JPS61242586A - Production of ketopantoic acid salt and/or ketopantolactone - Google Patents

Abstract

PURPOSE:To produce the titled compound useful as a synthetic intermediate for pantothenic acid, CoA, etc., economically on an industrial scale, by oxidizing L-pantoic acid salt or L-pantolactone with microorganism. CONSTITUTION:The objective substance is produced by oxidizing L-pantoic acid and/or L-pantolactone (preferably contained in DL-pantoic acid salt and/or DL-pantolactone) with microorganism (preferably belonging to Nocardia genus, Rhodococcus genus, Nocardioides genus, Mycobacterium genus, Streptosprangium genus or Corynebacterium genus) preferably at 5-10pH.

Description

[Detailed description of the invention] Ketopantolactone is converted into bantothenic acid, C
This leads to D-pantolactone, which is an important synthetic intermediate such as oA. Conventionally, ketopantolactone is chemically synthesized from chemically synthesized DL-pantolactone 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-pantolactone, which is a starting material for pantothenic acid, CoA, etc., is also oxidized at the same time.

In order to improve these drawbacks, the present inventors focused on the stereoselectivity of microorganisms, and searched for strains that oxidize only L-pantolactone to give the keto form. As a result of screening, a specific strain was found. It has been found that only L-pantolactone can be converted into ketopantolactone.

Using the strain of the present invention, DL-pantolactone or L-
When pantolactone is oxidized, only L-pantolactone changes to ketopantolactone without any change in D-pantolactone.

The present invention can be expressed by the following formula -■, and the obtained ketopantolactone can be easily converted to (■)-pantolactone by microbiological reduction.
0) As a result, D-pantolactone is obtained from I) [,-pantolactone.

To explain the present invention in detail, for example, glycerol 1.0
%, peptone 1.5%, yeast extract 0.3%.

The cells were cultured aerobically on a slant shaker in a liquid medium 5-5 consisting of 40.3% K2HPO, 0.2% NaCji and 0.5X L-pantolactone. The thus obtained culture solution is centrifuged to remove bacterial cells and then treated with local acid to cyclize pantoic acid or ketobandic acid into pantolactone or ketopantolactone. The amount of D-pantolactone produced and the proportion of D-pantolactone in pantolactone were analyzed by gas chromatography.

The proportion of D-pantolactone was determined by converting DL-pantolactone into diastereomers using menthyl chlorocarbonate and then analyzing the diastereomer by gas chromatography. (Anal, IIHochem,, IJ, 1
According to the method of the present invention, strains that give ketopantolactone in high yield are strains of the genus Nocardia, genus Rhodococcus, .
It is said that the song belongs to the genus Nocardioides, Mycobacterium, Streptospurangichnos, and Corynebacterium. Furthermore, it was found that in these strains, a reduction reaction of ketopantolactone to D-pantolactone occurred simultaneously with the oxidation reaction. When the pH of the reaction system was set to 5 to 10, reduction of the produced ketopandolactone was suppressed, so that ketopandolactone was produced in a good yield.

Bacterial culture conditions vary somewhat depending on the strain used, but generally speaking, carbon sources include carbohydrates such as glucose, 7-lactose, sucrose, and maltose, and nitrogen sources include ammonium sulfate, ammonium chloride, potassium nitrate, urea, amino acids, Peptone, casamino acid, corn stew licker, bran, rice bran, yeast extract, etc., inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, phosphoric acid-water potassium phosphate, potassium dihydrogen phosphate, etc.
Other nutritional sources include malt extract and meat extract.

A medium containing Pharmamedia I and the like may be used, but is not limited thereto.

The present inventors have discovered that the oxidative activity of bacterial cells is improved by adding a polyol, which 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 DL-pantolactone may be added from the early stage of the culture, or may give better results if added in several batches. Furthermore, by mixing the bacterial cells taken out from the culture solution and pantolactone, a reaction is carried out,
acetone powder 'p dry cel
In some cases, good results may be obtained by using microbial cells that have been treated with L. 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 pantolactone can be prepared as a solid solution or an aqueous solution, or
It is added in the form of its sodium salt, potassium salt, ammonium salt, etc. The pH of the culture is 4-9, and the pn of the reaction is 4-9.
12 is preferred. On the slightly acidic to alkaline side, the reduction of ketopantolactone is suppressed and the yield of ketopantolactone is increased.Next, specific examples of the production method of the present invention will be described with reference to Examples.

