propylene glycol 1% NH2CONH2 0.3%KH2PO
4 0.1 5%Na2HP04
o. 1 5%MgS04・7H20
0.05%FeSO ・7H20 0.0
01%4 CaCl2・2H20 0.001%MnSO
4.4H20 0.0002% yeast extract
0.02% PH 7.0 A culture medium containing components other than urea was previously steam sterilized at 121°C for 15 minutes, urea that had been sterilized by filtration was added, the pH was adjusted with caustic soda, and the pH was finally adjusted to the above composition.
The medium was adjusted so that 50 ml of the medium was dispensed into a 500 ml shake flask with a shoulder.

In this medium, the bacterial species Nocardia tartaricans (No.
cardia tartaricans NOV. S
One platinum loop of P) (Feikoken Bacterium No. 28820) was inoculated and cultured at 30°C for 100 hours.

After culturing, the bacterial cells were collected by centrifugation, propylene glycol was removed from the above medium, and the dried bacterial cells were added to a medium containing 0.2 mol/l of sodium cis-epoxysuccinate at a concentration of 5 g/l. Suspend at a concentration of 30
Enzyme induction was performed at ℃ for 20 hours.

Collect bacteria again by centrifugation and add 0.7 mOl/l cis-sodium epoxy succinate solution (PH7.0)10
ml to a concentration of 10 g/l as dry cells, cooled to 0 to 5°C, and sonicated (20 g/l).
KHZ, 60W, 10 minutes).

After ultrasonication, the surfactants shown in Table 1 were added to the supernatant from which the cell wall fraction had been removed at a predetermined concentration, and the reaction was carried out at 37° C. with shaking.

After 5 hours, the L-tartaric acid produced was quantified and the results are shown in Table 1.

A significant acceleration of the reaction rate is observed by the addition of surfactant.

Comparative Example 1 Into 10 ml of the enzyme solution of sodium cis-epoxysuccinate (PH 7.0) prepared in Example 1, cetyltrimethylammonium bromide, a cationic surfactant, and lauryl, a sulfate of anionic surfactants, were added. Sodium sulfate and sodium laurylbenzenesulfonate at 0.1, 0.05, 0.025, respectively.
A similar reaction was carried out by adding 0.0125 wt%, but no production of L-tartaric acid was observed in 5 hours.

Example 2 In Example 1, the surfactants DDP-8 and Pluronic L-6 1 were added to 0.02
For the system with 5% addition, after 5 hours, 1. 4mol/l (PH
7.0) 10 ml was added and the reaction was carried out in the same manner.

After 22 hours, L-tartaric acid was 156g/7,100g/
It was produced at a concentration of 1.

Claims (1)

[Claims] 1. When producing L-tartaric acid from cis-epoxysuccinic acid or its derivatives in the presence of an enzyme capable of cleaving and hydrolyzing an epoxy ring, (1) a higher fatty acid salt is present in the reaction solution. , at least one anionic surfactant selected from the group consisting of alkyl phosphate esters and alkyl polyoxyethylene phosphate esters, or (2) polyoxyethylene alkyl ethers, polyoxyethylene sorbicun fatty acid esters, and polyoxy A method for producing L-tartaric acid, which comprises adding at least one nonionic surfactant selected from the group consisting of ethylene polyoxypropylene block polymers.