LU80536A1 - PROCESS FOR PRODUCING 2,5-DICETOGLUCONIC ACID - Google Patents

Description

D. 5o.o62 GRAND-DUCHY OF LUXEMBOURG Patent N ° ........... 'A Λ P “i Λ du 1.6 noYeôb M Μ7.η u qm Minister, 0ν§ * Ξ & de l '' National Economy and the Middle Classes

Title issued: ........................................ 5¾¾¾. . x gWrjw Industrial Property Service

^ LUXEMBOURG

/ {_ Application for Patent of invention I. Request ..... The. ..... EFIYER ..... IUC .. 23.5 .. East.A2nâ Street., ..... à..iiEiÎ-YOPJôi ..... F / cafe. from New York ..... United States ..... dlMcrique., ..... representas .. par ..................... .........

..... Iions.ie.ur ..... Jacques ..... de.Jiu.yser .., ..... acting, in ... .quality .... de ... siândataire (2) remove ........ this ..... sixteen. .nQ.y.eiabr.e ..., 19oQ ..... seventy-seventh ........................... ....... © at ......... 15 .......... .... hours, at the Ministry of National Economy and the Middle Classes, in Luxembourg: 1. the present request for obtaining a patent of invention concerning: ...... "ErocSda ..... of ... production ... of acid ... ^., S- dicatogluconicue "........................................ (4) declares, assuming the responsibility for this declaration, that the Inventor (s) is (are): ..... 1 * -..... Donald ... JLlbart ... ü.I.TA / ..... Ueadcir.ïOOds ..Rosd, ...... à .... iSSEX-f ---- Crjàté..cQ ........ (5) ......... ............. Hidd the SX., .... State ... of ..... CQnnact.ic.ut., ..... United States ... ..à.Utàsriqna ..........................

...... 2 .. »..- r ..... Karl.ene .... Lli2ab.Cith .... EM.; L. / ...... 1.7 .... Granite ..... Street, ...... H. „USS_.L025LQSl / .... C.oint ..................... ..àe ... KÂ:; i .... London. ^ ... State ... c3 ... CorniJciciït / .... E ^ Jc2d & i3.àti ^^ idi2 ........ ................................

2. the delegation of power, dated Ιχ ^ νίτ.τ.ΥΟΒΚ., ................ on .2-5._JLOÛt ..... 19.7.3. ................

3. description in language ........ XD5.I1Ç2.ÀE & ....... ...................... of the invention in two copies; 4 ........ DD ................. drawing boards, in two copies; 5. the receipt of the taxes paid to the Luxembourg Registration Office, ...... 16 ... .novtnr: br.e. ... 19.7 δ ............................................. .................................................. .................................................. .......................................

claims for the above patent application the priority of one (or more) application (s) of (6) ....................... patent ..... ......................................... posted © PvP (7) _aux .. ..llt, nfcs.-Un.i5 ..... dl-lrnSriqus ......................

le ...... XS .... noMÂîéhre ..... 19.71 ___________ (Kcu ..... £ 52,, 9.5a) -............... .................................................. .................................................. ......... (8) in the name of S .... inyfcntS.UPS .................... ....... .................................................. .................................................. .................................................. .......... (9) elect domicile for him / her and, if appointed, for their proxy, in Luxembourg ................... .....................

..... 35,. bld .BÖ.V.3.1 ........................................... .................................................. .................................................. .................................................. .............. (10) requests the grant of a patent for the invention for the subject described and represented in the abovementioned appendices, - with postponement of this grant to .... .... IB ............................. months.

The ..... çinSifcalre ....... X ......... ¢, ....... Ü I, JX. NOT A WORD .

II. Deposit Minutes

The aforementioned patent application has been filed with the Ministry of National Economy and the Middle Classes, Industrial Property Service in Luxembourg, dated: 16 r.oy-ylrre 1973

Pr. The Minister at 1 5 a.m./Je.· s't * i * ÇYv of the National Economy and Movennes Classes, (/ r ~ \ »* /

U? J) (r; / J

A 6SQ07 \ J ν '' - 1 D. 5o.o62

CLAIM OF PRIORITY

, of the patent / txt application in the UNITED STATES OF AMERICA of NOVEMBER 18, 1977 Brief Description filed in support of an application for

PATENT

at

Luxembourg on behalf of: PFIZER INC.

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

4

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

2,5-Diketogluconic acid is an interesting intermediate compound for the synthesis of vitamin C. Until now, this acid has been produced by various bacterial species such as Acetobacter melanoqenum, Acetobacter aurantium, Gluconoacetobacter rubiginosus, Gluconoacetobacter liquifaciens and Pseudomonas sesami. However, the use of these microorganisms is not entirely desired from the industrial point of view, because of the production of large quantities of brown or yellowish-brown pigments as by-products of culture, which lower the purity 2,5-diketogluconic acid produced at the same time.

U.S. Patent No. 3,790,444 relates to the production of 2,5-diketogluconic acid without concomitant production of brown pigment, by a new species called Acetobacter fraqum.

The invention relates to an economical process for the production of 2,5-diketogluconic acid which uses strains of a microorganism which is readily available and available to the public; this microorganism is called Acetobacter cerinus. Two of these strains, IFO 3263 and 3266, produce 2,5-diketogluconic acid in yields of more than 95% based on glucose.

