JPS62210994A - Production of dihydroxyacetone - Google Patents

Abstract

PURPOSE:To efficiently produce dihydroxyacetone, by cultivating a specific microorganism belonging to the genus Micromonospora in a culture medium containing glycerol. CONSTITUTION:A microorganism, belonging to the genus Micromonospora and having the ability to convert glycerol into dihydroxyacetone, e.g. Micromonospora sp. 6168-A deposited as FERM BP-987 with the Fermentation Research Institute, is cultivated in a culture medium containing glycerol. The cultivation is preferably carried out by a liquid cultivation method, particularly submerged spinner cultivation method (100-300 r.p.m.) The cultivation is preferably carried out under aerobic condition at 25-32 deg.C and neutral-weak alkaline condition while adding glycerol to give 0.5-5wt% concentration thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing dihydroxyacetone using a microorganism belonging to the genus Streptomyces Micromonospora.

Dihydroxyacetone can be used in a wide range of fields such as pharmaceuticals and the chemical industry, including as a surfactant, a raw material for cosmetics, and an intermediate for X-ray contrast agents.

BACKGROUND ART Conventionally, as a method for producing dihydroxyacetone using microorganisms, methods using microorganisms belonging to the genus Acetobacter or Gluconobacter, which are Durum-negative bacteria, have been known (Japanese Patent Publications No. 49-11433 and No. 59-23794). It is being

Problems to be Solved by the Invention There is a desire for the development of a better method for producing dihydroxyacetone, which is useful as pharmaceuticals, chemicals, and the like.

Means for Solving the Problems The inventor conducted research to obtain better microorganisms that have the ability to produce dihydroxyacetone. As a result, the present invention was completed by discovering that a microorganism belonging to the genus Micromonospora newly isolated from soil has the ability to convert glycerol into dihydroxyacetone.

The present invention will be explained in detail below.

The present invention involves culturing a microorganism that belongs to the genus Micromonospora and has the ability to convert glycerol into dihydroxyacetone in a medium containing glycerol.
or dihydroxyacetone, which is characterized in that dihydroxyacetone is produced in the culture or aqueous solution by reacting bacterial cells obtained by culturing in a medium or a processed product thereof in an aqueous solution containing glycerol, and the dihydroxyacetone is collected. Provides a manufacturing method for seven tons.

As the microorganism used in the present invention, any microorganism can be used as long as it belongs to the genus Actinomycete Micromonospora and has the ability to convert glycerol to dihydroquinacetone. A specific example is Micromonospora s.
p, 6168-A strain. This strain is a microorganism that is clearly distinguished from bacteria of the genus Acetobacter and Gluconobacter, which are known to have the ability to convert glycerol to dihydroxyacetone.

Below, Streptomyces micromonospora St1.6168-A
The morphological, physiological, and culture characteristics of the strain (hereinafter also referred to as 6168-A) will be described.

(1) Morphological characteristics Hyphae: Substrate hyphae that do not form: Spores that do not divide Stalk: Very short, branching from basal hyphae Spores: Single bottom spore Surface: Smooth to rough spore shape Dispherical size (diameter 0.8-1.0 μm) Spore motility: None 2) Color base Hyphae: Orange Soluble pigment: None Melanoid-like pigment: Produced (3) Chemical composition Diamino-pimelic acid Stereotype: meso type Whole bacterial cell sugar:
Xylose, arabinose (4) Physiological characteristics For items other than growth temperature and effects on skim milk and cellulose, the results are shown after 2 weeks at 28°C; The results are shown after one month.

