JPH0655156B2 - Optical resolution method of DL-pantolactone - Google Patents

Description

TECHNICAL FIELD The present invention relates to an optical resolution method for DL-pantolactone.

(Prior Art) Conventionally, D-pantolactone has been obtained by optically resolving chemically synthesized DL-pantolactone. However, this division has a drawback that an expensive organic base such as quinine or brucine is required, and its recovery is not easy.

On the other hand, as a biochemical resolution method of racemic pantolactone, JP-B-47-19745 and JP-A-57-1528.
There is a method described in Japanese Patent Publication No. 95. The former, by completely digesting L-pantolactone by the action of microorganisms,
It obtains D-pantolactone and has a drawback that half of the substrate is lost.

The latter causes the racemate to act on a microorganism belonging to the genus Rhodotorula and specifically hydrolyze only the L-form.
-A method of separating and collecting the body. In the latter method, L-pantoic acid which is a hydrolyzate is racemized and reused as a substrate to obtain D-pantolactone in good yield.

(Problems to be Solved by the Invention) However, when L-pantolactone is hydrolyzed, L-pantoic acid is produced, the pH in the reaction solution is lowered, and the enzymatic reaction rate is significantly lowered.

Therefore, a method of using a phosphate buffer solution to maintain the pH has been proposed. (JP-A-57-152895) However, when such a buffer solution is used, the target D-pantolactone is also hydrolyzed by hydrolysis due to a chemical reaction, and there is a drawback that the yield is lowered.

(Means for Solving the Problems) Therefore, as a result of intensive studies to solve the above-mentioned problems, the present inventors have found that L-punt which is produced by dropping an aqueous solution of an inorganic basic compound. The inventors have found that L-pantolactone can be specifically decomposed by a hydrolase without neutralizing the acid and almost chemically hydrolyzing the desired D-pantolactone, and arrived at the present invention.

That is, the gist of the present invention is that L-pantolactone belongs to the genus Rhodotorula and L-pantolactone is added to L-pantolactone by using an aqueous solution of an inorganic basic compound in DL-pantolactone while maintaining the pH at 4 to 7.5.
An optical resolution method of DL-pantolactone is characterized in that a hydrolase of a microorganism having an ability to hydrolyze to pantoic acid is caused to act.

Hereinafter, the present invention will be described in detail.

Any microorganisms belonging to the genus Rhodotorula and having an enzyme capable of specifically hydrolyzing L-pantolactone are useful. A representative example thereof is Rhodotorula minuta Var texensis IFO 0412 Rhodotorula rubra IFO 0696.

The medium used for culturing these microorganisms is not particularly limited. Examples of carbon sources include various carbohydrates and organic acids, and examples of nitrogen sources include organic ammonium salts and
Inorganic ammonium salts, urea and the like can be used. If necessary, various phosphates, magnesium salts and the like can be used as the inorganic substance, and various organic nutrients can be added as necessary.

Culturing is usually performed under aerobic conditions for about 12 hours to 7 days. The pH of the medium is selected from 3 to 10 and the temperature is selected from about 20 to 40 ° C.

In the present invention, DL-pantolactone can be treated with a hydrolase of the above-mentioned microorganism by treating the culture obtained by culturing the strain in a liquid medium, the cells separated from the culture medium, or the cells or the culture. Any form such as the obtained dried bacterial cells or solidified bacterial cells, enzyme solution, or immobilized enzyme can be used.

The reaction can be carried out batchwise, semi-batchly, or continuously. In the reaction, a racemic pantolactone concentration of about 10 to 300 w / v% is usually adopted. The reaction temperature is usually 10 to 50 ° C. and the pH is maintained at 4 to 7.5, preferably about 6 to 7. The inorganic base for maintaining the pH is usually K,
Inorganic alkalis such as alkali metals such as Na, hydroxides and carbonates of alkaline earth metals such as Ca and Ba, preferably hydroxides of alkali metals are used. The concentration of the alkali for neutralization varies depending on the reaction conditions, etc., but is usually 0.1-
It is selected from 3 specified temperatures.

The reaction time varies depending on the reaction conditions and the like, but in the case of a batch system, it is usually selected from several hours to about 3 days. After the reaction is completed, D
-Pantolactone can be separated and obtained by operations such as fractional crystallization and solvent extraction. The L-pantoic acid remaining in the reaction solution is heated under acidic conditions to give L-pantolactone and then recovered by solvent extraction or the like. This L-pantolactone can also be recovered after being racemized by a conventional method.

(Examples) The present invention will be described in more detail with reference to the following examples.

Example 1 Rhodotorula minuta Vartexensis IFO 0412 was added to a 200 m Erlenmeyer flask containing 5 m of the medium having the following composition.
It was cultured at 8 ° C. for 48 hours.

Medium: Glucose 10 g Peptone 5 g Yeast extract 5 g Conste-Plica 5 g Water 100 m (pH 6.5) After culturing, the cells were collected by centrifugation. After washing once with distilled water, 1.0 g of DL-pantolactone was added and 50 m with distilled water
Then, the reaction was carried out at 30 ° C. for 24 hours while controlling the pH (pH 6.5 to 7.0) by dropping a 0.3N NaOH aqueous solution.

After removing bacterial cells from the reaction solution by centrifugation, the reaction solution was extracted with 200 m of benzene, and benzene was distilled off from the extract under reduced pressure to obtain 410 mg of D-pantolactone. On the other hand, the raffinate was adjusted to pH 2.0 with hydrochloric acid, heat-treated at 100 ° C. for 10 minutes, extracted with 200 m of benzene, and L-
420 mg of pantolactone was obtained.

The analysis of D and L-pantolactone was performed using gas chromatography.

(Anal. Biochem., The method 112 9-16 (1981) described YoTsuta.) Comparative Example 1 in the same manner as in Example 1 Rhodotorula Minuta Barr Tekusenshisu (Rhodotorula minuta Vartexensis) were cultured IFO 0412. Next, the reaction and post-treatment were carried out in the same manner as in Example 1 except that the reaction solution was reacted with 0.5 M phosphate buffer instead of controlling the pH with NaOH.

Results Only 350 mg of D-pantolactone was obtained.

Example 2 Rhodotorula minuta Vartexensis IFO 1102 was cultured in the same manner as in Example 1. Next, the reaction and post-treatment were carried out in the same manner as in Example 1 except that the alkali was changed to 0.2N KOH aqueous solution.

Results D-pantolactone 407 mg, L-pantolactone 4
30 mg was obtained.

(Effect of the Invention) According to the method of the present invention, the desired D-pantolactone can be obtained in high yield.

Claims (1)

[Claims] 1. A microorganism belonging to the genus Rhodotorula having the ability to hydrolyze L-pantolactone to L-pantoic acid while maintaining the pH at 4-7.5 by using an aqueous solution of an inorganic basic compound in DL-pantolactone. A method for optically resolving DL-pantolactone, which comprises allowing a hydrolase to act.