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WO1992018637A1 - Procede de production de l'acide d-gluconique - Google Patents

Procede de production de l'acide d-gluconique Download PDF

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Publication number
WO1992018637A1
WO1992018637A1 PCT/EP1992/000775 EP9200775W WO9218637A1 WO 1992018637 A1 WO1992018637 A1 WO 1992018637A1 EP 9200775 W EP9200775 W EP 9200775W WO 9218637 A1 WO9218637 A1 WO 9218637A1
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WO
WIPO (PCT)
Prior art keywords
bacillus
glucose
gluconic acid
badllus
process according
Prior art date
Application number
PCT/EP1992/000775
Other languages
English (en)
Inventor
Akira Asakura
Tatsuo Hoshino
Setsuko Masuda
Yutaka Setoguchi
Original Assignee
F.Hoffmann-La Roche Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F.Hoffmann-La Roche Ag filed Critical F.Hoffmann-La Roche Ag
Publication of WO1992018637A1 publication Critical patent/WO1992018637A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

  • This invention relates to a method for producing D-gluconic acid from glucose by fermentation.
  • D-gluconic acid and its derivatives have many commercial uses, such as being agents for the regulation of solidifying concrete, agents in textile printi and textile bleaching, as agents for preventing milkstone and beerstone in th dairy industry and breweries, respectively, and as sequestrant. D-gluconic acid and its derivatives have also a wide use in food and pharmaceutical industry and in detergents.
  • D-gluconic acid Many fermentation processes for the production of D-gluconic acid are known. Many microorganisms, such as Acetobacter, Pseudomonas, Gluconobacter, Aspergillus and Penicillium are known to be able to accumulate D-gluconic acid. However, the known fermentation processes are not fully satisfactory as commercial processes for the production of D-gluconi acid in terms of yields.
  • D-gluconic acid at a satisfactorily high yield, i.e. as a commercial process by using the mutants of bacteria of the genus Bacillus. It has been found that mutants of bacteria of the genus Bacillus which lack gluconokinase activity and have hig glucose dehydrogenase activity have an unusually high ability to accumulate D-gluconic acid. The present invention has been accomplished based on this finding.
  • the microorganisms belonging to the genus Bacillus exhibit several advantages.
  • the fermentation process by the genus Bacillus is simple because such microorganism can grow fast even in a simple and cheap medium, and because it is easy to handle, for instance, in the preparation of seed culture, an in the removal of the bacterial cells in the fermentated broth.
  • the isolation procedure for gluconic acid from the broth is easy, due to little accumulation of other organic acids, which may cause difficulty in separation, unlike gluconic acid.
  • the genus Bacillus has high stability in geneti characteristics, thus it can be stocked easily without decrease of its activities for the production.
  • the present invention is thus concerned with a process for producing D- gluconic acid which comprises cultivating a microorganism belonging to the genus Bacillus, which is capable of producing D-gluconic acid from D-glucose, which lacks gluconokinase, and which has high glucose dehydrogenase activity, in the presence of D-glucose in a culture medium, and recovering the resulting D-gluconic acid from the culture broth.
  • the lack of gluconokinase activity means, for example, tha when, by the following method, the discipline of which is described in Biochim. Biophys. Acta. 798, 88-95 (1984), the amount of reduction of oxidation form of nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP) is measured and the particular enzymatic activity is calculated for said cell free extract prepared by the procedure as described below ( See Example 3),the value is not more than 0.001 unit/mg-protein.
  • NADP nicotinamide adenine dinucleotide phosphate
  • the reaction mixture (0.5 ml) contained 100 ⁇ mole of Tris-HCl buffer (pH 8.0), 6.6 ⁇ mole of MgC-2, 3.2 ⁇ mole of adenosine triphosphate, 0.4 ⁇ mole of NADP, 1.0 ⁇ mole of sodium gluconate, 20 ⁇ l of the cell free extract and 0.005 unit of authentic 6-phosphogluconate dehydrogenase (Enzyme code 1.1.1.44, Sigma Chemical Co.,Ltd.).
  • the reaction was initiated by the addition of the substrate.
