DD140459A5 - PROCESS FOR PREPARING 2,5-DIKETOGLUCONE ACID - Google Patents

Description

Process for the preparation of 2,5-diketogluconic acid

Field of application of the invention:

Pie invention relates to the preparation of 2,5-diketogluconic acid, of 2-ketogulonic acid and 2-Ketoglu.consäure.

Characteristic of the known technical solutions:

2,5-diketogluconic acid is a useful intermediate in the synthesis of vitamin C. So far, 2,5-diketogluconic acid has been produced by several different types of bacteria such as Acetobacter melanogenum, Acetobacter aurantiuin, Gluconoacetobacter rubiginosus, Gluconoacetobacter liquifaciens and Pseudomonas sesami. However, the use of these microorganisms is not technically

completely satisfactory because of the large amounts of brown or yellowish-brown pigments as by-products of the cultivation, whereby the purity of the co-formed 2,5-diketogluconic acid is reduced.

U.S. Patent 3,790,444 discloses the formation of 2, 5-diketogluconic acid without accompanying brown pigment by a novel species called Acetobacter fragunr

.The aim of the invention ;

The present invention relates to an economical method auj? Preparation of 2,5-diketogluconic acid using readily available, publicly stored strains of Acetobacter cerinus. Two of these strains, namely IFO 3263 and J266, form 2,5-diketogluconic acid in yields of > 95% with respect to glucose.

Darlegun g of W esens de r Fiction invention:

2,5-2-ketogluconic acid is useful as an intermediate for the production of ascorbic acid. An aqueous solution of 2.5 ~ ^I) iketcgluconsäure can be selectively reduced to form a mixture of 2 ~ ketogulonate and 2-Xetogluccnat, Werder can be converted which (to ascorbic acid and erythorbic isoascorbic acid). , ,

2,5-diketogluconic acid is readily formed by the bacterial action on glucose, using easily accessible strains of Acetobacter cerinus according to the method of the invention. All of the listed publicly available strains of Acetobacter cerinus were examined and showed in this study that they form keto acids in a yield of 50 to 95% based on glucose. When Acetobacter cerinus IFO 3263 or 3266 is used, the keto acid formed consists entirely of the desired 2,5-diketogluconic acid in yields of> 95% with respect to glucose. The accessible, publicly held strains of Acetobacter cerinus are as follows:

08 86

IFO 5262 (ATCG 12503)

5265.

5264-

3265 5266 3267 5268 3269

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

The concentration of glucose in the medium varies between 2.5 and 20%, preferably between 10 and 12%, in order to obtain 2,5-diketogluconic acid in a particularly economical manner. The Ferroentationstemperatur is between 20 ⁰ C and 35 ⁰ C and preferably between 25 and 30 ° C, very particularly preferably in the 'vicinity of 28 ⁰ C. Der.Anfangs pH of the culture medium can Vert · of "5λ5 to 7> 5 ¹ Preferably, the pH varies from 5 to 6. In the course of the fermentation, the pH is maintained at about 5> 5 by the addition of iodonium hydroxide solution. Calcium carbonate can be used for pK control and is applied in a batch medium in autoclave treatment an amount of 30 g per 110 g added.

for seeding, the pemtation medium is stirred with a mechanical stirrer at about 17ΟΟIJpm, and it is aerated at a rate of 0.5 to 1 volume of air per volume of broth per minute.

the use of Acetobacter cerinus IFO 3263 or 3266, the fermentation-ion is carried out until a yield

of 2,5-diketogluconic acid of at least 90%, based on glucose, which usually requires 56 to 40 hours.

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

Glucose-δ * 2-ketogluconic acid ~> 2,5-diketogluconic acid glucose-9 * 5-ketogluconic acid -> 2 _i p-diketogluconic acid.

The chromatographic paper used was commercial filter paper (Whatman Mon. 1 and No. 4), as the solvent system methyl ethyl ketone / acetone / formic acid / water (80: 6: 2: 12). The spots of the acid was carried out by spraying with a 0.2% ethanolic solution of o-phenylenediamine, containing 1% nitric acid, and heating to about? 0 ⁰ C localized to give 5-Eetogluconsäure a blue color, 2-ketogluconic a yellow Staining and 2,5-diketogluconic acid a green color. For identification, the high pressure liquid chromatography can also be used.

2 _S 5-I ⁾ ogluconic acid can be separated and recovered from the finally obtained fermentation broth by any conventional procedure known per se to the person skilled in the art. Pie filtered fermentation broth can be further processed as such by treatment with a borohydride, and the resulting mixture of 2-zetogluconic acid and 2-ketogulonic acid can be hydrolyzed to form ascorbic acid and erythorbic acid.

F ettings:.

The invention will be explained in more detail with reference to the following examples

The following aqueous seed medium was prepared:

Component · g / l

Glucose 25

Corn steep liquor · 5

PO ₄ . 0.5

K ₂ HPO ₄ 0.5

0.2 pH = 6.2

A Schütuelflasche containing 1 1 of the medium was treated for 30 minutes, "at 121 ⁰ C in the autoclave. The pH of the cooled medium was 5iO. Cells of Acetcbacter cerinus IFO 3263 from a Iiähragar / bevel plate (5 nil of a 20 ml sterile aqueous suspension) were added to the bottle, which then on a Drehschuttier at about 28 ⁰ C for etv: a 2M hours was shaken.

A subset of the culture broth, sufficient to provide a 5 v / v% inoculum, was added to a 4-1 permenta port containing 2 l of the following production level:

Component g / l

Glucose 110 Shark water 0.5

(NH ₄ ) ₂ HP0 ^ 0.58

KH ₂ PO ₄ 1.5

MgSO ₄ .7H ₂ O 0.5

Urea 0.5

CuSO ₄ .5H ₂ O * 1 mg

Nicotinic acid 500'ug ,. pH e. 6.0

The fermentation was carried out at a temperature of about 28 ⁰ C under stirring at i? 00 rpm and aeration at the rate of 0.75 volume

After a fermentation period of about 20 hours, sterile glucose (5 g / i) was added. The pH was kept at 5-5 by the addition of sodium hydroxide solution. The fermentation was continued. until a yield of 2.5 ~ diketogluconic acid of> 95%, based on glucose, was reached. ,

The work of Example 1 was rut. similar results using Acetobacter cerinus IFO 5266 was repeated. ·

Claims (2)

Erfindungsanspriich 1. A process for the preparation of 2,5-diketogluconic acid, characterized in that Acetobactei · cerinus is aerobically propagated in a glucose medium and then the obtained 2,5 ~ diketogluconic acid is obtained or the fermentation broth by selective reduction to form 2-ketogulonic acid and 2-Xetogluconic acid is processed, Method according to claim 3, characterized in that the glucose concentration in the medium is from 2.5 to 20%, the fermentation temperature is from 20 ^° C. to 35 ^° C., the initial pH is from 3 * 5 "to 7 _? 5 and the pH-Vert is maintained at about 5.5 during the perforation. J. Method according to item 1 or 2, characterized in that the strain IFO 3263 is used as Acetobacter cerinus. 4. Method according to. Item 1 or 2, characterized in that the strain IFO 3266 is used as Acetobacter cerinus.