JP2782438B2 - Method for producing pantothenic acid or a salt thereof - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel method for producing pantothenic acid or a salt thereof useful as a medicine.

[Prior Art] A method for deriving pantothenic acid by chemically hydrolyzing a pantothenate ester has been reported, for example, by Williams et al. (J. Am. Chem. Soc., 454 (1939)). According to this report, although the concentration of pantothenic acid ester is not described, it is estimated that pantothenic acid is produced in a yield of 95 to 99% at 30 ° C for 1 to 2 hours using 0.05N sodium carbonate. I have.

However, in the hydrolysis of pantothenic acid ester by a chemical method, an alkali must be used in excess, so that the amide bond in the chemical structure of pantothenic acid may be hydrolyzed, and Since isolation of pantothenic acid or a salt thereof requires complicated purification steps such as desalting and salt exchange, this method cannot be said to be a practical method.

DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies on the hydrolysis of pantothenate, and as a result, using a microorganism, the pantothenate was converted into an ester without cleaving an amide bond in its chemical structure. It has been found that hydrolysis allows quantitative conversion to pantothenic acid or a salt thereof. The present invention has been made based on such findings.

That is, the present invention relates to the genus Citrobacter, Alkali Genes, Flavobacterium, Bacillus, Agrobacterium, Corynebacterium, Staphylococcus, Arthrobacter, Brevibacterium,
Acinetobacter, Pseudomonas, Chromobacterium, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, Pseudonocardia, Mucor, Rhizopus, Aspergillus, Penicillium, Neurospora, Pullularia Pantothenic acid using a microorganism having at least one ester hydrolytic ability selected from the group consisting of microorganisms belonging to the genera, Fusarium, Gibberella, Hansenula, Sporoboromyces, Torulopsis, Cyandeida, Rhodotorula. An object of the present invention is to provide a method for producing pantothenic acid and / or a salt thereof, which comprises subjecting an ester to ester hydrolysis.

 Hereinafter, the present invention will be described in detail.

The present inventors inoculated one loopful of a seed bacterium from a slant medium into 5 ml of various liquid media, aerobically shake-cultured at 28 ° C. for 2 to 6 days, and then obtained cells by centrifugation or filtration. 0.2 to 0.5 ml of 15 to 50 mM of methyl or ethyl ester of pantothenic acid was added to the body, and the mixture was shaken at 30 ° C. overnight, and the resulting reaction mixture was subjected to TLC or HPLC to reduce the amount of pantothenate and to generate pantothenic acid. As a result of measuring the amount, as a microorganism that converts pantothenate to pantothenic acid, Citrobacter, Alkaligenes, Flavobacterium, Bacillus, Agrobacterium, Corynebacterium, Staphylococcus, Arthrobacter, Brevibacterium, Acinetobacter, Pseudomonas, Chromobacterium,
Mycobacterium, Nocardia, Rhodococcus, Streptomyces, Pseudonocardia,
Those having ester hydrolytic ability belonging to the genus Mucor, Rhizopus, Aspergillus, Penicillium, Neurospora, Pullularia, Fusarium, Gibberella, Hansenula, Sporoboromyces, Torulopsis, Cyandeida, Rhodotorula Found to be appropriate.

Microorganisms with particularly good ester hydrolysis ability include Alkali Genes, Bacillus, Agrobacterium, Corynebacterium, Arthrobacter, Brevibacterium, Acinetobacter, Pseudomonas and Chromobacterium. Can be seen.

Conditions for cultivation of the bacterium vary depending on the strain used.However, as for the culture medium, sugars such as glucose, fructose and sucrose and alcohols such as glycerol, and alcohols such as glycerol as a carbon source, and ammonium sulfate, ammonium chloride, peptone and casamino as a nitrogen source are used. Acids, corn stay liquor, bran, yeast extract, etc., inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, etc. Malt extract, meat extract etc. as other nutritional sources Use a medium containing Culture is performed aerobically, usually from 0.5 to
The culture is carried out for about 7 days at a medium pH of 3 to 10 and a culture temperature of 15 to 60 ° C, more preferably 20 to 40 ° C.

