JPH02286089A - Production of dihydroxyacetone - Google Patents

Abstract

PURPOSE:To efficiently obtain the subject compound useful as a raw material for cosmetics, medicines, etc., in a short time by culturing a thermophilic microorganism, belonging to the genus Bacillus and capable of converting glycerol into dihydroxyacetone in a glycerol-containing culture medium. CONSTITUTION:A thermophilic microorganism [e.g. Bacillus.sp. TB-516 (FERM P-10524)], belonging to the genus Bacillus and having the ability to convert glycerol into dihydroxyacetone is cultured in a glycerol-containing culture medium to produce the objective compound in the above-mentioned culture. The resultant compound is then collected. Furthermore, the culturing is preferably industrially carried out by a spinner culture method with aeration.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for biochemically producing dihydroxyacetone using a thermophilic microorganism belonging to the genus Bacillus.

[Prior Art] Dihydroxyacetone has become increasingly important in recent years as a raw material for cosmetics, pharmaceutical products, surfactants, and the like.

Conventionally, as a method for producing dihydroxyacetone using microorganisms, there is a method using microorganisms belonging to the genus Acetobacter or Gluconobacter (Japanese Patent Publication No. 49-114
33. 59-23794), but this method requires 1 to 3 days for the growth of microorganisms and further 2 to 3 days for the subsequent production of dihydroxyacetone. Other methods for producing dihydroxyacetone using microorganisms include a method using microorganisms belonging to the genus Actinobacteria Micromonospora (Japanese Patent Application Laid-Open No. 62-2109414);
This method also requires 3 to 7 days to produce dihydroxyacetone.

[Problems to be Solved by the Invention] All of the above-mentioned known methods require a long time of 3 to 7 days for the growth of microorganisms that convert glycerol into dihydroxyacetone and the production of dihydroxyacetone, which is said to improve industrial productivity. From this point of view, it is desired to shorten manufacturing time.

[Means for Solving the Problems] The present inventors have overcome the above-mentioned problems, multiplied in a short time,
In addition, we conducted extensive research to obtain microorganisms capable of efficiently converting glycerol to dihydroxyacetone in a short period of time. As a result, it was discovered that a thermophilic microorganism belonging to the genus Bacillus newly isolated from soil had the desired properties, and the present invention was completed based on this knowledge.

The present invention will be explained in detail below.

The present invention relates to bacterial cells obtained by culturing thermophilic microorganisms belonging to the genus Bacillus and having the ability to convert glycerol into dihydroxyacetone in a medium containing glycerol or by culturing them in a suitable medium, or a processed product thereof. The present invention provides a method for producing dihydroxyacetone, which comprises producing dihydroxyacetone in a culture or an aqueous solution by aerobically acting on an aqueous solution containing glycerol, and collecting the dihydroxyacetone.

As the microorganism used in the present invention, any thermophilic microorganism that belongs to the genus Bacillus and has the ability to convert glycerol into dihydroxyacetone can be used. A specific example is Bacillus sp.
sp,) TB-516 (FEBM P-10524)
(hereinafter referred to as TB-516 bacteria), TB-927 (FE
BM P-10525) (hereinafter referred to as TB-927 bacteria). A thermophilic microorganism belonging to the genus Bacillus is the genus Acetobacter, which is known as a microorganism that has the ability to convert glycerol to dihydroxyacetone.

Bacteria such as the genus Gluconobacter and actinobacteria such as the genus Micromonospora are microorganisms that are clearly distinguished taxonomically.

Next, the mycological properties of TB-5113 bacteria and TB-927 bacteria are shown. The method and culture medium composition described in "Microorganism Identification Method" (Sanitation Technology Society) were used.

Mycological properties of TB-516 bacteria [morphological findings] (cultured at 55°C for 1-2 days) 1. Cell shape and size: rod-shaped, 0.25-0.4 x 0.7-0.
9 μm 2, Pleomorphism: None 3, Motility: None 4, Spores: Round endospores are formed at the center or tip of the cell. Spores and insults do not swell.

5. Durham staining: Positive 6. Acid-fast; None [Growth status] (55°C, cultured for 1 to 2 days) 1. Juicy agar plate culture Shape: Bi-circular periphery: Lobes or asymmetric tooth bud-like ridges: Squamous luster: Decorative light surface: Rough and crushed color Two-hand transparent 2, Meat juice agar slope culture Growth rate: Good shape, two major types 3, Meat juice liquid culture Surface growth: Film formation Turbidity: Cloudy Sedimentation: Small amount of coloring, Decolorization: None 4 , Meat juice gelatin puncture culture (30% gelatin added, 5
After incubating at 5°C for an appropriate time, cool and determine the solidification state) Liquefy the gelatin.

