JPS5923794B2 - Manufacturing method of dihydroxyacetone - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for enzymatically producing dihydroxyacetone using microorganisms belonging to the genus Acetobacter or Gluconobacter.

Conventional methods for producing dihydroxyacetone using microorganisms include culturing microorganisms belonging to the genus Acetobacter in a medium containing glycerol, yeast extract, potassium phosphate, etc., and producing and accumulating dihydroxyacetone in the medium [Industrial・Microbiology;S
, C. Prescott, C.G. Dunn, P., 459-46.
0 (1959)], or as an improvement of the method, a method using a medium consisting of glycerol, corn steep liquor, yeast extract, potassium phosphate, calcium carbonate, etc. (US Pat. No. 2,948,658), glycerol, corn steep liquor, etc. , a method using a medium consisting only of urea (West German Patent No. 1136994), glycerol,
A method using a medium consisting of corn steep liquor, calcium carbonate, organic acid ammonium salt, etc.
No. 44485), etc. are known.

All of the above-mentioned known methods are methods for producing dihydroxyacetone by fermentation, but the fermentation method inherently has the following drawbacks.

In other words, since the fermentation-finished liquid inevitably contains impurities such as medium components and by-products, these impurities must be removed when dihydroxyacetone is isolated and purified from the fermentation-finished liquid. This requires very complicated operations and requires a great deal of expense and effort.

The present inventors overcame the above-mentioned difficulties inherent in the fermentation method and conducted various studies on an industrially advantageous method for producing dihydroxyacetone. It has been found that when cultured using sorbitol, mannitol, glucose, or dextrin instead of glycerol, which has been conventionally used, the conversion ability of the bacterial cells to dihydroxyacetone from glycerol is significantly increased. If the obtained bacterial cells or their processed product are aerobically applied to an aqueous solution containing substantially only glycerol with a salt concentration of 0.1% or less, high-concentration glycerol can be almost quantitatively converted to dihydroxyacetone. The present inventors have discovered that the present invention can be carried out without requiring any additives including coenzymes.

That is, the present invention involves culturing a microorganism belonging to the genus Acetobacter or Gluconobacter and having the ability to convert glycerol into dihydroxyacetone in a medium containing sorbitol, mannitol, glucose, or dextrin as a carbon source, and then culturing the microorganism in a medium containing sorbitol, mannitol, glucose, or dextrin as a carbon source. A process for producing dihydroxyacetone, which involves aerobically acting on bacterial cells separated from a liquid or a processed product of the bacterial cells on an aqueous solution containing essentially only glycerol with a salt concentration of 0.1% or less, and collecting the dihydroxyacetone produced. It is a manufacturing method.

As the microorganism used in the present invention, any microorganism that belongs to the genus Acetobacter or the genus Gluconobacter and has the ability to convert glycerol to dihydroxyacetone can be used. For example, Acetobacter spp.
Preferred examples include Acetobacter suboxidans ATCC621, Acetobacter suboxidans IFO3254, Acetobacter suboxidans IFO3255, Gluconobacter selinus IAM1832, and Gluconobacter melanogenus IF03294.

The medium composition for culturing the above microorganisms is as follows:
A carbon source containing a carbon source, a nitrogen source, an organic nutrient source, an inorganic substance, etc. is used as appropriate, but it is necessary to use sorbitol, mannitol, glucose, or dextrin as the carbon source.

As the nitrogen source, any substance can be used as long as it can be assimilated by the strain used, such as inorganic nitrogen sources and organic acid ammonium salts.

As an organic nutrient source, yeast extract, corn steep liquor, meat extract, polypeptone, etc. can be used, and the first two are particularly preferred.

Moreover, ordinary inorganic salts are used as the inorganic substance.

The above-mentioned microorganisms may be cultured by a conventional method, for example, the culture medium is
It is preferable to set the strain to H5-8 and culture the strain aerobically at 25-30°C for 1-3 days after inoculation.

In addition, by adding calcium carbonate to the culture medium during culturing, it is possible to prevent the enzyme activity of the strain from decreasing due to a decrease in the pH of the culture solution.