Example 1゜Crycerol 1%, peptone 1.5%, yeast extract 0.
3%, K2npo4o, 3%, NaCA O,2
% and L-pantolactone 0.5%, pH 7.0, inner diameter 1.4c1n, length 16eIn.
into test tubes and autoclaved at 121℃ for 15 minutes.
Heat sterilized for 1 minute. One platinum loop 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 predetermined method, and the results shown in Table 1 were obtained. The products other than the produced ketopantolactone are pantolactones. The proportion of D-pantolactone in pantolactone is D-rat
io (%).

Table 1 Example 2゜Example 1.0 liquid medium containing no L-pantolactone was used. One platinum loop of the inoculum shown in Table 2 was inoculated from the slant culture medium and cultured aerobically on a rotary shaker at 28°C for 2 days. L-pantolactone was added thereto to give a concentration of 1 wt%, and the mixture was further shaken for 4 days to obtain the results shown in Table 2.

Table 2 Example 3: 4% glucose, 1 tX polypeptone, 0.
5%, dihydrogen phosphate 0.5% and magnesium sulfate 0.2%.
Example 2. The same procedure as above was carried out, and the results shown in Table 3 were obtained.

Table 3 Example 4 Example 1 except that 0.5% of 1.2-propanediol was added to the medium. The same procedure as above was carried out, and the results shown in Table 4 were obtained.

Table 4 Example 5゜Example 4. The reaction was carried out using bacterial cells cultured for 2 days in the following medium. That is, 0.0% of L-pantolactone was added to two test tubes (5rdX 2 ) of bacterial cells obtained by centrifugation.
1g (2%), 0.2M potassium phosphate buffer (1) H7
,0)5 m/ was added and shaken at 28°C for 4 days. This reaction solution was analyzed 12 and the results shown in Table 5 were obtained.
The reaction was carried out using the bacterial cells. Example 5 except that the reaction time was 6 days. It is similar to As a result of the analysis, the reaction solution contained 92.1% of ketopantolactone, D-pantolactone, and L-pantolactone, respectively.7. O
N and contained in a yield of 0.997;

Example 7゜Example 4. The reaction was carried out using bacterial cells obtained in the same manner as described above. Add 0.1 g (2%) of L-pantolactone to 2 test tubes (5agX2) of Nocardia asuyaroides (IF03384) cells and 0.2M of 2HPO.
45d was added and shaken at 28°C for 4 days.

Analysis of this reaction solution revealed that the ketopan trough yield was 1.
It was 00%.

Example 8゜Example 2. The reaction was carried out using cells of Nocalde□iaasteroides (IF03384) that had been cultured for 2 days in the same medium. 1.2- to the bacterial cells of 2 test tubes (5v/X2)
Propanediol o, 05 g (I%).

L-pantolactone 0.1 g (25X), 0.2
M. potassium phosphate buffer (pH 7) (5 ml) was added, and the mixture was shaken at 28° C. for 4 days. As a result of the analysis, ketopantolactone, D-pantolactone, and 1. - Pantolactone 87.1%, 7.5% and 5.4% respectively
contained in a yield of

Example 9 Example 7 was repeated, except that 0.25 g of L-pantolactone, a pH 8 solution containing 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-pantolactone, and I4-pantolactone were each 833π.
, with a yield of 7.5% and 9.2%.

Example 10° Example 9 except that 0.50 g of DL-pantolactone was used instead of L-pantolactone. I did the same thing. Analysis of the reaction solution revealed that ketopantolactone, D-pantolactone, and L-pantolactone were each 40.6
%, with a yield of 53.1 jt and 6.3 k.

Claims (9)

[Claims] (1) A method for producing ketopantoate and/or ketopantolactone, which comprises oxidizing L-pantoate and/or L-pantolactone using a microorganism. (2) The method according to claim (1), wherein L-pantoate or/and L-pantolactone is included in DL-pantoate or/and DL-pantolactone. (3) The microorganism is a species of the genus Nocardia, the genus Rhodococcus, the genus Nocardioides, the genus Mycobacterium, or the genus Streptosprangium.
ium), Corynebacterium
Claims (1) using at least one type of microorganism selected from the group consisting of microorganisms belonging to the genus Terium
) method described. (4) The method according to claim (1), which uses a culture method when oxidizing using microorganisms. (5) The method according to claim (1), wherein when oxidizing using microorganisms, the bacterial cells separated from the culture solution are added to a freshly prepared substrate solution. (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.