5. Meat juice agar puncture culture Shape: Only surface growth Surface growth: Vigorous 6. Lidmus milk Lidmus no discoloration, no pH change, some coagulation of milk.

[Physiological properties] (Culture at 55°C for 1 to 2 days) 1. Nitrate reduction and denitrification reactions: Both nitrate reduction and denitrification reactions exist 2. MR test: negative 3, VP test: positive 4, Indole generation: None 5, Hydrogen sulfide generation: None 6, Starch hydrolysis: Yes 7, Citric acid usage: Yes 8, Inorganic nitrogen source usage: Both nitrates and ammonium salts 9, Pigment production: None 10, Urease activity: No 11, Oxidase activity: Yes 12, Catalase activity: Yes 13, Growth pH: [1,0-9.5, Optimal pH range =
6.5-8.0 14, Growth temperature = 18-58℃, Optimal growth temperature range: 33-50℃ tS, Attitude towards oxygen: Aerobic, grows well,
Growth can be observed even under anaerobic conditions.

16.0-F Test: Fermentation17
, growth on Sabouraud dextrose agar:
Growing 18, growth in a medium containing 0.02% sodium azide, 55°C culture; Growing 19, growth in the presence of 0.001% lysozyme (
Tested at 45℃): Grows 20, Phenylalanine deamination reaction: None 21, Sodium chloride tolerance = 1O% growth 22, Vitamin requirement: None 23, Tyrosine degradability: None [From carbon source acid and gas production] D-xylose, D-glucose, D-mannose, D
-Fructose, D-galactose.

ays, shakerose, trehalose, D-mannitol, D-sorbitol, inositol.

It grows by assimilating starch and glycerin and produces acid, but does not produce gas. Availability of arabinose and lactose is weak or absent.

Based on the mycological properties of microorganisms mentioned above, PURGE AIDS Manual of Systematic Bacteriology (
Bargey's manual of system
(1984)
The TB-516 bacterium was identified as a Bacillus genus by searching according to the classification method of From Bacillus licheniformis and Bacillus coagulans
agulans), but TB-5
Bacteria 16 differs from both of the above in that they are not motile. Furthermore, Bacillus licheniformis differs from Bacillus licheniformis in that it does not produce acid from arabinose and grows in the presence of 0.02% sodium azide, whereas Bacillus coagyulans differs from O, OOT% in the presence of lysozyme and in the presence of 5 to 10% sodium azide. %NaC
They differ in that they grow in the presence of j and in that they decompose gelatin.

As is clear from the above, since the TB-516 bacterium is distinct from known bacterial species, it was concluded that it is appropriate to designate it as a new bacterial species.

The TB-516 bacterium was submitted to the National Institute of Microbial Technology, Agency of Industrial Science and Technology under the contract No. 10524 (FEBN P-10524).
It has been deposited as.

T! Mycological properties of -927 bacteria [morphological findings] (55°C, 18 hour culture) 1. Cell shape and size: rod-shaped, 0.3-0.4 X017-1.3
μm 2, pleomorphism: none 3, motility: none 4, spores Two circular endospores are formed in the center of the cell.

5. Durham staining: Positive 6. Acid-fastness: None [Growth condition] (Cultivated at 55°C for 18 hours! 1. Cultured on meat juice agar plate Shape: Circular Rim: Gold edge to slightly wavy ridges: Flat Gloss: Slightly present Surface: Slightly coarse color tone, two-hand transparent 2. Meat juice agar slant culture Growth rate: Good shape 1 Thick filament 3, Meat juice liquid culture Surface growth: None Turbidity: Slightly turbid Sediment: None Coloring, bleaching: None 4, Meat juice gelatin puncture culture (gelatin 30% addition, 5
After culturing at 5°C, cool and determine the solidification state) Due to poor growth, a small amount of gelatin is submerged, and the gelatin does not liquefy.

5. Meat juice agar puncture culture Shape: Growth only on the surface Surface growth: Vigorous 6. Lidmus milk No fading, no pH change, no coagulation or liquefaction of milk [Physiological properties] (Culture at 55°C for 1 to 2 days) 1. Nitrate reduction and denitrification reaction: Nitrate reduction.