In the method of the present invention, the microorganism used is a culture obtained by culturing the strain in a liquid medium, cells isolated from the culture solution, or dried cells obtained by treating the cells or the culture,
Alternatively, any form such as immobilized cells can be used.

As pantothenic acid esters, alkyl esters,
Any ester such as an aralkyl ester can be used. Preferred examples include methyl esters,
Lower alkyl esters such as ethyl ester and propyl ester are exemplified.

The operation can be performed in any of a batch system, a semi-batch system, and a continuous system. The concentration of the pantothenate used is usually between 10 and 250 g / l. The temperature of the system is usually
The operation time is usually several hours to 3 days in the case of a batch system. The pH of the reaction system is usually about 3 to 11.

Free pantothenic acid is produced by ester hydrolysis of the pantothenic acid ester by the microorganism, but due to the produced pantothenic acid, the pH of the reaction solution is lowered, and at the same time, the reaction rate is also reduced. In order to increase the reaction rate, it is preferable to maintain the pH of the reaction solution at the optimum pH of the ester hydrolase of each microorganism. At that time, as the inorganic base for maintaining the pH, hydroxides or carbonates of alkali metals, alkaline earth metals can be used, and by appropriately selecting these, the alkali metal salts of pantothenic acid or Alkaline earth metal salts can be produced directly. That is, for example, when calcium hydroxide or calcium carbonate is used to maintain the pH of the reaction solution, calcium pantothenate can be obtained, and when sodium hydroxide or sodium carbonate is used, sodium pantothenate can be obtained. .

Among microorganisms having the ability to hydrolyze pantothenate esters, those belonging to the genera Arthrobacter, Brevibacterium, and Pseudomonas are particularly highly selective for the D-form of pantothenate, so that the D, L-pantothenate can be used. Kinetic partitioning is possible. That is, when the above microorganisms are allowed to act on D, L-pantothenate, the D-pantothenate is preferentially subjected to ester hydrolysis to become D-pantothenate, and the L-pantothenate remains as it is. This L-pantothenate can be easily racemized with sodium methylate or the like after separation and recovery, and D, L-pantothenate can be obtained. This DL body can be used again as a raw material.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Examples 1 to 12 1% glucose, 1.5% peptone, 0.3% dipotassium hydrogen phosphate, 0.2% sodium chloride, 0.2% magnesium sulfate.
A liquid medium (pH 7.0) consisting of 02% and yeast extract 0.1% was dispensed into test tubes (φ1.4 cm × 16 cm) in 5 ml portions and sterilized by heating at 121 ° C. for 15 minutes in an autoclave. One loopful of each of the various inoculum shown in Table 1 was inoculated from the slant medium and cultured aerobically on a reciprocating shaker at 28 ° C. for 3 days. Each cell was obtained by centrifuging or passing the culture. To the cells, add 0.5 ml of a 50 mM ethyl pantothenate solution (250 mM phosphate buffer, pH 7.0), and add 30 ml.
Shake at ℃ overnight. After the reaction, HPLC (Cosmosil 5C ₁₈ φ4.6
× 100mm, eluent 20% methanol (pH2.5), flow rate 1ml /
min, detection wavelength 210 nm), the remaining amount of ethyl pantothenate and the amount of generated pantothenic acid were measured. The results are as shown in Table 1.

Examples 13 to 17 In place of the liquid medium used in Examples 1 to 12, a liquid medium (pH 7.0) composed of 1.0% peptone, 0.5% meat extract, 0.1% yeast extract, and 0.5% sodium chloride was used. Was operated under the same conditions as in Examples 1 to 12 to obtain various cells shown in Table 2. Using each of the cells, 0.2 ml of a 15 mM methyl pantothenate solution (100 mM buffer phosphate, pH 7.0) was added, and the mixture was shaken at 30 ° C. overnight. Next, the remaining amount of methyl pantothenate and the amount of generated pantothenic acid were analyzed in the same manner as in Examples 1 to 12. The results shown in Table 2 were obtained.

