JPH04234995A - Production of d-pantolactone - Google Patents

Abstract

PURPOSE:To produce the subject D-pantolactone of high optical purity from a high-concentration DL-pantolactone in a short time. CONSTITUTION:DL-pantolactone is brought into contact with a microorganism belonging to Arthrobacter genus or Agrobacterium genus and L-pantolactone is stereoselectively hydrolyzed, thus carrying out optical resolution.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a method for producing D-pantolactone by biochemically optically resolving DL-pantolactone. D-pantolactone is a substance useful as an important synthetic intermediate for D-pantothenic acid (vitamin B5) and CoA.

0002

[Prior Art] A conventional method for producing D-pantolactone by biochemical optical resolution of DL-pantolactone is as follows:
(b) A method for obtaining D-pantolactone by oxidizing DL-pantolactone to α-ketopantolactone by organic synthesis or microorganisms, and then asymmetrically reducing it by microorganisms (Japanese Patent Publication No. 61-14797, JP Showa 61-29
3386, JP-A No. 62-187426, etc.), (b) DL-pantolactone was brought into contact with microorganisms such as Fusarium genus, and only D-pantolactone was hydrolyzed, and the remaining L-pantolactone was extracted with a solvent. After, acidification treatment,
Method for obtaining D-pantolactone by solvent extraction (Japan Agricultural Chemistry Society 1990 Conference Abstracts, pages 678-679)
(c) A method for obtaining D-pantolactone by contacting DL-pantolactone with microorganisms such as Rhodotorula sp. -152895,
JP-A No. 62-294092, etc.).

0003

[Problems to be Solved by the Invention] The above method (a) requires a two-step reaction and is complicated, and the activity of the bacteria is low, so it is difficult to obtain D-pantolactone in high yield and high optical purity. There are disadvantages such as difficulty in Furthermore, in the method (b), it is difficult to suppress the hydrolysis of the L-isomer during the reaction, and it is difficult to obtain D-pantolactone with high optical purity. Furthermore, the method (c) has the disadvantage that optical resolution of DL-pantolactone at a high concentration is difficult because the hydrolyzing ability of the bacterial strain used is low.

0004

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have made extensive studies and have found that microorganisms belonging to the genus Arthrobacter and Agrobacterium have been specifically isolated from racemic pantolactone. It was found that the activity of hydrolyzing only the L-form is high. That is, the present invention is characterized in that DL-pantolactone is brought into contact with microorganisms belonging to the genus Arthrobacter and Agrobacterium, and only L-pantolactone is stereoselectively hydrolyzed and optically resolved. - Provides a method for producing pantolactone.

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

embedded image The present invention will be explained in detail below. The stereoselective hydrolysis reaction of the present invention includes Arthrobacter sp.
acter) and Agrobacterium (Agrob
A group of microorganisms belonging to the genus Acerium can be effectively used. A typical example is Arthrobacter ureafaciens.
aciens) IFO 12140, Agrobacterium radiobacter (Agrobacterium
radiobacter) IFO 12664 and the like. This strain is included in the "List of Culture" published by the Fermentation Research Institute.
s) 8th edition, 1988, and is easily available. In addition, strains that have been artificially mutated by UV irradiation or treatment with mutagens, and other microbial cells that have been artificially extracted and incorporated the gene fragments necessary for expressing the hydrolytic activity. can be used in the method of the present invention.

[0006] These microorganisms can be cultured continuously or intermittently by conventional strain culture, aeration and agitation culture, etc. The medium used may have a normal composition that allows the microorganisms used to grow, and the carbon source may include carbohydrates,
Those that can be assimilated are appropriately selected from fats and oils, fatty acids, and alcohols, and used alone or in combination. Nitrogen sources include organic nitrogen sources such as peptone, soybean flour, cottonseed flour, corn steep liquor, yeast extract, meat extract, malt extract, and urea, as well as ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium phosphorus. Inorganic nitrogen sources may be used alone or in combination as appropriate. In addition to a carbon source and a nitrogen source, essential growth factors and growth-promoting substances such as minerals, amino acids, and vitamins necessary for growth are preferably added to the medium. For the purpose of controlling pH and foam during culture, commonly used buffers such as Tris-HCl buffer and basic substances such as sodium hydroxide, ammonia, and sodium carbonate can be added as appropriate, and antifoaming agents can also be added. The addition of is also effective. Furthermore, it is also effective to aerate oxygen for the purpose of making the environment aerobic. The culture temperature may be selected to be suitable for the growth of the microorganism used, and it is usually advantageous to culture at a temperature of 15°C to 65°C, preferably 25°C to 40°C. Also,
The cultivation time is continued for a sufficient period of time for the growth of the microorganism used, but is usually 5 hours to 240 hours, preferably 10 hours to
It is 170 hours.

[0007] As a method of bringing the microorganism into contact with the raw material DL-pantolactone and allowing its hydrolytic enzyme to act, there is a method of adding the raw material before the start of culture of the bacterial strain or at an appropriate time during the cultivation, or a method of adding the raw material to the culture solution. A method can be used in which a raw material is added at an appropriate time to a liquid containing bacterial cells isolated from a microorganism. Furthermore, dried bacterial cells, immobilized bacterial cells, enzymes, immobilized enzymes, etc. obtained by treating cultured bacterial cells or cultures may be used. The raw material is added as a solution or suspension in a suitable solvent such as water, or as a powder, all at once or continuously or intermittently over a certain period of time. The concentration of DL-pantolactone in the reaction solution in the reaction carried out in contact with bacterial cells is preferably 0.1.
% to 40%, more preferably 0.5% to 15%. Generally the reaction is carried out from 10°C to 70°C, preferably from 20°C to 4°C.
It is carried out under rotary shaking at a temperature of 0° C. for 6 to 96 hours. The pH is preferably 3 to 9.5, preferably 6 to 8. To maintain the pH within this range, commonly used buffers such as Tris-HCl buffer, meth buffer, sodium hydroxide, ammonia, and sodium carbonate are used. It is preferable to add a basic substance such as the like as appropriate. Additionally, minerals and vitamins may be added to promote the reaction.

