DK152679B - METHOD OF PREPARING 2,5-DICETOGLUCONIC ACID OR A MIXTURE OF 2-KETOGULONIC ACID AND 2-KETOGLUCONIC ACID - Google Patents

Description

DK 152679 B

The invention relates to a process for the preparation of 2,5-diketogluconic acid or a mixture of 2-ketogulonic acid and 2-ketogluconic acid by growing a microorganism of the genus Acetobacter in a medium containing glucose at a concentration of 2.5 to 20%, followed by by extracting the resulting 2,5-diketogluconic acid from the medium or working it up by selective reduction to obtain a mixture of 2-ketogulonic acid and 2-ketogluconic acid.

2,5-Diketogluconic acid is a suitable intermediate in the production of vitamin C. To date, 2,5-diketogluconic acid has been produced with a variety of bacteria such as Acetobacter melanogenum, Acetobacter aurantium, Gluconoacetobacter rubiginosus, Gluconoaceto-bacter liquifaciens and Pseudomonas sesami. Use of these mi

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however, the organisms are not ideal from an industrial point of view, as large amounts of brown or yellow-brown pigments are formed as by-products in culture, thereby reducing the purity of the 2,5-diketogluconic acid formed.

U.S. Patent No. 3,790,444 discloses the preparation of 2,5-diketogluconic acid without accompanying brown pigment using a novel species called Acetobacter fragum. This yields a yield of up to 87% (based on glucose).

It has been found that Acetobacter cerinus generates 2,5-diketogluconic acid in high yield by growing in a medium containing glucose as the main carbon source. Two of the strains tested, IFO 3263 and 3266, form 2,5-diketogluconic acid in yields greater than 95% (based on glucose).

The process according to the invention is characterized by the characterizing part of claim 1 *.

2,5-Diketogluconic acid is, as mentioned, suitable as an intermediate in the preparation of ascorbic acid. An aqueous solution of 2,5-diketogluconic acid can be selectively reduced to give a mixture of 2-ketogulonate and 2-ketogluconate which can be converted to ascorbic acid and erythrobic acid.

In the method of the invention readily available strains of Acetobacter cerinus can be used. All of the publicly deposited strains of Acetobacter cerinus listed below have been tested and found to give keto acids in a yield of 50-95% or more (based on glucose). When Acetobacter cerinus IFO 3263 or 3266 is used, the keto acid formed is solely the desired 2,5-diketogluconic acid in yields greater than 95%, based on glucose. The publicly available strains of Acetobacter cerinus available are as follows: IFO 3262 (ATCC 12303) 3263 3264 3265 3266 3267 '3268 3269.

These microorganisms do not require expensive organic nitrogen sources such as peptone or meat extract. When urine is used 3

DK 152679 B

substance or inorganic nitrogen sources such as ammonium sulphate, ammonium nitrate or ammonium phosphate add nicotinic acid as an essential growth factor.

The glucose concentration in the medium preferably varies between 10 and 12% to obtain the 2,5-diketogluconic acid most economically. The fermentation temperature is between 20 and 35 ° C, preferably between 25 and 30 ° C, most preferably approx. 28 ° C. The initial pH of the culture medium may be from 3.5 to 7.5, preferably from 5 to 6. During the course of fermentation, the pH is maintained at ca. 5.5 by adding sodium hydroxide solution. Calcium carbonate can be used for pH control and added to the amount of medium used for autoclaving backfill in an amount of 30 g / ml. 110 g glucose.

After inoculation, the fermentation medium is stirred e.g. with a mechanical stirrer at 1700 rpm. and is aerated with from 0.5 to 1 volume of air per minute. volume of culture medium per minute.

Using Acetobacter cerinus IFO 3263 or 3266, fermentation is carried out until a yield of 2.5 diketo-gluconic acid of at least 90%, calculated on glucose (36-40 hours) is obtained.

It is determined by paper chromatography that the conversion of glucose to 2,5-diketogluconic acid occurs in the following ways:

Glucose -> 2-ketogluconic acid-> 2,5-diketogluconic acid, and glucose-5-ketogluconic acid * 2,5-diketogluconic acid.

Whatman No. 1 and No. 2 paper and a solvent system of methyl ethyl ketone: acetone: formic acid: water are used (80: 6: 2: 12). The acid spots are localized by spraying with 0.2% o-phenylenediamine in ethanolic solution containing 1% nitric acid and heating to ca. 70 ° C (5-ketogluconic acid gives blue, 2-ketogluconic acid gives yellow and 2,5-diketogluconic acid gives green). High pressure liquid chromatography can also be used for identification.

2,5-Diketogluconic acid can be separated and recovered from the finished fermentation liquid by any conventional method as will be known to those skilled in the art. The filtered fermentation liquid can be worked up, for example, by treatment with a borohydride, and the resulting mixture of 2-ketogluconic acid and 2-ketogulonic acid is hydrolyzed to form ascorbic acid and erythrobic acid.

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DK 152679 B

Example 1 The following aqueous inoculation medium was prepared:

Ingredient Gram / liter

Glucose 25

Ma j s s tap water 5 KH2PO4 0.5 K2HPO0 0.5

MgSO4.7H2O 0.2

CaCO3 6.3 pH 6.2

A shake flask containing one liter of medium was autoclaved for 30 minutes at 121 ° C. The pH of the cooled medium was 5.0. Cells of Acetobacter cerinus IFO 3263 from a slant culture of nutrient agar (5 ml of a 20 ml sterile aqueous suspension) were added to the flask which was then shaken on a rotary shaker table at ca. 28 ° C for approx. 24 hours.

A sample of this culture, sufficiently large to give an inoculum of 5 v / v%, was added to a 4 liter stirred fermenter containing 2 liters of the following production medium:

Ingredient Gram / liter

Glucose 110

Maize molding water 0.5 (NH4) 2HPO4 0.58 KH2PO4 1.5

MgSO4.7H2O 0.5

Urea 0.5

CuSO4.5H2O 1 mg

Nicotinic acid 300 γ pH 6.0 The fermentation was carried out at a temperature of approx. 28 ° C with stirring at 1700 rpm. per minute and aeration with an amount of 0.75 volume of fermentation liquid per minute. minute. After a fermentation period of approx. For 20 hours, sterile glucose (55 grams / liter) was added. The pH was kept at 5.5 by the addition of sodium hydroxide solution. The fermentation was continued to yield 2,5-diketogluconic acid

Claims (4)

A process for preparing 2,5-diketogluconic acid, or a mixture of 2-ketogulonic acid and 2-ketogluconic acid, by culturing a microorganism of the genus Acetobacter in a medium containing glucose at a concentration of 2.5 to 20%, followed by extraction of the resulting 2,5-diketogluconic acid from the medium or working it up by selective reduction to give a mixture of 2-ketogulonic acid and 2-ketogluconic acid, characterized in that such as a microorganism, one belonging to Acetobacter cerinus is used. Process according to claim 1, characterized in that the fermentation temperature is from 20 to 35 ° C, the initial pH is from 3.5 to 7.5 and the pH during fermentation is kept at 5.5. Method according to claim 1 or 2, characterized in that said Acetobacter cerinus is of the strain IFO 3263. Method according to claim 1 or 2, characterized in that said Acetobacter cerinus is of the strain IFO 3266.