WO1992018637A1

WO1992018637A1 - Method for the production of d-gluconic acid

Info

Publication number: WO1992018637A1
Application number: PCT/EP1992/000775
Authority: WO
Inventors: Akira Asakura; Tatsuo Hoshino; Setsuko Masuda; Yutaka Setoguchi
Original assignee: F.Hoffmann-La Roche Ag
Priority date: 1991-04-16
Filing date: 1992-04-06
Publication date: 1992-10-29

Abstract

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 activity, 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.

Description

Method for the production of D-gluconic acid

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.

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.

According to the present invention, it is possible to produce 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. In addition, 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. Moreover, 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.

In this context, 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.

Procedure for Assay of Gluconokinase

The reaction mixture (0.5 ml) contained 100 μmole of Tris-HCl buffer (pH 8.0), 6.6 μmole of MgC-2, 3.2 μmole of adenosine triphosphate, 0.4 μmole of NADP, 1.0 μmole of sodium gluconate, 20 μl 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 μmole of NADP per minute.

In this context, 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.

Procedure for Assay of Glucose Dehydrogenase

The assay mixture (0.5 ml) contained 50 μmole of D-glucose, 2 μmole of NADP, 0.3 mmole of Tris-HCl buffer(pH 8.0), 5 nmole of MnS0 and 20 μl 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 μmole 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.

Examples of the strains most preferably used in the present invention ar

Bacillus pumilus RMX1 and the like. This microorganism has been deposited in Agency of Industrial Science and Technology, Fermentation Research Institute, Japan under the following Number:

Bacillus pumilus RMX1 FERM-BP No. 3335 (date of deposit: March 29, 1991)

In the preferable embodiment of the present invention, 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.

It is usually required that 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.

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.

It is also possible to use immobilized 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.

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. To the concentrate is added 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. Whether the above or any other appropriate and known method, e.g. for the separation of D-gluconic acid, e.g. of a culture broth is employed, D-gluconic acid can always easily be recovered.

EXAMPLES

The following examples are further illustrative of this invention, it being understood, however, that the invention is by no means limited thereto.

Preparation of the Mutant Strain RMX1

One loopful of 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 μg/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.

D-Sorbitol 0.5 %

Bacto peptone ( Difco ) 1 % Yeast extract ( Oriental Yeast ) 0.2%

NaCl 0.2%

Agar 1.5 %

( pH 7.0 )

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 for 5 days at 36.5°C. Then the supernatant of the fermented broth was obtained by centrifugation and analyzed for D-gluconic add production level by thin layer chromatography. One μl of the supernatant was spotted on a silica gel plate ( ieselgel 6OF254, Merck) and developed by a solvent system consisting of n-propanol, 95% ethanol, and 0.05M potassium phosphate buffer ( 55:25:20 ) . Then silica gel plates were sprayed with KIO4- Tetrabase reagent to visualize the spot of D-gluconic add. Out of the mutants cultivated, the strain RMX1 was selected as a D-gluconic add high producer which was superior to its parent strain. EXAMPLE 1

One loopful of agar culture of Badllus pumilus ATCC 31093 (the parent strain of RE5), the parent strain RE5 and the mutant RMXl were inoculated into 5 ml of production medium in test tubes, respectively. The composition the medium is 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.

Table 1

D-gluconic acid Productivity of Bacillus pumilus ATCC 31093, RE5 and RMXl

* Values are expressed in terms of the free form. (D-gluconic add was actually accumulated as caldum salt in the culture broth.)

EXAMPLE 2

One loopful of agar culture of Badllus pumilus ATCC 31093, the parent strain RES and the mutant RMXl were inoculated into 7 ml of seed culture medium in test tubes, respectively. The composition of the medium is shown below.

D-Sorbitol 2.0 %

Corn steep liquor 2.0 % KH₂P0₄ 0.1 %

K2HPO4 0.3 %

L-Phenylalanine 0.0025 %

L-Tryptophan 0.0025 %

( pH 6.7 )

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 mutant RMXl produced 217.6 g/1 of D-gluconic acid from 250 g/1 of D-glucose (molar conversion, yield = 80 %) in 6 days fermentation.

