JPS61293386A - Production of d-pantoic acid salt or/and d-pantolactone - Google Patents

Abstract

PURPOSE:To obtain the aimed substance readily, by oxidizing an L-pantoic acid salt or/and L-pantolactone with a microorganism to give a ketopantoic acid salt or/and ketopantolactone and reducing the resultant compound with a microorganism to afford the titled substance. CONSTITUTION:A strain, selected from microorganisms belonging to the genus Nocardia, Rhodococcus, Nocardioides, Mycobacterium, Streptosporangium, Corynebacterium, etc., and having both oxidative and reductive ability is cultivated in a culture medium. An L-pantoic acid salt or L-pantolactone or both are reacted with a culture of the above-mentioned microorganism or separated microbial cells, etc., passed through a ketopantoic acid salt or/and ketopantolactone and converted into the aimed d-pantoic acid salt or/and D- pantolactone. In the process, a polyol, e.g. 1,2-propanediol, is added to the culture medium or substrate solution to improve the oxidative activity.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] The present invention relates to a method for producing D-vantolactone from L-vantolactone via ketopantolactone.

(Industrial Application Field) D-bantolactone is an important synthetic intermediate for pantothenic acid, CoA, etc.

(Prior art) (Problems to be solved by the invention) Ketopantolactone is converted into pantothenic acid and C by asymmetric reduction.
This leads to D-bantolactone, which is an important synthetic intermediate such as oA. Conventionally, ketopantolactone is chemically synthesized from chemically synthesized DL-vantolactone using an oxidizing agent such as bromine or calcium hypochlorite.

However, in this method, the oxidizing agent is expensive,
This is not a good method because D-bantolactone, which is a starting material for pantothenic acid, CoA, etc., is also oxidized at the same time.

On the other hand, D-bantolactone is obtained by (1) a method of extracting only D-unit from chemically synthesized DL-vantolactone using an optical resolution agent; (2) A method of asymmetric reduction of ketopantolactone using a rhodium catalyst having a chiral ligand is known, but (1) requires an expensive resolving agent such as quinine or brucine.

(2) requires the use of large amounts of expensive catalyst;
・Since the reaction was carried out under hydrogen pressure, it had drawbacks such as being difficult to handle.

The present inventors have studied various industrially advantageous methods for producing monopantoate salts and/or D-bantolactone.

That is, focusing on the stereoselectivity of microorganisms, we searched for strains that oxidized only L-vantolactone to give it a keto form, and
As a result of screening, it was found that a specific strain converts only L-vantolactone into ketopantolactone. (
DL-vantolactone or L.
- When vantolactone is oxidized, only L-vantolactone changes to ketopantolactone without any change in D-vantolactone.

Furthermore, it was discovered that ketopantoic acid, a salt thereof, or ketopantolactone could be advantageously converted into D-banthoic acid, a salt thereof, and/or D-vantolactone by utilizing the reducing power of microbial cells, and the patent was granted. I applied.

(Japanese Patent Application Laid-Open No. 59-25690) Adding ketopantolactone as a solid or as an aqueous solution to a culture obtained by culturing bacteria, a culture medium, a suspension of bacteria, or a bacterial suspension taken from a culture solution. Ketopantolactone exists in equilibrium with its open ring form. When this ring-opened product is stereospecifically reduced by microorganisms, it becomes D-banto acid salt. Further, when ketopantolactone is directly stereospecifically reduced, it becomes D-vantolactone. Here, D-banto acid salt and D
- Equilibrium also exists between vantolactones, and if you want to obtain only D-bantolactone, acidification will cause ring closure, and 9D
- Banto acid salt becomes D-bantolactone.

However, this method requires two microbial treatments because different microorganisms are used in the oxidation reaction and the reduction reaction. This inevitably increased the cost of culturing and the number of steps, making it still not an economical method.

[Structure of the invention]

The gist of the present invention is a method for producing a ketopantoate salt or/and a ketopantolactone, which is characterized by oxidizing the L-banto salt salt or/and L-bantolactone using a microorganism, and D-banto acid salt or/and D- which is characterized by being reduced using microorganisms
In the former case, the produced ketopantolactone can be reduced by a conventionally known method, but according to the latter method,
L-banto acid salt or/and L-bantolactone can be oxidized and reduced in the same system using the same microorganism using L-banto acid salt or/and L-bantolactone as a starting material, and D-banto acid salt or/and D-bantolactone can be obtained once. This is more preferable because only microbial treatment is required.