  • the change of absorbance at 340 nm was measured with a spectrophotometer Model UVT ON 810 (Kontron K.K.) at room temperature.
  • One unit of the enzyme activity was defined as the amount of the enzyme catalyzing the reduction of 1 ⁇ mole of NADP per minute.
  • high glucose dehydrogenase activity means, for example, that when, by the following method, the discipline of which is described in Agric. Biol. Chem. 43, 271-278 (1979), the amount of reduction of NADP is measured at 340 nm with a spectrophotometer Model UVIKON 810, and the particular enzymatic activity is calculated for said cell free extract as described above, the value is not less than 0.1 unit/mg-protein.
  • the assay mixture (0.5 ml) contained 50 ⁇ mole of D-glucose, 2 ⁇ mole of NADP, 0.3 mmole of Tris-HCl buffer(pH 8.0), 5 nmole of MnS0 and 20 ⁇ l of the cell free extract.
  • the reaction was initiated by the addition of the substrate.
  • One unit of enzyme activity was defined as the amount of enzyme catalyzing the reduction of 1 ⁇ mole of NADP per minute.
  • the microorganisms used in the present invention embrace all the strains belonging to the genus Bacillus which lack gluconokinase and have high glucose dehydrogenase activity.
  • Such strains can be easily derived from microorganisms belonging to the genus Bacillus such as , Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus mesentricus, Bacillus pumilus, Bacillus subtili etc., by such means as irradiating the parent strains with radiation such as ultraviolet light, X-rays, gamma rays or the like, or exposing the parent strain to the action of chemical utagens, such as N-methyl-N'-nitro-N- nitrosoguanidine (hereinafter referred to as MNNG), nitrogen mustard, ethylmethanesulfonate, etc.
  • MNNG N-
  • the production D-gluconic acid is effected by cultivating the above microorganism in an aqueous medium containing D-glucose and supplemented with appropriate nutrients under aerobic conditions.
  • Said medium can contain D-glucose in a concentration of about 50 g/1 to about 300 g/1, preferably from about 100 g/1 to about 250 g/1.
  • the culture medium contains nutrients as assimilable carbon sources, e.g. D-glucose, D-fructose, D-mannose, D-sorbitol, D-mannitol, sucrose, molasses, starch hydrolyzates, starch, acetic acid and ethanol; digestible nitrogen sources such as organic substances, for example, peptone, yeast extract, soybean meal, corn steep liquor, cottonseed refuse, drie yeast and meat extract, and inorganic substances, for example, ammonium sulfate, ammonium chloride, ammonium phosphate, potassium nitrate and potassium phosphate; vitamins, metals, amino acids and trace elements, etc.
  • nutrients e.g. D-glucose, D-fructose, D-mannose, D-sorbitol, D-mannitol, sucrose, molasses, starch hydrolyzates, starch, acetic acid and ethanol
  • digestible nitrogen sources such as organic substances, for example,
  • the cultivation may be conducted at pH values of about 4.0 to about 9.0, preferably from about 4.5 to about 8.0.
  • a cultivation period varies depending upon the microorganisms and nutrient medium to be used, preferably about 10 to about 150 hours.
  • a preferred temperature range for carrying out for the cultivation is from about 20 to about 45°C, preferably from about 25 to about 40°C.
  • microorganism on appropriate supports, such as -carrageenan, calcium alginate and other polymers, for the production of D-gluconic acid, and this enables the microorganism to be used repeatedly.
  • appropriate supports such as -carrageenan, calcium alginate and other polymers
  • the D-gluconic acid thus accumulated can be easily recovered, for example, by the following procedure:
  • the culture broth is first adequately diluted with water to dissolve D- gluconic acid, which has precipitated in the culture broth, then filtered or centrifuged, whereby the cells can be removed with great ease. Then, the filtrat might be decolorized, e.g. by treatment with activated carbon, and then, concen trated.
  • an appropriate organic solvent such as ethanol, whereupon D-gluconic acid crystals separate in the salt form, such as t sodium salt and the calcium salt, for example.
  • D-gluconic acid can always easily be recovered.
  • Bacillus pumilus RE5 (FERM-BP NO. 2833) grown on an agar medium was inoculated into a 100 ml of seed culture medium whose composition is shown below.
  • the flask was incubated at 30°C for 18 hours.
