JPS61293384A - Production of d-pantoic acid or/and d-pantoic acid salt - Google Patents

Abstract

PURPOSE:To industrially and advantageously produce D-pantoic acid capable of making D-pantolactone which is an important synthetic intermediate for pantothenic acid, CoA, etc., by reducing ketopantoic acid or/and a salt thereof with a microorganism. CONSTITUTION:Ketopantoic acid or a salt thereof or both are reduced with at least one microorganism selected from microorganisms belonging to the genuses Nocardia, Rhodococcus, Nocardioides, Mycobacterium, Strepto sporangium and Streptomyces. The microorganism is previously cultivated and the cultivated microbial cells are added to a substrate solution at 6-10 pH. The supernatant after removing the microbial cells is then acidified and heated to cyclize ketopantoic acid and pantoic acid into ketopantolactone and pantolactone respectively.

Description

Detailed Description of the Invention In the following description, ketobantoic acid or/and its salt will be referred to as KPA, pantoic acid or/and its salt as PA, ketobantolactone as KPL, and pantolactone as PL.
It is abbreviated as Moreover, when PA and PL have optical activity, they are written as DPA, LPA, DPL, and LPL.

The present invention relates to a method for manufacturing DPA.

DPL, which can be more easily derived than DPA, is an important synthetic intermediate for pantothenic acid, CoA, and the like. Traditionally, DP
(1) A method for extracting only D from chemically synthesized DL-PL using an optical resolving agent.

(2) A method of asymmetric reduction of KPL using a rhodium catalyst having a chiral ligand is known, but (1) requires an expensive resolving agent such as quininepursin. (2)
However, this method had drawbacks such as requiring the use of a large amount of expensive catalyst and requiring the reaction to be carried out under hydrogen pressure.

As a result of examining various industrially advantageous methods for producing PA, the present inventors have discovered that K P can be produced by utilizing the reducing power of microorganisms.
It was discovered that A can be advantageously derived into DPA, leading to the present invention.

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

KPA is KPL (Japanese Unexamined Patent Publication No.
It can be easily synthesized by hydrolysis of 8-198480).

Add KPA to the bacterial culture, medium or bacterial cell suspension.
When added, stereospecific reduction occurs and DPA and 5S
0 This product undergoes ring closure easily under acidic conditions, resulting in DP
It becomes L.

To date, there have been almost no reports on the reduction of KPA to υDPA using microorganisms. As a related matter, Wilken et al. reported that KP was applied to processed bacterial cells of Satucharomyces cerevisiae and Escherichia corino.
It merely states that A reductase activity is observed.

The Journal of Biological Chemistry (Th
e Journal of Biological Ch.
emistry) 25th, 2311-2314
The present inventors have repeatedly screened various cultures and soils, and have found that there are a wide range of bacterial strains that reduce KPA and give DPA, and these include molds, yeasts, bacteria, actinobacteria, and basidiomycetes. I learned that bacteria and lactic acid bacteria are present, especially actinomycetes, especially Nocardia spp.
Bacterial strains belonging to the genera Rhodococcus, Nocardioides, Mycobacterium, Streptosprangium, and Streptomyces showed strong reducing ability.

Bacterial culture conditions vary somewhat depending on the strain used, but generally speaking, carbon sources include carbohydrates such as glucose, fructose, shakerose, and maltose, and alcohols such as ethanol and glopanol.

Hydrocarbons, organic acids, etc., and ammonium salts such as ammonium sulfate and ammonium chloride as nitrogen sources.

Nitrates such as potassium nitrate, urea, amino acids, peptone, casamino acids, corn steep licker.

Bran, rice bran, yeast extract, etc.; inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, potassium hydrogen phosphate, potassium dihydrogen phosphate, etc.; malt extract, meat extract as other nutritional sources.

A culture medium containing Pharmamedia etc. is used, but there are special cases.
It is not limited to these. The bacterial strain is inoculated into this medium and cultured aerobically or anaerobically at a culture temperature of 15 to 60°C, more preferably 20 to 60°C.
The temperature is 40°C. In addition, the substrate KPA is added after the bacteria are grown for 1 to 4 days, but KPA
You can add it from the beginning, or you may get better results by adding it in several parts. The substrate KPA is solid, aqueous solution, or its sodium salt, potassium salt,
Added in the form of ammonium salt, etc.

Generally, reactions are carried out under rotary shaking for 12 to 96 hours after substrate addition. The pH of the culture solution or enzyme reaction solution during the reaction varies depending on the bacterial strain used, but is generally pH 6 to 10.
．． Preferably a pH range of 7 to 8 gives good results.