No denitrification reaction 2, MR test: positive 3, VP test negative 4, indole production: none 5, hydrogen sulfide production: none 6, starch hydrolysis: yes 7, citric acid utilization: yes 8, inorganic Utilization of nitrogen source: Both nitrate and ammonium salt are weak 9, Pigment production: None 10, Urease activity: None 11, Oxidase activity: Yes 12, Catalase activity: Yes 13, Growth pH: 4.5-7.5 , Optimum pH range: 5.0-6,5 14, Growth temperature: 35-60℃, Optimum growth temperature range: 43-58℃ 15, Attitude towards oxygen: Aerobic 1 [i, O-F test: Fermentation1
7. Growth in Sabouraud dextrose agar medium: Good 18. Growth in culture medium containing 0.02% sodium azide at 55°C: None 19. Growth in the presence of 0.001% lysozyme (
Tested at 45℃): None 20, Phenylalanine deamination reaction: None 21, Sodium chloride tolerance: Does not grow even at 0.5% 22, Vitamin requirement: None 23, Tyrosine degradability: None [carbon source Generation of acid and gas from D-xylose, D-glucose, D-mannose, D
-Fructose, D-galactose.

Wheat-f” Grows by assimilating sugar, sucrose, trehalose, D-mannitol, D-sorbitol, starch, and glycerin and produces acid, but no gas. Utilization of arabinose, lactose, and inositol is weak. Or none.

Based on the mycological properties of microorganisms mentioned above, PURGE AIDS Manual of Systematic Bacteriology (
8ergey's manual of system
(1984)
The TB-927 bacterium was identified as a Bacillus genus by searching according to the classification method of Philus (Bacil)
lusStearothermo hilus), Bacillus coagulans (Bacillus coag)
ulans) and Bacillus plevis (Bacillus
s brevis), but TB-
The 927 bacterium differs from all of the above in that it is not motile. Furthermore, Bacillus stearothermophilus grows on Sabouraud dextrose medium, does not hydrolyze casein, and produces dihydroxyacetone, whereas Bacillus coagyulans grows on anaerobic agar medium and
．． It is different from Bacillus plebis in that it does not grow in the presence of 2% sodium azide or 2% NaCl, and that it produces acid from xylose, does not decompose casein, gelatin and tyrosine, and produces dihydroxyacetone. Each one is different.

As is clear from the above, since the TB-927 bacterium is distinct from known bacterial species, it was concluded that it is appropriate to designate it as a new bacterial species.

The TB-927 bacterium was submitted to the Institute of Microbiological Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 10525 (FERM P-10525).
It has been deposited as.

The nutrient medium used in the present invention includes a carbon source.

Any natural or synthetic medium may be used as long as it contains a suitable amount of nitrogen source, inorganic substances, and if necessary, micronutrients.

As a carbon source, glucose, mannitol, sorbitol, dextrin, +11 powder, glycerin, etc. can be used. Nitrogen sources include nitrogen-containing natural products such as peptone, yeast extract, meat extract, soybean flour, cornstarch liquor, and ammonium sulfate.

Organic chemicals such as ammonium chloride and ammonium nitrate,
Amino acids such as glutamic acid can be used. Other inorganic substances such as various phosphates, magnesium sulfate, calcium chloride, and calcium carbonate are added as necessary.

As a culture method, both solid culture and liquid culture are possible, but the aerated agitation culture method is the most suitable from an industrial perspective.

Culture at 15-58℃ for TB-516 bacteria, TB-927
For bacteria, it can be carried out at a temperature in the range of 35 to 60°C, preferably 33 to 50°C and 43 to 58°C, respectively. The pH is preferably neutral to weakly acidic.

In the present invention, dihydroxyacetone is produced and accumulated in the culture by culturing the microorganism in a medium containing moderate amounts of glycerol at a concentration of 0.5 to 15%, the aforementioned nitrogen source, and inorganic substances.

In addition, cultured bacterial cells or processed products thereof (for example, washed bacterial cells, dried bacterial cells, crushed processed products, protein fractions of crushed processed products, enzyme-treated products, surfactant-treated products, immobilized products of treated products, or immobilized bacterial cells) When dihydroxyacetone is produced in an aqueous solution by aerobically acting on an aqueous solution containing glycerol, the above-mentioned medium components and culture conditions are used for culturing the microorganism. The concentration of glycerol aqueous solution is usually 5-3
It is preferably about 0%, and a trace amount of inorganic salts may also be added. The reaction temperature is preferably 20 to 80°C, particularly 40 to 60°C.