  • the cells were collected by centrifugation and suspended into 20 ml of 50 mM phosphate buffer (pH 8.0). A portion of the cell suspension ( 0.75ml ) was added by 100 ⁇ g/ml ( final concentration ) of MNNG and treated for 30 minutes at 30°C.
  • the treated cells were collected by centrifugation, washed once by sterile water, resuspended into 5 ml of seed culture medium and incubated for 2 hours at 30°C.
  • the culture thus prepared was appropriately diluted by sterile water and spread on the agar culture medium as shown below.
  • the plates were incubated at 36.5°C for 2 days. Colonies well grown on th plates were streaked on a fresh agar medium as shown below.
  • the plates were incubated at 36.5°C for 24 hours to obtain enough amoun of cell mass for tube culture as described below.
  • Each of the agar cultures thus prepared was used to inoculate 5 ml of production medium as shown below.
  • the tubes were incubated at 36.5°C for 5 days.
  • the D-gluconic acid productivities of Badllus pumilus ATCC 31093, the parent strain RE5 and the mutant RMXl are shown in Table 1.
  • the mutant RMXl showed about 16 and 37 times higher productivity of D-gluconic add than Bacillus pumilus ATCC 31093 and the parent strain RE5, respectively.
  • the inoculated test tubes were incubated at 36.5°C for 6 hours on a tube shaker.
  • the seed cultures thus prepared ( 4 ml ) were inoculated into production media made up to 40 ml after inoculation in 500 ml Erlenmeyer flasks.
  • the composition of the production medium was as follows.
  • the flasks were incubated at 36.5°C and 220 rpm for 6 days.
  • the D- gluconic add productivities of Badllus pumilus ATCC 31093 of the strain RE5 and the mutant RMXl are shown in Table 2.
  • the mutant RMXl showed much higher productivity of D-gluconic acid than did Badllus pumilus ATCC 31093 or the strain RE5.
  • the cells were removed by filtration after the broth was diluted with water to dissolve D-gluconic acid which was precipitated in the culture broth, and the filtrate was concentrated to half the original volume. Then, about one-quarter of its volume of ethanol was adde and the predpitate was discarded. The supernatant was decolorized on a column of activated carbon. The decolorized solution was concentrated, and about 4 times its volume of ethanol was added, whereby 7.3 g of crystalline D- gluconic acid ( calcium salt ) was obtained ( 96 % purity ).
  • Bacillus pumilus ATCC 31093, the parent strain RE5 and the mutant RMXl were cultivated in 500 ml Erlenmeyer flasks .
  • 50 ml of cultured broth was withdrawn from each flask .
  • the broth was centrifuged at 6,000 x g for 10 minutes, and the predpitated cells were suspended in 10 ml of 50 mM Tris-HCl buffer(pH 7.5).
  • the cell suspension wa centrifuged at 6,000 x g for 10 minutes, and the precipitated cells were washed again by the same procedure as described above.
  • the obtained cells were froze at -20°C until use.
  • the frozen cells were thawed, suspended in 10 ml of 50 mM Tris-HCl buffer (pH 7.5) and centrifuged at 6,000 x g for 10 minutes.
  • the predpitated cel were re-suspended in 10 ml of the same buffer and added by lysozyme ( Sigm Chemical Co. ) to the final concentration of 500 ⁇ g/ml.
  • the mixture was then incubated at 37°C for 1 hour with agitation ( 240 rpm ) to lyse the cells.
  • the lysate thus obtained was centrifuged at 6,000 x g for 10 minutes.
  • the resulting supernatant was used as cell free extract.
  • the enzyme activities of glucose dehydrogenase (GDH) and glucono- kinase (GAK) in the cell free extracts of Bacillus pumilus ATCC 31093 and the parent RE5 and the mutant RMXl were measured. The results are shown in Table 3. It was found that the mutant RMXl possessed about 4.5 times higher spedfic activity of glucose dehydrogenase than the parent.
  • the seed culture of the mutant RMXl was prepared and inoculated into the production medium whose composition was as follows.
  • Example 5
  • the inoculated flasks were incubated at 36.5°C for 7.5 hours.
  • the seed culture thus prepared (300 ml) was inoculated into the production medium made up to 3 L after inoculation in a 5 L jar fermentor (B.E. Marubishi Co., Ltd.).
  • the composition of the production medium was as follows.
  • the fermentation was carried out at 36.5°C, with agitation at 500 rpm and aeration at 0.5 wm.
  • the pH value was controlled with 6N NaOH (not to be lowered below 5.3).