(Means for solving the problem) (Effect) Using the strain of the present invention, DL-bantolactone or L-
When vantolactone is oxidized, only L-vantolactone changes to ketopantolactone without any change in D-vantolactone.

The obtained ketopantolactone can be easily converted into D-bantolactone by microbiological reduction (Japanese Unexamined Patent Application Publication No. 1989-1992).
9-25690) As a result, D-vantolactone is obtained from DL-vantolactone.

That is, the present invention can be expressed by the following formula.

D-vantolactone microorganism D-vantolactone (L-
Vantolactone 'v'r' (Ketopantolactone) Furthermore, the present inventors extensively screened for strains that possess both the above-mentioned oxidizing and reducing abilities. When a bacterial strain is found and used, it has both oxidation and reduction abilities, and therefore L-vantolactone can be converted to D-vantolactone all at once in the same system.

The microorganism used in the method of the present invention is Nocardia (Noc.
ardia), Rhodococcus
us ) jJi.

Nocardioides
Genus, Mycobacterium (Mycobacterium iu)
m) genus, Streptosprangium (Strepto
sprangium), Streptomyces (St
reptomyces), Corynebacterium (C
At least one microorganism selected from the group consisting of microorganisms belonging to the genus Orynebacterium), and furthermore, for these strains, a reduction reaction of ketopantolactone to D-vantolactone occurs simultaneously in parallel with the oxidation reaction. I found out that

Bacterial culture conditions vary somewhat depending on the strain used, but generally speaking, carbohydrates such as glucose, fructose, shakerose, and maltose are used as carbon sources, and ammonium sulfate, ammonium chloride, potassium nitrate, urea, amino acids, and peptone are used as nitrogen sources. , casamino acids, corn stave licker, bran, rice bran, yeast extract, etc., and inorganic salts such as magnesium sulfate, sodium chloride, and calcium carbonate.

Culture media containing malt extract, meat extract, Pharmamedia, etc. as other nutritional sources such as potassium hydrogen phosphate and potassium dihydrogen phosphate are used, but are not limited to these.

The present inventors have discovered that the oxidative activity of bacterial cells is improved by adding a type of polyol that is rare in nature, such as 1,2-propanediol, to the culture medium or reaction system. The polyol added here can be used as a 1,2-
It is not limited to propanediol.

The bacterial strain is inoculated into this medium and cultured aerobically or anaerobically. The temperature suitable for culturing is 15 to 60°C, more preferably 20 to 40°C. Bacteria are usually grown for 2 to 6 days, but the substrate vantolactone may be added from the early stage of culture, or may give better results if added in several batches.

Furthermore, a reaction was carried out by mixing the bacterial cells taken out from the culture solution with pantolactone, acetone, po
In some cases, good results may be obtained by using bacterial cells treated with wder or dry cells, or by adding a surfactant.

Disintegrated microbial cells or immobilized microbial cells may be used, and high activity may be obtained by mutation treatment.

The substrate vantolactone can be a solid or an aqueous solution or
It is added in the form of its sodium salt, potassium salt, ammonium salt, etc.

The pH when converting the substrate using the above-mentioned culture method or bacterial cell method is preferably in the range of 6 to 1O, more preferably 7 to 1O.
Good results can be obtained by keeping it in the range of 8. At this time, good results can be obtained by adjusting the pH with an acid such as hydrochloric acid or sulfuric acid, or a base such as sodium hydroxide, potassium hydroxide, or ammonia, if necessary.

For example, in one embodiment of the present invention, peptone 1.5%, yeast extract 0.3%, and meat extract 1%.

The cells were cultured aerobically on an inclined shaker in a liquid medium 5- consisting of 0.3% K2HPO4, 2% NaCf1O and 1.5% 1.2-propanediol. The culture solution obtained in this way is centrifuged to remove bacterial cells, and then treated with hydrochloric acid to cyclize pantoic acid or ketopantoic acid into vantolactone or ketopantolactone, resulting in the production of ketopantolactone. The proportion of D-vantolactone in pantolactone was analyzed by gas chromatography, and the proportion of OD-vantolactone was determined by
L-Bant lactone can be added in the process of the present invention by D-chloromenthyl carbonate L-Bant 2
The concentration of chotons or DL-Bant 2 chthons is usually between 1 and 1
A range of 0% is appropriate. At high concentrations, ketopantolactone is produced in good yields.

In addition, ketopantolactone, which is produced when the concentration is low and the amount of bacterial cells is increased and the treatment time is prolonged, is further reduced and converted to D-bantolactone at a good yield.

Next, a specific example of the manufacturing method of the present invention will be explained using Examples.