25 2,5-Diketogluconic acid is an interesting intermediate compound to be used for the production of ascorbic acid. An aqueous solution of 2,5-diketogluconic acid can be selectively reduced to produce a mixture of 2-ketogulonate and 2-ketogluconate which can be converted into ascorbic and erythorbic acids.

2,5-Diketogluconic acid is easily prepared by bacterial action on glucose using, in accordance with the process of the present invention, strains of Acetobacter cerinus which are easy to obtain. All the strains indicated for Acetobacter cerinus, 35 made available to the public, were subjected to tests by which they were found to give keto acids in a yield of 50 to 95% on the basis of glucose. When using Acetobacter cerinus. [2 IFO 3263 or 3266, the keto acid produced is all the desired 2,5-diketogluconic acid, which is obtained in yields of more than 95% over the glucose base. The existing strains of Acetobacter cerinus, available to the public 5, are as follows: IFO 3262 (ATCC 12303) 3263 3264 3265 10 3266 3267 3268 3269

These strains of Acetobacter cerinus are grown in a medium whose main source of carbon is glucose. These microorganisms do not require expensive organic sources of nitrogen such as peptone or deviant 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 glucose concentration of the medium varies between 2.5 and 20%, preferably between 10 and 12%, 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 it is very advantageously of the order of 28 ° C. The initial pH of the culture medium can range from 3.5 to 7.5 and is preferably between 5 and 6.

During fermentation, the pH is maintained at about 5.5 by the addition of a sodium hydroxide solution. Calcium carbonate can be used to adjust the pH and this salt is added as a make-up to the medium after autoclaving, in an amount of 30 g per 110 g of glucose.

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

3 ί m

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

5 It has been determined by paper chromatography that the conversion of glucose to 2,5-diketogluconic acid is carried out according to the following schemes: glucose— ·> 2-ketogluconic acid — 1> 2,5-diketogluconic acid glucose — 5 * acid 5-ketogluconic—? 2,5-diketogluconic acid.

1 10 We use No. 1 and No. 4 "Whatman" paper and use a mixture of solvents consisting of methyl ethyl ketone, acetone, formic acid and water in the proportion of 80: 6: 2: 12.The spots corresponding to the acid are localized by spraying with an ethanolic solution containing 0.2% of o-phenyl-15-enenediamine containing 1% of nitric acid and heating to approximately 70 ° C (5-ketogluconic acid - blue color; 2-ketogluconic acid - yellow color; 2,5-diketogluconic acid - green color). Identification can also be carried out by high pressure liquid chromatography.

2,5-Diketogluconic acid can be separated and collected from the final fermentation broth by any conventional operation known to those skilled in the art. The fermentation broth which has been filtered can be treated for example with borohydride and the resulting mixture of 2-ketogluconic acid and 2-ketogulonic acid can be hydrolyzed to give the ascorbic and erythorbic acids.

EXAMPLE 1

The following aqueous medium is prepared for inoculation: 30 Ingredients Grams / liter

Glucose 25

Soluble corn extract 5 KH2P04 0.5 K2HP04 0.5 '35 MgS04.7H20 0.2

CaC03 6.3 pH 6.2 it 4% · »

A shake flask containing one liter of medium is autoclaved for 30 minutes at 121 ° C.

The pH of the cooled medium is 5.0. Acetobacter cerinus IFO 3253 cells from a tilt-5 culture born on nutrient agar (5 ml of a sterile aqueous suspension with a volume equal to 20 ml) are added to the flask which is then shaken on a shaker rotating at 28 ° C for approximately 24 hours.

A growing culture sample sufficient to represent a proportion of 5% by v / v of inoculum is added to a fermenter with stirring of 4 liters of capacity containing 2 liters of the following production medium:

Ingredients Grams / liter 15 Glucose 110

Soluble corn extract 0.5 (NH.KHPO, 0.58 KH2P04 1.5

MgS04.7H20 0.5 20 Urea 0.5

CuS04.5H20 1 mg

Nicotinic acid 300 Ύ pH 6.0

The fermentation is carried out at a temperature of about 28 ° C with stirring at 1700 rpm and aerating at a rate of 0.75 volume per volume of broth per minute. After a fermentation period of approximately 20 hours, sterilized glucose (55 g / l) is added. The pH is maintained at 5.5 by the addition of a sodium hydroxide solution. Fermentation is continued until the yield of 2,5-diketo-gluconic acid is greater than 95% based on glucose.

EXAMPLE 2 The procedure of Example 1 is repeated using the Acetobacter cerinus IFO 3266j, results ‘35 which are comparable are obtained.

Claims (5)

1. A process for the production of 2,5-diketogluconic acid, characterized in that it consists in carrying out the aerobic culture of Acetobacter cerinus in a glucose medium and then in collecting the 2,5- acid. resulting diketogluconic or to treat the fermentation broth by selective reduction to obtain 2-ketogalonic and 2-ketogluconic acids. 2. Method according to claim 1, characterized in that the glucose concentration of the medium has a value of 2.5 to 20%, the temperature of the fermentation ranges from 20 to 35 ° C, the initial pH ranges from 3 , 5 to 7.5 and the pH during fermentation is kept at around 5.5. 3. Method according to one of claims 1 and 2, characterized in that the strain of Acetobacter cerinus 15 is the strain IFO 3263. 4. Method according to one of claims 1 and 2, characterized in that the strain of ♦ Acetobacter cerinus is the strain IFO 3266. 5. 2,5-Diketogluconic acid, 2-keto-gulonic acid or 2-ketogluconic acid obtained by a process according to any one of the preceding claims. "