As mentioned above, it is necessary to use sorbitol, mannitol, glucose, or dextrin as a carbon source in the culture medium composition, but by using such a carbon source, dihydroxyacetone is more abundant than glycerol in bacterial cells, compared to when glycerol is used as a carbon source. The conversion ability to is significantly increased.

In order to clarify this point, Table 1 below shows the results of an investigation of the ability of the bacterial cells to convert dihydroxyacetone when the strains were cultured in media containing various carbon sources.

Note 1) Medium: 8% carbon source, 2% corn steep liquor, 0.3% calcium carbonate (2% if glucose or dextrin is used as the carbon source) Note 2) Strain used: Acetobacter suboxy Dance ATCC621 Note 3) Culture conditions: Shaking culture at 30°C for 48 hours Note 4) Reaction conditions: Suspend the bacterial cells in a 10% glycerol aqueous solution and shake at 30°C for 1 hour Reactivity 5) Activity (unit): 1 The unit is the activity of producing 1 μmol of dihydroxyacetone per minute, or the bacterial cells separated by centrifugation from the culture solution obtained as described above, or the processed product of the bacterial cells (e.g., washed bacterial cells, dried bacterial cells, etc.). Dihydroxyacetone can be produced by aerobically reacting immobilized bacterial cells (immobilized bacterial cells by an acrylamide gel method, etc.) with an aqueous solution containing essentially only glycerol with a salt concentration of 0.1% or less. .

The concentration of the glycerol aqueous solution is usually preferably about 10 to 30%.

The reaction temperature is preferably about 20 to 50°C, particularly about 30 to 40°C.

The pH of the reaction solution is preferably about 4 to 7, particularly about 5 to 6.

The pH of the reaction solution obtained by suspending the bacterial cells obtained from the culture solution in an aqueous glycerol solution is usually in the range of pH 4 to 7, and there is no need to particularly adjust the pH, but if necessary, acid or alkali may be added. pH can be adjusted by

At this time, the amount of acid or alkali added should be as small as possible.

As mentioned above, an aqueous glycerol solution containing essentially only glycerol with a salt concentration of 0.1% or less should be used; however, if the salt concentration exceeds 0.2%, the ability of bacterial cells to convert glycerol into dihydroxyacetone will decrease. decreases, which is not desirable.

In order to clarify this point, we investigated the ability of bacterial cells to convert dihydroxyacetone when various salts were added to a glycerol aqueous solution at various concentrations.
Shown in the table.

Note 1) Medium: Sorbitol 20%, corn steep liquor 2%, calcium carbonate 0.3% Note 2) Bacterial strain used: Acetobaccu suboxidans ATCC621 Note 3) Culture conditions: 30°C, shaking culture for 48 hours Note 4) Reaction conditions: Suspend the bacterial cells in a 10% aqueous glycerol solution to which salts have been added, and shake at 30°C for 1 hour. Activity: Relative activity when the activity without the addition of salts is set as 100. Completion of the reaction. After that, since the reaction solution contains essentially only the bacterial cells or the treated bacterial cells and dihydroxyacetone, after removing the bacterial cells or the treated bacterial cells from the reaction liquid, it can be easily removed by means such as concentration. Crystals of dihydroxyacetone can be obtained.

Moreover, even if purification means such as ion exchange resin operation must be performed, the amount of ion exchange resin used can be reduced because there are few contaminants mixed in.

As is clear from the above description, the method of the present invention provides the following advantages.

(2) By using sorbitol, mannitol, glucose, or dextrin as a carbon source, bacterial cells with an extremely superior ability to convert glycerol to dihydroxyacetone can be obtained.

(2) Highly concentrated glycerol can be almost quantitatively converted to dihydroxyacetone by allowing the bacterial cells or the treated bacterial cells to act on an aqueous solution containing essentially only glycerol.

(2) After the reaction, an almost pure dihydroxyacetone aqueous solution can be obtained by simply removing the bacterial cells or the treated bacterial cells, and the purification operation for extracting dihydroxyacetone from the reaction solution is extremely simplified.