Examples 18 to 30 Instead of the liquid medium used in Examples 1 to 12, a liquid medium consisting of 5.0% malt extract and 0.3% yeast extract (pH 5
Using the same procedures as in Examples 1 to 12, operations were performed under the same conditions as in Examples 1 to 12, and various bacterial cells shown in Table 3 were obtained. To each of these cells, a 50 mM ethyl pantothenate solution (25
0.5 ml of 0 nM phosphate buffer (pH 7.0) was added, and the mixture was shaken at 30 ° C. overnight. Next, the remaining amount of ethyl pantothenate and the amount of generated pantothenic acid were analyzed in the same manner as in Examples 1 to 12. The results are shown in Table 3.

Example 31 Liquid medium 15 having the same composition as the medium used in Examples 1 to 12
Brevibacterium linens (IFO12141) was shake-cultured at 28 ° C. for 5 days using a 500 ml shake flask containing 0 ml. After the culture, the cells are collected by centrifugation, and washed with distilled water. 20 mM D-ethyl pantothenate aqueous solution was added to the obtained cells.
15 ml was added, and the reaction was carried out at 30 ° C. for 24 hours. After the cells were removed from the reaction solution by centrifugation, the reaction solution was purified by reverse phase chromatography and freeze-dried to obtain 56 mg of D-pantothenic acid (Y = 85%, [α] _D + 37 °). .

Example 32 To a cell of Brevibacterium linens (IFO12141) obtained by the same operation as in Example 31, 20 ml of a 0.2 MD-ethyl pantothenate aqueous solution is added. By adding calcium hydroxide little by little to the reaction solution, the reaction was carried out at 35 ° C. for 12 hours while maintaining the pH at 6.5 to 9.0. The same post-treatment as in Example 31 was performed, and 890 m of D-calcium pantothenate was used.
g (Y = 93%, [α] _D + 28.0 °).

Example 33 10 ml of a 100 mM aqueous solution of D-ethyl pantothenate is added to the cells of Brevibacterium linens (IFO12141) obtained in the same manner as in Example 31. Further calcium carbonate 15
0 mg was added, and the reaction was carried out at 30 ° C. for 36 hours. Example 3
After the same post-treatment as in 1, the reverse phase chromatographic eluate was adjusted to pH 8.0 with an aqueous solution of calcium hydroxide, lyophilized, and dried with 219 mg of calcium D-pantothenate (Y
= 92%, [α] _D + 28.0 °).

Example 34 The same procedure was performed as in Example 32, except that the reaction pH (6.5 to 9.0) was maintained using 0.01 N NaOH instead of calcium hydroxide. 907 mg of sodium D-pantothenate (Y = 95%,
[Α] _D + 27.1 °).

Example 35 Brevibacterium obtained by culturing in the same manner as in Example 31
0.2 MD, 10 ml of ethyl L-pantothenate and 100 mg of calcium carbonate were added to the cells of Linnens (IFO12141), and the mixture was added at 35 ° C for 30 minutes.
A time response was performed. After the cells were removed from the reaction solution by centrifugation, the reaction solution was separated and purified by reversed-phase chromatography, freeze-dried, and dried with 145 m of calcium D-pantothenate.
g ([α] _D + 26.5 °). In addition, 331 mg of unreacted ethyl pantothenate was recovered.

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Claims (1)

(57) [Claims] (1) a genus of Citrobacter, a genus of Alcaligenes,
Flavobacterium, Bacillus, Agrobacterium, Corynebacterium, Staphylococcus,
Arthrobacter, Brevibacterium, Acinetobacter, Pseudomonas, Chromobacterium, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, Pseudonocardia, Mucor, Rhizopus, At least one selected from the group consisting of microorganisms belonging to the genera Aspergillus, Penicillium, Neurospora, Pullularia, Fusarium, Gibberella, Hansenula, Sporoboromyces, Torulopsis, Cyandeida, Rhodotorula.
A method for producing pantothenic acid and / or a salt thereof, wherein the pantothenic acid ester is subjected to ester hydrolysis using a microorganism having a kind of ester hydrolytic ability.