JPH02227085A - Production of d-pantothenic acid ester - Google Patents

Abstract

PURPOSE:To enable quantitative production of the subject ester by carrying out the asymmetric reduction of a 2-ketopantothenic acid ester with a microorganism having reducing power and belonging to genus Kluyveromyces, Hansenula, Sporobolomyces, etc. CONSTITUTION:At least one kind of microorganism having reducing power and selected from the group of microorganisms of genus Aerobacter, Erwinia, Agrobacterium, Corynebacterium, Arthrobacter, Xanthomonas, Kluyveromyces, Saccharomyces, Hansenula, Sporobolomyces, Candida, Rhodotorula, Pichia and Mortierella is aerobically cultured in a liquid medium. The obtained culture product, microbial cell separated from the culture liquid, etc., are made to react at 10-50 deg.C with a new 2'-ketopantothenic acid ester derived from ketopantolactone and a beta-alanine ester. A D-pantothenic acid ester can be quantitatively produced by this process.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention provides D-pantothenic acid, which is an intermediate in the production of D-pantothenic acid, which is useful as a medically or physiologically important vitamin.
This invention relates to a new method for producing pantothenic acid ester.

[Background Art] The present inventors have previously developed a method for industrially producing D-pantothenic acid, using D-pantothenic acid ester as a raw material and commercially available enzymes and microorganisms to quantitatively produce D-pantothenic acid.
-Provided a method for converting pantothenic acid or its salt (Japanese Patent Application No. 63-53571, Patent Application No. 63-53572)
reference).

However, D-pantothenic acid ester, which is the raw material, can be obtained by subjecting D-pantolactone obtained by optical resolution, which requires chemically complicated steps, to amithrisis with β-alanine ester. separately reported that 21-ketopantothenic acid ester, which is a 10-chiral form of D-pantothenic acid, was easily synthesized by the reaction between cutopantolactone, which is much more susceptible to 7-minolysis than D-pantolactone, and β-alanine ester. It was found that this 2'
It has been discovered that -ketopantothenic acid ester can be quantitatively converted to D-pantothenic acid ester by asymmetric reduction using microorganisms.6 The present invention was made based on this finding.

[Disclosure of the Invention] The present invention relates to the genus Aerobacter, the genus Erwinia, the genus Agrobacterium, the genus Corynebacterium, the genus Arthrobacter, the genus Xanthomonas, the genus Kluyveromyces, the genus Satucharomyces, the genus Hansenula, the genus Sporobolomyces, and the genus Candeida. At least one selected from the group consisting of microorganisms belonging to the genus Rhodotorula, the genus Pichia, and the genus Morteierella.
D- characterized in that asymmetric reduction of 2°-ketopantothenic acid ester is carried out using a microorganism having the ability to reduce seeds.
A method for producing pantothenic acid ester is provided.

The present invention will be explained in detail below.

The present inventors inoculated 1 platinum loop of various bacteria from a slant culture into 21 J of various liquid media, cultured them aerobically with shaking at 28°C for 2 days, and then added ethyl 2°-ketopantothenate to this culture solution. 20. Esters. Add each portion and shake at 28℃ for 2 days.
For the resulting reaction solution, the amount of pantothenic acid ethyl ester produced was measured by TLC and HPLC, and the optical purity of vantolactone was measured by GLC.
Aerobacterium is a microorganism that converts ketopantothenic acid ethyl ester into D-pantothenic acid ethyl ester.
Genus, Erwinia, Agrobacterium, Corynebacterium, Arthrobacter, Xanthomonas,
It has been found that compounds having asymmetric reduction ability belonging to the genera Kluyveromyces, Satucharomyces, Hansenula, Sporobolomyces, Candeida, Rhodotorula, Vitia, and Morteierella are suitable.

Microorganisms with particularly excellent asymmetric reduction ability are found in the genera Kluyveromyces, Hansenula, Sporobolomyces, Candeida, Vitia, and the like.