After a certain period of time, it was confirmed that pantoic acid was produced in the culture solution or enzyme reaction solution by using paper chromatography. The Journal of Biology (4'heJournal of Biology)
(logical chemistry) 249゜The supernatant obtained by removing the bacterial cells from these liquids by centrifugation is
After making the mixture acidic with hydrochloric acid or the like, KPA and PA were ring-closed into KPL and PI, respectively.

The amount of KPL and PL produced was determined by gas chromatography (
It was quantified by GC). Furthermore, after converting this PL into diastereomers with D-menthyl chlorocarbonate, G
Analytical Biochemistry (Analytical Biochemistry)
) uJ, 9-16 (1981), the ratio of DPL contained therein was accurately determined. As a result, it was found that all PLs produced in any of the above-mentioned strains were DPLs.

To explain embodiment i of the present invention and an example, for example, peptone 1.5%, yeast extract 0.3X, meat extract 19c.

K2HPO40.3%, NaCj! 0.2%, 1.
1 Nocardia asteroides IF03 in a pH 7.0 liquid medium consisting of 145% 2-propanediol.
384 inoculum was inoculated and cultured aerobically at 28°C for 2 days on a rotary shaker. This culture solution was centrifuged to obtain bacterial cells.

3 g of this wet bacterial body, 100 μg of ketobantic acid, and 0.1 g of calcium carbonate, a solution adjusted to 7
- The reaction was carried out by rotating and shaking at 28°C for 4 days. The product was confirmed to be PA by paper chromatography of the reaction solution. according to the prescribed method υ
After the treatment, analysis revealed that DPL74η was produced.

As the enzyme solution for the reaction, in addition to the bacterial cells, an enzyme-active fraction obtained from the bacterial cells by a known treatment method can also be used.

For example, immobilized bacterial cells or immobilized enzymes obtained by known methods are also effective.

Carrageenan is used as a carrier for immobilization.

Natural compounds such as alginic acid, agar, collagen, gelatin, pectin, or synthetic polymers such as polyacrylamide, polyacrylic acid, ethylene acrylic acid copolymers, photocrosslinkable resins, etc. can be used.
In some cases, good results may be obtained by using bacterial cells treated with powder or dry cells, or by adding a surfactant. Even bacterial cells subjected to mutation treatment are included in the present invention as long as they have the enzyme activity.

The present invention will be specifically explained below with reference to Examples.

Example 1゜Peptone 1.59<, yeast extract 0.3'X, meat extract 1'X, K2HPO40,39, N
aCX O, 2%, 1,2-propanediol 165%
The strains shown in Table 1 were inoculated into a liquid medium at pH 7.0 consisting of the following, and cultured aerobically on a rotary shaker at 28°C for 2 days. Bacterial cells were obtained from this culture solution by centrifugation. 3g of this wet bacterial body.

A solution 10- adjusted to pH 7 consisting of 100η of ketobantic acid and 0.1 g of potassium carbonate was heated at 28°C for 2 days.
The reaction was carried out by rotary shaking. Analysis of the reaction solution by paper chromatography revealed that banthoic acid was produced in all strains. After ring closure by a predetermined method, analysis revealed that D-pantolactone and ketobantolactone were produced in the yields shown in Table 1.

Table 1 Example 2゜Example 1.0 was further added with 1% concentration of ketobantic acid to adjust the pH to 7.0, and Rhodococcus erythropolis IFO12540 was used as the strain at 28°C. The cells were cultured aerobically on a rotary shaker for 5 days. Analysis of the reaction solution by paper chromatography revealed that banded acids were produced.

D-vantolactone and ketovantolactone were produced in 77% and 23% yield, respectively, when analyzed by routine methods.

Applicant: Steel Chemical Industry Co., Ltd. Representative Hiroshi Sasaki

Claims (4)

[Claims] (1) D-banthoic acid or/and which is characterized by reducing ketobanthoic acid or/and a salt thereof using microorganisms.
and a method for producing its salt. (2) The microorganism is a species of the genus Nocardia, the genus Rhodococcus, the genus Nocardioides, the genus Mycobacterium, or the genus Streptosprangium.
ium) and Streptomyces (Streptm)
The method according to claim (1), wherein the method is at least one microorganism selected from the group consisting of microorganisms belonging to the genus P. ices. (3) The method according to claim (1), which uses a culture method when performing the reduction using microorganisms. (4) The method according to claim (1), wherein the bacterial cells separated from the culture solution are added to a newly prepared substrate solution during the reduction using microorganisms. (Enzyme method) (5) The method according to claim (1), wherein the pH during the reduction using microorganisms is 6 to 10.