The method of the present invention will be specifically explained below with reference to Examples.
In the examples, dihydroxyacetone was confirmed and quantified using the dinitrophenylhydrazine coloring position by paper chromatography, the p-anisidine coloring position by thin layer chromatography, and the modified method of W, S, Hoffman (J, Bi
o#, Chem, t120.51 (1937)).

Example 1 Acetobacter suboxidans ATCC621 was inoculated into a medium having the following composition (100 TL, 11,500 ml volume Sakaro flask), and cultured with shaking at 30°C for 20 hours.

Medium composition (in 100ml): Sorbitol 20g Corn steeple strength - 2g Calcium carbonate 0.3g Cells were collected by centrifugation from 100ml of the culture solution obtained in this way, and suspended in 100ml of 15% glycerol aqueous solution, pH: 5. 5), and the reaction was carried out at 30'C for 40 hours with shaking.

As a result, 149g of dihydroxyacetone was found in the reaction solution.
Generated and accumulated.

The bacterial cells were removed from this reaction solution by centrifugation, the supernatant was decolorized and concentrated, and ethanol was repeatedly added to obtain 10.2 g of crude crystals of dihydroxyacetone.

Example 2 Gluconobacter melanogenes IFO3294 was cultured in a medium having the same composition as in Example 1 under the same conditions.

The bacterial cells were collected from 100 ml of the obtained culture solution by centrifugation, and suspended in 100 ml of a 20% glycerol aqueous solution.
The reaction was allowed to proceed at 30°C for 66 hours with shaking.

As a result, 19.3% of dihydroxyacetone was present in the reaction solution.
．． p production and accumulation.

The bacterial cells were removed from this reaction solution by centrifugation, and the supernatant was concentrated to yield 17.5 g of crude dihydroxyacetone.
I got it.

Example 3 Acetobacter suboxidans IFO3254, Acetobacter suboxidans IFO3255, and Gluconobacter selinus IAM1852 were each inoculated into a medium with the following composition (100 ml/500 rul Sakaro flask) and shaken at 30°C for 24 hours. It was cultivated for a long time.

Medium composition (in 100 ml) Mannitol 8 g Yeast extract 2 g Calcium carbonate 0.3 g From 100 ml of the thus obtained culture solution, collect bacterial cells by centrifugation.
Suspend in 15% glycerol aqueous solution and incubate at 30℃ for 40 minutes.
The reaction was allowed to take place while shaking for hours.

As a result, the amount of dihydroxyacetone (DHA) produced and accumulated in the reaction solution was as shown in Table 3 below.

Example 4 Acetobacter suboxidans ATCC621 was cultured in a medium with the same composition as in Example 1 under the same conditions.

Bacterial cells were collected from 100 ml of the obtained culture solution by centrifugation and suspended in pure water to make 5 ml.

To this were added 0.94 g of acrylamide, 0.05 g of N,N'-methylenebisacrylic acid amide, 0.6 ml of 5% β-(dimethylamino)propionitrile, and 0.6 ml of 1% potassium persulfate. It was left standing at room temperature for 10 minutes.

Next, the resulting gel was crushed and washed with pure water to obtain immobilized bacterial cells 7.5I.

7.5I of these immobilized bacterial cells were added to 10% glycerol aqueous solution.
00ml and reacted at 30°C for 40 hours with shaking.

As a result, 8 g of dihydroxyacetone was produced and accumulated in the reaction solution.

Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Acetobacter or Gluconobacter and having the ability to convert glycerol to dihydroxyacetone is cultured in a medium containing sorbitol, mannitol, glucose or dextrin as a carbon source, and then the culture solution dihydroxyacetone, which is characterized in that the bacterial cells isolated from the bacterial cells or the processed product of the bacterial cells are allowed to act aerobically on an aqueous solution containing essentially only glycerol with a salt concentration of 0.1% or less, and the generated dihydroxyacetone is collected. manufacturing method. 2. The production method according to claim 1, wherein the microorganism is Acetobacter suboxidans, Gluconobacter serinus, or Gluconobacter melanogenes, which have the ability to convert glycerol into dihydroxyacetone.