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

On décrit un procédé de production de l'acide D-gluconique qui consiste à cultiver un micro-organisme appartenant au genre Bacillus capable de produire de l'acide D-gluconique à partir de D-glucose, qui ne présente pas d'activité gluconokinase et qui présente une activité de glucose déhydrogénase élevée en présence de D-glucose, dans un milieu de culture. Enfin, on récupère l'acide D-gluconique résultant à partir du bouillon de culture.
PCT/EP1992/000775 1991-04-16 1992-04-06 Procede de production de l'acide d-gluconique WO1992018637A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP91106009.3 1991-04-16
EP91106009 1991-04-16

Publications (1)

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WO1992018637A1 true WO1992018637A1 (fr) 1992-10-29

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1373480A4 (fr) * 2001-04-04 2005-02-02 Genencor Int Mecanismes d'action productifs et cataboliques non couples des cellules hotes
US7033804B2 (en) 2001-04-04 2006-04-25 Genencor International, Inc. Methods for the production of products in host cells
JP2007513043A (ja) * 2003-12-01 2007-05-24 ダブリュー・アール・グレイス・アンド・カンパニー−コネチカット セメント及びコンクリ−ト混和物のためのグルコン酸塩ブロス
WO2015151009A1 (fr) * 2014-03-31 2015-10-08 Lubrizol Advanced Materials, Inc. Extrait de ferment de souche bactérienne pour l'augmentation des teneurs en adiponectine

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOCHIMICA ET BIOPHYSICA ACTA vol. 798, 1984, ELSEVIER ;AMSTERDAM,NL. pages 88 - 95; Y. FUJITA ET AL: 'Catabolite repression of inositol dehydrogenase and gluconate kinase synthese in Bacillus subtilis' cited in the application *
CHEMICAL ABSTRACTS, vol. 104, no. 13, 31 March 1986, Columbus, Ohio, US; abstract no. 103309C, FUJITA,YASUTARO ET AL: 'The characterization and cloning of a gluconate -gnt- operon of Bacillus subtilis' page 158 ;column L ; *
CHEMICAL ABSTRACTS, vol. 105, no. 7, 18 August 1986, Columbus, Ohio, US; abstract no. 57740G, OTANI,MIEKO ET AL: 'Predominance of gluconate formation from glucose during germination of Bacillus megaterium' page 353 ;column R ; *
CHEMICAL ABSTRACTS, vol. 107, no. 7, 17 August 1987, Columbus, Ohio, US; abstract no. 55522E, OTANI,MIEKO ET AL: 'Gluconate metabolism in germinated spores of Bacillus megaterium QM B1551:primary roles of gluconokinase and the pentose cycle' page 417 ;column R ; *
CHEMICAL ABSTRACTS, vol. 92, no. 23, 9 June 1980, Columbus, Ohio, US; abstract no. 194200H, MARUYAMA,TOMOKI ET AL: 'Glucose catabolism during germination of Bacillus megaterium spores' page 325 ;column R ; *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1373480A4 (fr) * 2001-04-04 2005-02-02 Genencor Int Mecanismes d'action productifs et cataboliques non couples des cellules hotes
US7033804B2 (en) 2001-04-04 2006-04-25 Genencor International, Inc. Methods for the production of products in host cells
US7241587B2 (en) 2001-04-04 2007-07-10 Genencor International, Inc. Method of uncoupling the catabolic pathway of glycolysis from the oxidative membrane bound pathway of glucose conversion
US7407780B2 (en) 2001-04-04 2008-08-05 Genencor International, Inc. Process for producing glycerol in recombinant bacterial host cells
EP2055773A1 (fr) 2001-04-04 2009-05-06 Genencor International, Inc. Découplage des voies anaboliques et cataboliques dans des cellules hôtes
JP2007513043A (ja) * 2003-12-01 2007-05-24 ダブリュー・アール・グレイス・アンド・カンパニー−コネチカット セメント及びコンクリ−ト混和物のためのグルコン酸塩ブロス
US7462236B2 (en) 2003-12-01 2008-12-09 W. R. Grace & Co.-Conn. Gluconate broth for cement and concrete admixture
WO2015151009A1 (fr) * 2014-03-31 2015-10-08 Lubrizol Advanced Materials, Inc. Extrait de ferment de souche bactérienne pour l'augmentation des teneurs en adiponectine
US10159641B2 (en) 2014-03-31 2018-12-25 Lubrizol Advanced Materials, Inc. Ferment extract of a bacterial strain for the increase of adiponectin levels
AU2015242227B2 (en) * 2014-03-31 2020-04-16 Lubrizol Advanced Materials, Inc. Ferment extract of a bacterial strain for the increase of adiponectin levels

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