Growth temperature range = 20-37℃ Optimum temperature range: 30-32℃ Liquefaction of gelatin: Negative Decomposition of fibrin: Negative Hydrolysis of starch Coagulation and peptonization of skimmed milk: Both negative melanin-like pigments Production: Assimilation of positive carbon sources: L-arabinose + D-xylose + D-glucose + D-fructose + sucrose + inositol - L-rhamnose + raffinose ± D-mannite + D-galactose + α-melibiose + Salicin + D-mannose + Erythritol - D-ribose - Glycerol - Lactose ± (5) Growth status in each medium Table 1 shows the results of culturing the 616g-A strain in each medium at 28°C for 21 days. show. Color display is Color Harmony Manual (Co1or)IarmonyManu
al, Container Corporation
of America). In the table, G is growth;
SM indicates the color tone of the basal hyphae, and P indicates the soluble pigment.

Table 1 616 The B-A strain contains meso-diaminopimelic acid in its cell wall, and contains xylose and arabinose as characteristic sugars in the total cell sugar composition.

Furthermore, morphologically, this strain does not form aerial hyphae, but forms a single spore on an extremely short sporophyte that is simply branched from a long hyphae, so this strain belongs to the genus Micromonospora within the order Actinomycetes. being classified. Therefore, this strain was named Micromonospora 911.6168-A, and was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology (Feikoken) on February 13, 1986.
It has been deposited as FεRM BP-987.

Micromonospora strain S11.6168-A, as seen in the case of other actinomycetes, is susceptible to changes in its properties;
For example, they can be mutated by artificial mutagenesis methods such as ultraviolet rays, X-rays, radiation, and chemicals. Therefore, any strain of the genus Micromonospora that has the ability to convert dihydroxyacetone can be used in the present invention, even if it is an artificially obtained mutant strain.

The medium for culturing these microorganisms may be either a natural medium or an artificial medium as long as it contains carbon sources, nitrogen sources, inorganic substances, etc. in moderation.

Glucose, mannitol, dextrin, starch, etc. can be used as carbon sources. Soybean flour, wheat,
Germ, peptone, meat extract, yeast extract, cornstarch liquor, etc. can be used. In addition, inorganic salts such as calcium carbonate, potassium chloride, and phosphate are added as necessary.

In addition, organic and inorganic substances that promote the growth of the strain used and the ability to convert glycerol to dihydroxyacetone can be appropriately added.

The most suitable culture method is the liquid culture method, especially the deep stirring culture method (100 to 300 r, pm), as in the case of general actinomycetes. Cultivation is performed under aerobic conditions. The appropriate temperature for culturing is 25 to 32°C, but it is usually cultured at around 30°C. The pH is preferably neutral to weakly alkaline.

In the present invention, culture is carried out for 3 to 7 days by adding or adding glycerol to a medium commonly used for culture at a concentration of 0.5 to 5%. In this way, dihydroxyacetone is produced and accumulated in the culture.

When dihydroxyacetone is obtained by reacting microorganism cells or a processed product thereof with glycerol (referred to as an enzymatic conversion method), a medium and culture conditions having the above composition are used for culturing the microorganism.

The obtained microbial cells can be used as they are for the reaction, or the processed products obtained by subjecting the microorganisms to various treatments may be used for the reaction.

As the microbial cells, the microbial cells themselves or a culture solution containing the microbial cells are used.

Examples of bacterial cell-treated products include mechanically ground bacterial cells, ultrasonic-treated products, freeze-dried products, enzyme-treated products, dry-treated products, surfactant-treated products, protein fractions of bacterial cells, Immobilized products of cells and treated bacterial cells are used.

The reaction is carried out by allowing the bacterial cells obtained above or a treated product thereof to act on glycerol in an aqueous solution.

The bacterial cells or their processed material are suspended in a 1-30% glycerol aqueous solution or a reaction solution to which inorganic salts have been added, and reacted under aerobic conditions. The appropriate temperature for the reaction is 20
~35°C, but usually the reaction is carried out at around 30°C.

In this way, dihydroxyacetone is produced in the aqueous solution.

To isolate and purify the dihydroxyacetone produced and accumulated in the culture, adsorbents such as carbon bundles, Sephadex,
Chromatography using a gel filtration agent such as cellulose and purification by adding an organic solvent such as ethanone ethyl acetate are efficient.