Example 1 Glycerol 196', peptone 1.5%, yeast extract 0.3%, K2HPO40.3%, NaC10.2
A liquid medium consisting of 0.5%
The mixture was dispensed into In test tubes and sterilized by heating at 121° C. for 15 minutes in an autoclave. One platinum loopful of the inoculum shown in Table 1 was inoculated from the slant medium, and cultured aerobically on a rotary shaker at 28°C for 4 days. The culture fluid thus obtained was analyzed by a prescribed method, and the results shown in Table 1 were obtained. Except for the ketopantolactone produced, it is vantolactone. The proportion of D-vantolactone in vantolactone is D −r
It is shown in atio (%).

Table 1 Example λ Example 1 without L-bantolactone. A liquid medium was used. One platinum loopful of the inoculum shown in Table 2 was inoculated from the slant medium and cultured aerobically on a rotary shaker at 28°C for 2 days. Then, L-bantolactone was added to Klwt so that the following was obtained, and the mixture was further shaken for 4 days to obtain the results shown in Table 2.

Table 2 Example 3 Glucose 4%, Polypeptone IN, Yeast Extract 0.5
%, except that a pH 7 medium consisting of 0.5% potassium dihydrogen phosphate and 0.2% magnesium sulfate was used.
The same procedure as in Example 2 was carried out to obtain the results shown in Table 3.

Table 3 Example 4 Example 1 except that 0.5% of 1.2-propanediol was added to the medium. I did the same thing and got the result of 4th tail.

Table 4 Example 5゜Example 4. The reaction was carried out using bacterial cells cultured for 2 days in the following medium. That is, two test tubes (5
0.1 g of bacterial cell r/cL-bantolactone of dX2)
(2%), 0.2M potassium phosphate buffer (pH 7.0
) 5- was added and shaken at 28°C for 4 days. This reaction solution was analyzed and the results shown in Table 5 were obtained.

Table 5 Example 6 A reaction was carried out using cells of Nocardia asnaloides (IFO3384). Example 5 except that the reaction time was 6 days. It is similar to As a result of the analysis, ketopantolactone, D-bantolactone, and L-vantolactone were present in the reaction solution.
Example 7. Example 4. Vantolactone was contained in yields of 92.1%, 7.0% and 0.9%, respectively. The reaction was carried out using bacterial cells obtained in the same manner as described above. Add 0.1 g (2%) of L-bantolactone to 2 test tubes (5dX2) of Nocardia asnaloides (IF03384) cells, and add 0.2M of 2HPO4.
5- was added and shaken at 28°C for 4 days.

When this reaction solution was analyzed, the yield of ketopanto 2 chtons was 1 (
10)%.

Example 8 A reaction was carried out using Nocardia asteroides (IF03384) cells cultured in the medium of Example 2 for 2 days. Add 0.05 g (IN) of 1,2-propanediol to 2 test tubes (5 mj x 2) of bacterial cells.

0.1 g (2%) of L-vantolactone and 0.2 M potassium phosphate buffer (pH 7) were added, and the mixture was shaken at 28° C. for 4 days. As a result of analysis, the reaction solution contained ketopantolactone, D-vantolactone, and L-vantolactone in yields of 87.1%, 7.5%, and 5.4%, respectively.

Example 9 Example 7 except that a pH 8 solution containing 0.25 g of L-vantolactone and 0.3 M potassium phosphate buffer was used, and the reaction time was 2 days. I did the same thing.

Analysis of the reaction solution revealed that ketopantolactone, D-vantolactone, and L-vantolactone were each 83.3%.
, 7.5 and 9.2% yield.

Example 10° Example 9 except that 0.50 g of DL-vantolactone was used instead of L-vantolactone. I did the same thing. When the reaction solution was analyzed, ketopantolactone, D-vantolactone, and L-vantolactone were found to be 40% each.
．． 6%, 53.1% and 6.3% yields.

Example 11゜Peptone 1.5%, yeast extract 0.3%, meat extract 1%
, K2HPO40.3%, NaCA O.2%, 1
, 2-propanediol 1.5% pH 7.0
Nocardia asteroides IFO3 in liquid medium
384 was inoculated and cultured aerobically at 28°C for 2 days on a rotary shaker. Bacterial cells were obtained from this culture solution by centrifugation. 2 g of this wet bacterial body, 0.0 g of DL-bantolactone.
A reaction was carried out by rotating and shaking a solution of 8 g and 0.1 g of calcium carbonate adjusted to pH 7 at 28° C. for 2 days. As the reaction progressed, the pH of the reaction system decreased, so the pH was adjusted to 7 with NaOH solution every 6 hours. Analysis of the resulting reaction solution revealed that D-vantolactone, L-vantolactone, and ketopantolactone were produced in yields of 57%, 3%, and 40%, respectively.