After the reaction as described above, 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 form L-pantoic acid.
- Pantolactone is recovered as racemic DL-pantolactone through operations such as solvent extraction and racemization treatment, and can be used again as a raw material for the present invention.

[0009]

Effects of the Invention The present invention has the advantage that D-pantolactone with high optical purity can be produced in a short time from highly concentrated DL-pantolactone by carrying out the reaction using the above-mentioned microorganisms.

[Examples] The present invention will be explained in more detail with reference to Examples below. Example 1 A liquid medium consisting of 5% glucose, 5% corn steep liquor, 0.2% ammonium sulfate, and 1% calcium carbonate was adjusted to pH 7.0 with an aqueous sodium hydroxide solution and
The mixture was dispensed into 20 ml Erlenmeyer flasks and sterilized by heating in an autoclave at 121° C. for 20 minutes. Agrobacterium radiobacter IF from slant culture here
One platinum loop of O 12664 was inoculated and cultured aerobically at 28°C on a rotary shaker. 24 hours after the start of culture, DL-pantolactone was added to the flask at a concentration of 2% and Tris-HCl buffer at 0.2M relative to the culture solution.
The mixture was further shaken aerobically for 24 hours. D-pantolactone, L-pantolactone, D-pantoic acid, and L-pantoic acid in the reaction solution thus obtained were quantified as follows. After removing the bacterial cells by centrifugation,
Two times the amount of ethyl acetate was added to the supernatant to extract D-pantolactone and L-pantolactone. (Pantoic acid is not extracted.) These are separated using an optical resolution column SUMICHIRA.
High performance liquid chromatography using L OA-4100 (25 cm x 4 mm ID) (mobile phase: hexane/
1,2-dichloroethane/ethanol = 90/8/2
;Flow rate: 1.2ml/min ;Detection: UV230nm
) was used for fractional quantification. Furthermore, for D-pantoic acid and L-pantoic acid, the diluted solution of the reaction supernatant was subjected to optical resolution column MCI G.
EL CRS10W (50mm x 4.6mmI.D
．． ) high performance liquid chromatography (mobile phase: 2mM
CuSO4/acetonitrile = 9/1; flow rate: 0.5
ml/min; detection: UV 254 nm). By these methods, D-pantolactone, L
-The concentrations of pantolactone, D-pantoic acid, and L-pantoic acid are 9.21 mg/ml and 0.12 mg/ml, respectively.
, 0.48 mg/ml, and 9.55 mg/ml, and the hydrolysis rate of the L form calculated from these values was 98.8%, and the ratio of the D form in the remaining pantolactone was 98.7%.

Example 2 Arthrobacter ureafaciens I as a bacterial strain
Example 1 was repeated except that FO 12140 was used and the DL-pantolactone concentration at the start of the reaction was 4%. As a result, D-pantolactone, L-pantolactone, D
-The concentrations of pantoic acid and L-pantoic acid are each 19.12.
mg/ml, 0.49mg/ml, 0.33mg/ml
, 18.82 mg/ml, and the hydrolysis rate of the L form calculated from this value was 97.6%, and the ratio of the D form in the remaining pantolactone was 97.5%.

Example 3 20ml containing 20ml of sterile liquid medium with the same composition as Example 1
One platinum loopful of Arthrobacter ureafaciens IFO 12140 was inoculated from a slant medium into a 0 ml Erlenmeyer flask, and cultured aerobically on a rotary shaker at 30°C for 24 hours. Add 20 ml of this first seed culture to 1000 ml containing 200 ml of a sterile second seed medium of the same composition.
The cells were transplanted into Erlenmeyer flasks and cultured with rotational shaking at 30°C for 24 hours. 125 ml of this second shaken culture was mixed with 8% glucose, 5% corn steep liquor, and 0.5 dipotassium hydrogen phosphate.
%, monopotassium hydrogen phosphate 0.2%, ammonium sulfate 0.2%, magnesium sulfate 0.05%, citric acid 0.
2. Sterile medium consisting of 0.05% calcium carbonate and 0.1% calcium carbonate.
The cells were transplanted into a 5-liter jar fermenter containing 5 liters of the culture, and cultured with aeration and stirring at 28° C. for 48 hours. Bacteria were collected from 2 liters of this culture by centrifugation, and 1.8 liters of a solution containing the bacterial cells and 120 g of DL-pantolactone was aerated and stirred in a 5-liter jar fermenter to initiate a reaction. pH adjustment is 10% if it is below 6.8
A certain amount of concentrated sodium carbonate solution was automatically added. As a result, the pH of the reaction solution was 6.8.
It was maintained between ~7.0. After stirring for 14 hours, the final liquid volume was 2.3 liters, and the D in the reaction liquid was 2.3 liters.
-The concentration of pantolactone, L-pantolactone, D-pantoic acid, and L-pantoic acid is 26.03 mg/ml each.
, 0.11mg/ml, 0.48mg/ml, 26.2
It became 4 mg/ml. The hydrolysis rate of the L form calculated from this value was 99.6%, and the ratio of the D form in the remaining pantolactone was 99.6%.

Claims (1)

[Claims] 1. A method for producing D-pantolactone, which comprises contacting DL-pantolactone with a microorganism belonging to the genus Arthrobacter or the genus Agrobacterium.