The conditions for culturing the bacteria include glucose,
Carbohydrates such as fructose and sucrose, alcohols such as glycerol, nitrogen sources such as ammonium sulfate, peptone, casamino acids, cornstay 1 liquor, bran, and yeast extract, and inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, and phosphorus. A medium containing malt extract, meat extract, etc. is used as a nutrient source for the pond, such as potassium acid-hydrogen or potassium dihydrogen phosphate. Cultivation is carried out aerobically, usually for about 1 to 7 days, at a medium pH of 3 to 10, at a culture temperature of 15 to 50°C, more preferably at a temperature of 2.
Carry out at 0-40°C.

In the method of the present invention, the microorganisms used include a culture obtained by culturing bacterial strains in a liquid medium, bacterial cells isolated from the culture solution, Tcl1 cells, dried m-cells obtained by treating the culture, or immobilized bacterial cells. It can be used in any form.

As the 2°-ketopantothenic acid ester, any ester such as an alkyl ester or an aralkyl ester can be used. Preferred examples include lower alkyl esters such as methyl ester, ethyl ester, and propyl ester.

The concentration of 2'-ketopantothenic acid ester used as a raw material is usually 10 to 150 g/J.

The reaction temperature is usually 10 to 50°C, and the reaction time is usually several hours to 5 days. The Dll of the zero reaction system is usually about 3 to 9.

The present invention will be described in more detail below with reference to nine examples, but the present invention is not limited to these examples.

Although the 2'-ketopantothenic acid ester used in the method of the present invention is a new compound, it can be produced as described below.

That is, 2I-ketopantothenic acid ester.

It can be obtained by amithrisis using ketopantolactone and β-alanine ester, but when carrying out this reaction, the reaction solvent may be alcohol such as methanol, esters such as ethyl acetate, hexane, etc. Aliphatic hydrocarbons such as benzene, aromatic carbons such as arsenic, halogenated hydrocarbons such as chloroform, ketones such as acetone, acetonitrile, etc. are used.
The reaction temperature is within the range from Mll to about the boiling point temperature of each reaction solvent, and the reaction time is usually 1 hour to 1 day. 6 After the reaction is completed, the reaction solvent is distilled off, and ethyl acetate or methylene chloride The desired 21-ketopantothenic acid ester is extracted using an organic solvent such as When this is purified by washing with a dilute aqueous acid solution or a dilute alkali aqueous solution as the case requires, a single 2°-ketopantothenic acid ester can be obtained in a good yield.

An example of producing this ester is shown below.

Production Example 1 Production of 2'-ketopantothenic acid methyl ester 1.2 g (52 mmol) of sodium metal was dissolved in anhydrous methanol 2
The mixture was concentrated to 00 iJ, and 7.3 g (52 mmol) of β-alanine methyl ester hydrochloride and 0.4 g (5 Gmmol) of ketopantolactone were added in this order at room temperature. After stirring overnight at room temperature, methanol was distilled off. The reaction mixture containing by-produced sodium chloride is dissolved in water,
Extract with ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography using silica gel to obtain 10.1 g (yield: 87.3%) of 2°-ketopantothenic acid methyl ester as oil.
obtained as.

IR(Hat) v (11-': 338G, 2
955.2875.174G.

1715, 1680.1515.142G, 1370
．． 1260°1200, 1180.1050 ”H-NHR (CDCj s) δ: 1.28 (OH
,s) 2.57 (2H,t) 3.58 (2H,Q) 3,72 (511,S) 7.5 (III, bs) Production IM2 Production of 2'-ketopantothenic acid ethyl ester Sodium ethoxy 3.6 g (52.5 mmol) of β-alanine ethyl ester hydrochloride was dissolved in absolute ethanol 15017, and 8.1 g (52.5 mmol) of β-alanine ethyl ester hydrochloride and 6.4 g (50 mmol) of ketopantolactone were dissolved in 15017 absolute ethanol. ) in that order. After stirring overnight at room temperature, ethanol is distilled off. After ethanol distillation, the same operation as after methanol distillation in Production Example 1 was performed to obtain 10 Jg of 2°-ketopantothenic acid ethyl ester (yield: 88.1%).
It is advantageous to use it as oil.