Example 12゜L-vantolactone 0 instead of DL-vantolactone
．． Example 11 except that 8g was used. I did the same thing.

Analysis of the resulting reaction solution revealed that D-bantolactone, L
- 18% vantolactone and ketopantolactone;
It occurred in 1% and 81% yield.

Example 13 As a bacterial strain, Rhodococcus erythropolis IFO12
Example 11 except that 540 was used and 0.5 g of L-vantolactone was used instead of DL-vantolactone. I did the same thing. Analysis of the resulting reaction solution revealed that D-vantolactone, L-vantolactone, and ketopantolactone were produced in yields of 32%, 27%, and 41%, respectively.

Example 14゜The amount of bacterial cells during the enzyme reaction was doubled, and L-bantolactone 1
Example 13 except that (10)q was used and the reaction time was 4 days. I did the same thing. Analysis of the resulting reaction solution revealed that D-vantolactone, L-vantolactone, and ketopantolactone were produced in yields of 88%, 3%, and 9%, respectively.

Example 15 The same procedure as in Example 1H was carried out except that the strains shown in Table 6 were used, and the results shown in Table 6 were obtained.

Table 6 Example 16゜DL-vantolactone 1 instead of L-vantolactone
(10) Example 14 except that 7a9 was used. I did the same thing. Analysis of the resulting reaction solution revealed that D-vantolactone, L-vantolactone, and ketovantolactone were produced in yields of 96%, 1%, and 3%, respectively.

〔Effect of the invention〕

By carrying out the present invention, D-
Vantolactone can be produced industrially from L-vantolactone. In particular, the method of performing oxidation and reduction in the same system using one type of bacterial cell is unprecedented, and will greatly contribute to the pharmaceutical industry.

Claims (1)

[Claims] (1) A method for producing a ketobanto acid salt or/and a ketobantolactone, which comprises oxidizing the L-banto acid salt or/and L-bantolactone using a microorganism. (2) The method according to claim (1), wherein the L-banto acid salt or/and L-bantolactone is included in the DL-banto acid salt or/and DL-bantolactone. (3) The microorganism is a species of the genus Nocardia, the genus Rhodococcus, the genus Nocardioides, the genus Mycobacterium, or the genus Streptosprangium.
ium), Streptomyces
ces) genus, Corynebacterium (Coryne-ba
Claims (1) are at least one microorganism selected from the group consisting of microorganisms belonging to the genus Cterium).
) method described. (4) The method according to claim (1), which uses a culture solution when oxidizing using microorganisms. (Culture method) (5) When oxidizing using microorganisms, the bacterial bodies isolated from the culture solution are
Claims (1) added to freshly prepared substrate solution
) method described. (Bacterial cell method) (6) The method according to claim (1), wherein the pH during oxidation using microorganisms is 5 to 10. (7) The method according to claim (1), wherein the culture medium is cultured by adding a polyol. (8) Claims of adding a polyol to the substrate solution (
5) The method described. (9) The method according to claim (7) or (8), wherein the polyol is 1,2-propanediol. (10) A method for producing D-banto acid salt or/and D-bantolactone, which comprises converting L-banto acid salt or/and L-bantolactone using a microorganism. (11) The method according to claim (10), wherein the L-banto acid salt or/and L-bantolactone is included in the DL-banto acid salt or/and DL-bantolactone. (12) The microorganism is Nocardia.
Genus, Rhodococcus, Nocardioides, Mycobacterium,
Streptosprangium (Streptosprangium)
genus Streptomyces, Streptomyces
yces), Corynebacterium (Coryne-b
The claimed invention is at least one type of microorganism selected from the group consisting of microorganisms belonging to the genus Acterium (
10) The method described. (13) The method according to claim (10), which uses a culture solution during the conversion using microorganisms. (Culture method) (14
) The method according to claim (10), wherein when converting using microorganisms, bacterial cells separated from the culture solution are added to a freshly prepared substrate solution. (Bacterial cell method) (15) The method according to claim (10), wherein the pH during conversion using microorganisms is 6 to 10. (16) The method according to claim (10), wherein the culture medium is cultured by adding a polyol. (17) The method according to claim (14), in which a polyol is added to the substrate solution. (18) The method according to claim (16) or (17), wherein the polyol is 1,2-propanediol.