On the other hand, dihydroxyacetone produced in the reaction solution by enzymatic conversion can be easily obtained as crude crystals of dihydroxyacetone by a method such as concentration after removing the bacterial cells by centrifugation or the like.

The present invention will be specifically explained below with reference to Examples.

For confirmation and quantification of dihydroxyacetone in the examples,
p- by commonly used thin layer chromatography
In addition to conventional methods such as anisidine coloration and measurement of absorbance at 490 nm after reaction with resorcinol hydrochloride, a bioassay based on the antibacterial activity of dihydroxyacetone was also used. The minimum inhibitory concentration of dihydroxyacetone is
Bacillus subtilis (Bacillus 5ubti)
lis) 300■/ml for ATCC6051,
Pseudomonas cepacia
cepacia) 25 for ATCC25608
00■7 ml, and can be quantitatively determined by the paper disk method or the diluted agar method.

Example 1 Production of dihydroxyacetone by fermentation As a seed medium, 1% glucose, 1% soluble starch,
Beef extract 0.3%, yeast extract 0.5%.

A medium containing 0.5% peptone and 0.1% calcium carbonate (pH 7.0 before sterilization) was used. Nuclide medium 5
Micromonospora sp, strain 6168-A was inoculated into Qml (300 ml Erlenyer flask),
The cells were cultured at 28°C for 4 days. Next, 500 ffll of a production medium having the following composition was placed in four 21-capacity Lurenmeyer flasks, and 1 Qml of the above seed culture was inoculated.

Production medium composition Nigelcose 1%, glycerol 2%, cornstarch liquor 0.5%, dried yeast 1%, soy flour 2%, K2 HP 040.05%, magnesium sulfate 0.05%, calcium carbonate 0.5 % (pH 7 before sterilization,
0) Culture was performed at 28°C for 4 days with shaking. After culturing,
The culture solution was separated into a bacterial cell portion and a supernatant by centrifugation (6000×g, 20 minutes). The bacterial cell portion was used in Example 2. After adjusting the pH of the supernatant portion 1.81 with hydrochloric acid to 4.0, it was passed through a 10 Qml carbon bundle (manufactured by Chugoku Sangyo Co., Ltd., GLC) column and eluted with an 80% acetone aqueous solution. A fraction with a high concentration of dihydroxyacetone in the eluate was collected and concentrated under reduced pressure. The column was then passed through a 10 Qml cellulose column, eluted with water-saturated n-butanol, and the fractions with high concentrations of dihydroxyacetone were collected and concentrated under reduced pressure. 1 of this concentrate
0 Qml Sephadex LH-20 (Pharmacia)
Fine Chemicals, Inc.) and eluted with a 50% aqueous methanol solution, and fractions with high dihydroxyacetone concentration were collected and concentrated under reduced pressure. As a result, 3.2 g of dihydroxyacetone was obtained.

Example 2 Production of dihydroxyacetone by enzymatic conversion method 30 g of the bacterial cells cultured and isolated in Example 1 were suspended in 60 Qml of a 10% glycerol aqueous solution in a 21-volume Erlenmeyer flask, and incubated at 28°C. The reaction was performed while shaking. After 120 hours, 29.2 g of dihydroxyacetone was produced and accumulated in the reaction solution. The bacterial cells were removed from this reaction solution by passing, and the p solution was freeze-dried to obtain 26.6 g of crude dihydroxyatone.

Effects of the Invention According to the present invention, dihydroxyacetone can be efficiently obtained using microorganisms belonging to the genus Actinomycetes Micromonospora.

Claims (1)

[Claims] A microorganism that belongs to the genus Micromonospora and has the ability to convert glycerol to dihydroxyacetone is cultured in a medium containing glycerol, or a bacterial cell obtained by culturing in a medium or a processed product containing glycerol. A method for producing dihydroxyacetone, which comprises producing dihydroxyacetone in a culture or an aqueous solution by reacting in an aqueous solution, and collecting the dihydroxyacetone.