IR(neat) a/1m-”: 33g0.2
98G, 2945.28?5゜1735, 168G,
1525.1480.1380.1190゜1100
, 1050'H-NOR(CDCj s) δ: 1.28 (6H
, s) 1.28 (31, t) 2.57 (2H, t) 3.58 (2H, Q) 3.75 (2H, S) 4.18 (2H, Q) 7.5 (IH, bs ) Production Example 3 Production of 2'-ketopantothenic acid benzyl ester β-alanine benzyl ester-p-toluenesulfonate 3
．． 5 g (10 mmol) was dissolved in 10% sodium carbonate water and extracted with ethyl acetate. The ethyl acetate layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was dissolved in anhydrous methanol and added with 1.3 g of ketopantolactone.
g (10 mmol) is added. After boiling under reflux for 6 hours, methanol was distilled off. After methanol distillation, Production Example 1
2°-ketopantothenic acid benzyl ester 1. G g (yield: 52%) was obtained as oil.

IR(nflat) v >-': 34GG, 2
975.2940.28?5゜1735, 1680.1
52G, 1475.14G0.1390°1360,
1260.1180.1105.1050 “River-NHR
(C0Cj s) δ: 1.2G (6H, s) 2.6
2 (2H, t) 3.58 (2H, Q) 3.73 (2H, s) 5.14 (2H, s) 7.35 (5H, S') Examples 1 to 6 Glucose 5%, Cornsteel A liquid medium (pH 7.0) consisting of 5% E liquor and 1% calcium carbonate was dispensed into 6 test tubes at 2 ij each, and incubated in an autorape for 12 ml.
Heat sterilization was performed at 1° C. for 20 minutes. Into the medium in each test tube,
One platinum loopful of the various strains listed in Table 1 was inoculated from the slant culture medium, and cultured aerobically with shaking at 28°C for 2 days.

Add 20 μl of 2°-ketopantothenic acid ethyl ester to the culture solution in each test tube, and leave it at 28°C for 2 days.
i! After the 0 reaction, HPLC (Cosmosi15C
0, φ4.6 XI 100 tm, eluent 30% methanol (pi(2,5), flow rate 1 if /1n,
The amount of pantothenic acid ethyl ester produced in each test tube was measured at a detection wavelength of 230 nn. Furthermore, pantothenic acid ethyl ester was extracted from the reaction solution using ethyl acetate and hydrolyzed with hydrochloric acid.
ochemistry, 112 9-16 (1981
) The results are shown in Table 1.

In Table 1, IFOIIQ indicates the Fermentation Research Institute catalog number, and IAHk indicates the Institute of Applied Microbiology, University of Tokyo catalog number (the same applies hereinafter).

Examples 7 to 14 In place of the liquid medium used in Examples 1 to 6, a liquid medium (pHe, o>) consisting of 5% glucose and 5% cornstarch was used, and Examples Nt17 to 1 in Table 2 were used.
Using each of the various strains shown in each column of No. 14, the operations were carried out under the same conditions as described in Examples 1 to 6, and the results shown in Table 2 were obtained.

Example 15 Liquid medium 200+ej used in Examples 7 to 14
was used, and Quandida macedoniensis (IFo
96G) was cultured at 28° C. for 3 days with shaking in a Sakaro flask, and after culturing, the bacteria were collected by centrifugation. A solution of 2°-ketopantothenic acid ethyl ester and glucose dissolved in 0.2 M phosphate buffer to a concentration of 5% each (DH5,0>100mj
was added to the bacterial cells obtained above, and after 2 days of shaking at 28°C, the reaction mixture was salted out with sodium chloride, extracted with ethyl acetate, and the ethyl acetate layer was dried over anhydrous sodium sulfate. By concentrating under reduced pressure, 4.6 g of D-pantothenic acid ethyl ester (yield: 92%, optical purity: 98%
Obtain e, e, ).

Table 1

Claims (1)

[Claims] (1) Aerobacter, Erwinia, Agrobacterium, Corynebacterium, Arthrobacter, Xanthomonas, Kluyveromyces, Satucharomyces, Hansenula, Sporobolomyces, Candeida, Rhodotorula, D-pantothenic acid ester, characterized in that asymmetric reduction of 2'-ketopantothenic acid ester is carried out using at least one microorganism having a reducing ability selected from the group consisting of microorganisms belonging to the genus Vitia and Morteierella. manufacturing method.