NO783877L - PROCEDURE FOR THE PREPARATION OF 2,5-DIKETOGLUCONIC ACID - Google Patents

Description

"Process for the preparation of 2,5-diketogluconic acid".

This invention relates to the production of 2,5-diketogluconic acid.

2,5-diketogluconic acid is a useful intermediate in the synthesis of vitamin c. So far, 2,5-diketogluconic acid has been formed with several different types of bacteria such as Acetobacter melanogenum, Acetobacter aurantium, Gluconoacetobacter rubigino-sus, Gluconoacetobacter liquifaciens and Pseudomonas sesami. The use of these microorganisms is, however, not entirely desirable from an industrial point of view due to the large amount of brown or yellow-brown pigments that are formed as by-products during cultivation, thus reducing the purity of the simultaneously formed 2,5-diketogluconic acid.

U.S. patent document no. 3,790,444 has a claim to formation

of 2,5-diketogluconic acid, without accompanying brown pigment, using a new species designated Acetobacter fragum.

This invention relates to a new economic method for the production of 2,5-diketogluconic acid using readily available, published strains of Acetobacter cerinus.

Two of these strains, IFO 3263 and 3266, form 2,5-diketogluconic acid with yields of 95% (based on glucose).

2,5-diketogluconic acid is useful as an intermediate for the production of ascorbic acid. An aqueous solution of 2,5-diketogluconic acid can be selectively reduced to provide a mixture of 2-ketogulonate and 2-ketogluconate which can be converted to ascorbic and erythorbic acids.

2,5-diketogluconic acid is easily formed by bacterial action on glucose using, according to the present invention, readily available strains of Acetobacter cerinus. All the listed published strains of Acetobacter Cerinus have been tested, and in this invention have shown formation of the keto acids with a yield of 50-95% (based on glucose). When

Acetobacter cerinus IFO 3263 or 3266 is used, the keto acid formed is entirely the desired 2,5-diketogluconic acid with yields of 95% (based on glucose). The available, published strains of Acetobacter cerinus are the following:

These strains of Acetobacter cerinus are grown in a medium in which the main carbon source is glucose. These microorganisms do not require expensive organic nitrogen sources, such as peptone or meat extract. When urea and inorganic nitrogen sources, such as ammonium sulphate, ammonium nitrate or ammonium phosphate, are used, nicotinic acid is added as an essential growth factor.

The glucose concentration in the medium varies between 2.5 and 20%, preferably between 10 and 12%, in order to obtain 2,5-diketogluconic acid in the most economical way. The fermentation temperature is between 20 and 35°C, preferably between 25 and 30°C, and most preferably around 28°C. The initial pH in the culture medium can lie in the range from 3.5 to 7.5, preferably at 5 to 6. During the course of the fermentation, the pH is kept at approx. 5.5 by adding sodium hydroxide solution, Calcium carbonate can be used for pH regulation and added to the medium composition after autoclave treatment in a quantity of 30 grams per 110 grams of glucose.

After inoculation, the fermentation medium is stirred with a mechanical stirrer with approx. 1700 rpm per minute, and aerated at a rate of 0.5 to 1 volume of air per volume of broth per minute.

When using Acetobacter cerinus IFO 3263 or 3266, the fermentation is carried out until a yield of 2,5-diketogluconic acid of at least 90% (based on glucose) is achieved (36-40 hours).

It was determined by paper chromatography that the conversion of glucose to 2,5-diketogluconic acid takes place via the following pathways: glucose 2-ketogluconic acid 2,5-diketogluconic acid

glucose 5-ketogluconic acid 2,5-diketogluconic acid Whatman paper No. 1 and No. 4 are used and a solvent system of methyl ethyl ketone: acetone: formic acid: water is used

(80:6:2:12). The acidic spots are localized by spraying with

a 0.2% ethanolic o-phenylenediamine solution containing 1% nitric acid, and heating to approx. 70°C, (5-ketogluconic acid - blue; 2-ketogluconic acid - yellow; 2,5-diketogluconic acid - green).

High-pressure liquid chromatography can also be used for identification. 2,5-diketogluconic acid can be separated and recovered from the final fermentation broth by any conventional method known to those skilled in the art. The filtered fermentation broth can be processed as by treatment with borohydride, and the resulting mixture of 2-ketogluconic acid and 2-ketogulonic acid hydrolyzed to give ascorbic and erythorbic acids.

Example 1

The following aqueous seeding medium was prepared:

A shaking 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 cerinium IFO 3263 from a nutrient agar slant (5 ml of a 20 ml 1s sterile aqueous suspension) were added to the flask which was then shaken on a rotary shaker at approx. 28°C for approx. 24 hours.

An aliquot of the culture growth sufficient to provide a 5% volume/volume inoculum was added to a 4 liter stirred fermentation vessel containing 2 liters of the following production medium:

The fermentation was carried out at a temperature of approx. 28°C when stirring with 1700 rpm minute and aeration at a rate of 0.75 volume per volume of broth per minute. After a fermentation period of approx. 20 hours, bee sterile glucose added (55 g/liger), pH was maintained at 5.5 by addition of sodium hydroxide solution. The fermentation was continued until a yield of 2,5-diketoglucoric acid of 95% (based on glucose) had been obtained.

Example 2

The procedure from example 1 can be repeated with comparable results using Acetobacter cerinus IFO-3266.

Claims (4)

1.. Process for the production of 2,5-diketogluconic acid, characterized by . aerobic cultivation of Acetobacter cerinus in a glucose medium and then recovery of the resulting 2,5-diketogluconic acid or processing of the fermentation broth by selective reduction to produce 2-ketogulonic acid and 2-ketogluconic acid. 2. Method according to claim i, characterized in that a glucose concentration in the medium from 2.5 to 20%, a fermentation temperature from 20 to 35°C, an initial pH from 3.5 to 7.5 and a pH during the fermentation process of approx. 5.5. . 3. Method according to claim 1 or 2, characterized in that the strain IFO 3263 is used as the aforementioned A cetobacter cerinus. 4. Process according to claim 1 or 2, characterized in that the aforementioned A cetobacter cerinus is used in accordance with FO 3266.