From this fermentation broth, 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 ).

Table 2

- lu ni i rodu i i f Bacillus umilus ATCC 31093, RE5 and RMXl

* Values are expressed in terms of the free form. (D-gluconic acid was accumulated as caldum salt in the culture broth.)

** Molar conversion yield (%) Example 3

In the same manner as described in Example 2, Bacillus pumilus ATCC 31093, the parent strain RE5 and the mutant RMXl were cultivated in 500 ml Erlenmeyer flasks . When the D-glucose in medium had completely disappeared - as confirmed by a test-paper normally used for urine sugar analysis, 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 μg/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.

Table 3

Enzyme Activities of Cell Free Extracts of Bacillus pumilus ATCC31093, RE5 and RMXl

* unit / ml-broth ** unit / mg-protein

Example 4

In the same manner as described in Example 2, the seed culture of the mutant RMXl was prepared and inoculated into the production medium whose composition was as follows.

Molatein 0.5%

Corn steep liquor 0.45%

(NH4)₂S0₄ 0.55%

(NH4)2HP0₄ 0.2%

FeS0₄7H₂0 0.00032%

MnS0 «6H₂0 0.00019%

D-Glucose 25%

The flask was incubated at 36.5°C and 240 rpm for 4 days. 5% of D-glucose (4 ml of 50% solution) was added after 3 days of cultivation. As a result, the mutant RMXl produced 303.4 g/1 of D-gluconic acid from a total 30% of D-glucose (molar conversion, yield = 93%) after 4 days fermentation. Example 5

One loopful of agar culture of Bacillus pumilus RMXl was inoculated into 100 ml of seed culture medium in 500 ml Erlenmeyer flasks. The composition of the medium is shown below.

D-Sorbitol 6.0%

Corn steep liquir 6.0%

KH2PO4 0.3%

K2HPO4 0.9%

L-Phenylalanine 0.0075% L-Tryptophan 0.0075%

(pH 6.7)

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.

Molatein 0.5%

Corn steep liquor 0.9%

(NH4)2S0₄ 0.55%

(NH4)2HP0₄ 0.2% FeSθ₄7H₂θ 0.00032%

MnS0₄»6H₂0 0.00019%

D-Glucose 25%

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).

As a result, 229.3 g/1 of D-gluconic acid (255.0 g/1 of Na-D-gluconic add) was produced from 246.6 g/1 of D-glucose (molar conversion, yield = 85.4%).

Claims

1. A process for producing D-gluconic acid, which comprises cultivating microorganism belonging to the genus Bacillus, which is capable of producin D-gluconic add from D-glucose, which lacks gluconokinase activity, and whi has high glucose dehydrogenase activity, in the presence of D-glucose, in a culture medium, and recovering the resulting D-gluconic add from the culture broth.

2. A process according to claim 1, wherein the microorganism belonging to the genus Bacillus has at least 0.1 units of glucose dehydrogenase activity /mg protein.

3. A process according to claim 1, wherein the corresponding micro¬ organism has less than 0.001 units of gluconokinase activity /mg protein.

4. A process according to claim 1, wherein the microorganism is of the spedes Badllus brevis, Badllus cereus, Badllus circulans, Badllus coagulans, Badllus licheniformis, Bacillus megaterium, Badllus mesentricus, Bacillus pumilus and Bacillus subtilis.

5. A process according to daim 1, wherein the microorganism is Bacillus pumilus RMXl ( FERM BP-3335).

6. A process according to any one of claims 1 - 5, wherein the initial concentration of D-glucose in the culture medium is about 50 g/1 to about 300 g/1, preferably from about 100 g/1 to about 250 g/1.

7. A process according to any one of daims 1 - 6, wherein the cultivation is carried out at a pH between about 4.0 and 9.0, preferably between about 5.0 and 8.0.

8. A process according to any one of claims 1 - 7, wherein the cultivation is carried out at a temperature between about 20 and 45°C, preferably between about 25 and 40°C.

9. Bacillus pumilus RMXl (FERM-BP No. 3335) which is capable of producing D-gluconic acid from D-glucose, which Bacillus lacks gluconokinase activity and has high glucose dehydrogenase activity.