Dihydroxyacetone produced and accumulated in the culture is reduced by 1 minute!
II To purify, remove the bacterial cells by centrifugation, etc., and then extract with #1 solvent such as ethyl acetate or use various gels. Filtration chromatography and adsorption chromatography may be performed in combination.

On the other hand, when dihydroxyacetone is produced by allowing bacterial cells and their processed material to act on an aqueous glycerol solution, first remove the bacterial cells and their processed material by centrifugation, etc.
As above, separation and purification can be achieved efficiently by combining solvent extraction and various types of chromatography.

[Example] The method of the present invention will be specifically explained below with reference to Examples. In the examples, dihydroxyacetone was confirmed and quantified by thin-layer chromatography and gas chromatography using color development with triphenyltetrazolium chloride.

Example I Inoculate the T516 bacteria into a seed medium with the following composition.5011
) 1500mj Erlenmeyer flask), 50℃
The cells were cultured for 16 hours.

Polar medium composition: glycerol 0.5%, yeast extract 0.2
5%, peptone 0.25%, dipotassium phosphate 0.1%
, calcium chloride 0.03% and magnesium sulfate 0
．． 01% (pH 7.0 before sterilization) Next, the cultured cells thus obtained were placed in a dihydroxyacetone production medium (
Inoculate 1 ml each into 50mf1500mj) and
The culture was carried out aerobically at ℃ for 8 hours.

Dihydroxyacetone production medium composition: glycerol 2%
, cornstarch liquor 1%, yeast extract 0.5%,
Dipotassium phosphate 0.1%, calcium chloride 0.05%
, magnesium sulfate 0.01% (before sterilization 1) H7.0) After completion of the culture, the bacterial cells were removed by centrifugation.

As a result, the amount of dihydroxyacetone produced and accumulated in the culture solution was approximately 8 g/e.

Example 2 1 ml of the TB-516 bacteria cultured in the seed medium of Example 1 was added to a bacterial production medium (50 mN, 1500 mj) with the following composition.
The cells were inoculated and cultured aerobically at 50°C for 8 hours.

Culture medium for cell production: 0.5% glycerol, 1% cornstarch liquor, 0.5% yeast extract, 0.5% dipotassium phosphate, 60.5% ammonium chloride.

Calcium chloride 0.05%, magnesium sulfate 0.01
% (before sterilization pH 7, o) After the completion of culture, collect the bacterial cells by centrifugation and strain at 50 mN (50
The cells were suspended in a 10% aqueous glycerol solution in a 0ml Erlenmeyer flask and reacted aerobically at 50°C for 18 hours, and after the reaction was completed, the bacterial cells were removed by centrifugation. As a result, the amount of dihydroxyacetone produced and accumulated in the reaction solution was approximately 65 g/j.

Example 3 TB-927 bacteria cultured in the seed medium of Example 1
111j of each cellulose were inoculated into a dihydroxyacetone production medium and cultured aerobically at 52°C for 8 hours.

After the culture was completed, the bacterial cells were removed by centrifugation classification 1. As a result, the amount of dihydroxyacetone produced and accumulated in the culture solution was approximately 10 g/ff.

Example 4 TB-927 bacteria cultured in the seed medium of Example 1 was used in Example 2.
The cells were inoculated at 1 mR each into a bacterial cell production medium, and cultured aerobically at 52°C for 8 hours.

After culturing, collect the bacterial cells by centrifugation and place them in a 50 m6 (50
The cells were suspended in a 10% aqueous glycerol solution in a 10-volume Erlenmeyer flask) and reacted aerobically at 50°C for 16 hours. After the reaction was completed, the bacterial cells were removed by centrifugation. As a result, the amount of dihydroxyacetone generated and accumulated in the reaction solution was approximately 8
It was 9g/R.

[Effects of the Invention] According to the present invention, dihydroxyacetone can be efficiently produced in a short time using thermophilic microorganisms belonging to the genus Bacillus.

Procedures (overwriting (voluntary) June I6, 1999

Claims (2)

[Claims] (1) A thermophilic microorganism that belongs to the genus Bacillus and has the ability to convert glycerol to dihydroxyacetone,
By culturing in a medium containing glycerol, or by aerobically acting on an aqueous solution containing glycerol with bacterial cells obtained by culturing on a suitable medium or a processed product thereof, dihydroxyl can be added to the culture or aqueous solution. A method for producing dihydroxyacetone, which comprises producing acetone and collecting it. (2) The production method according to claim 1, wherein the thermophilic microorganism belonging to the genus Bacillus having the ability to convert glycerol to dihydroxyacetone is Bacillus sp. TB516 or Bacillus sp. TB927.