JP2876417B2 - Method for producing D-sorbose - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing D-sorbose, and more specifically, belongs to the genus Pseudomonas and is selected from D-galactitol and D-tagatose. A bacterium capable of producing D-sorbose from carbohydrates is converted from a carbohydrate selected from D-galactitol and D-tagatose by D-galactitol and D-tagatose.
The present invention relates to a method for producing sorbose.

[Conventional Method] D-sorbose is a monosaccharide classified as ketohexose, and is a rare saccharide that hardly exists in nature. As a production method, in principle, D-gulose or D-idose is isomerized by an organic chemical method in the coexistence of an alkaline drug such as caustic soda, pyridine, and the like.
It is possible to produce sorbose. However, it is actually difficult to obtain D-growth or D-idose as a raw material. Motiram.R.Dhawale, etc. are also carbohydrate
Research (Carbohydrate Research), Volume 155, Volume 262
265 (1986) report that D-sorbose was synthesized from L-glucitol using an unidentified bacterium. However, the raw material L-glucitol in this case is:
It is prepared by reducing L-glucose which cannot be obtained in large quantities. Thus, no suitable method for producing a sufficient amount of D-sorbose has been reported to date.

[Problems to be Solved by the Invention] In recent years, the biochemical industry has rapidly developed, and in the field of carbohydrate chemistry, the development of new carbohydrates is desired. Although D-sorbose is commercially available in small quantities as a reagent, its mass production method has not been established, and it has not yet been used as an industrial raw material in the food, pharmaceutical, and chemical industries.

[Means for Solving the Problems] The present inventors have conducted intensive studies for the purpose of producing D-sorbose in large quantities and at low cost by biochemical means.

As a result, a bacterium belonging to the genus Pseudomonas and having a D-sorbose-producing ability from a saccharide selected from D-galactitol and D-tagatose, a saccharide selected from D-galactitol and D-tagatose in an aqueous solution, It was found that D-sorbose was easily converted to D-sorbose, and by collecting this, it was confirmed that D-sorbose was produced in high yield, thus completing the present invention.

That is, in the present invention, the bacterium used for producing D-sorbose from a saccharide selected from D-galactitol and D-tagatose belongs to the genus Pseudomonas, and is selected from D-galactitol and D-tagatose. A bacterium having the ability to produce D-sorbose from one or both carbohydrates. One example is Pseudomonas cichorii ST-24 described below.
Alternatively, mutants thereof can be advantageously used.

Pseudomonas cichorii ST
-24 was deposited on Jan. 19, 1990 with the Institute of Microbial Engineering, National Institute of Advanced Industrial Science and Technology under Microorganism Accession Number FERM BP-2736.

This Pseudomonas chicory ST-24 (FERM BP-273
The bacteriological properties of 6) are described below.

A. Collection site and isolation source Collection site Miki-cho, Kida-gun, Kagawa Isolation source Soil B. Cell morphology (1) Cell shape and size Bacillus bacilli 0.6 to 1.0 x 0.9 to 1.6 µm (2) Cell multiplication No (3) Motility Yes (4) Presence of flagella Yes (5) Presence of spores No (6) Gram stain negative (7) Acid resistance No C. Growth in each medium (1) Gravy agar plate Culture (28 ° C, 2 days) The colonies are opaque, moist, yellowish white,
The surface is smooth and has a semi-lens-like ridge. The periphery is uniform at all edges. No pigment is formed.

(2) Broth gelatin puncture culture (20 ° C. and 28 ° C., 5 days) Colonies are formed around the puncture site on the surface of the medium and do not liquefy.

D. Physiological properties (1) Nitrate reduction positive (2) Denitrification negative (3) MR test negative (4) VP test negative (5) Indole formation negative (6) Hydrogen sulfide formation negative (7) Starch Hydrolysis of Negative (8) Use of citric acid Positive (9) Pigment not generated (10) Urease positive (11) Oxidase positive (12) Catalase positive (13) Growth range Growth pH 5 to 8 Growth temperature 20 (14) Attitude to oxygen Aerobic (15) OF test Oxidizes sugar (glucose) only under aerobic conditions.

(16) Generation of acid from saccharide L-arabinose-D-xylose + D-glucose + D-mannose + D-fructose-D-galactose-maltose-sucrose-lactose-trehalose-D-sorbitol-D-mannitol-D -Galactitol-inositol-glycerol-(17) assimilation of carbon source D-glucose + D-xylose + D-sorbitol + D-fructose + acetic acid + This strain was obtained from Williams &・ Wilkins & Wilkins, “Bergey's Manual of Systematic Bacterio
Classification according to Volume 1 (1984), it is a gram-negative, aerobic, short rod, positive for both catalase and oxidase, and produces acid from D-glucose. ) Bacteria belonging to the genus. Furthermore, Pseudomonas chicory (Pseudomonas cichori)
i) and designated Pseudomonas chicory ST-24.

In the present invention, D-galactitol and D-tagatose belong to the genus Pseudomonas and are derived from D-tagatose.
-Contacting a sorbose-producing bacterium with an aqueous solution containing a saccharide selected from D-galactitol and D-tagatose to produce D-sorbose, and collecting this means that D-galactitol and D-galactitol; For example, Pseudomonas chicory ST-24 (FERM BP) having the ability to produce D-sorbose from sugars selected from tagatose
-2736) or cells of this mutant strain or the like are cultured in a nutrient medium containing a saccharide selected from D-galactitol and D-tagatose, preferably under aerobic conditions such as shaking and aeration and stirring. After culturing, D-sorbose may be produced in the culture solution and collected. Furthermore, the saccharide selected from D-galactitol and D-tagatose in the aqueous solution is converted into D-sorbose by using the bacteria (live cells) obtained by culturing in this manner, and D-sorbose produced Can also be collected.

As a culture method, D-galactitol is added to a nutrient source required by bacteria belonging to the genus Pseudomonas, for example, a medium containing a carbon source, a nitrogen source, an inorganic salt, etc., preferably a slightly acidic to slightly alkaline liquid medium. And a saccharide selected from D-tagatose and a bacterium capable of producing D-sorbose may be inoculated and cultured under aerobic conditions at a temperature of about 20 to 35 ° C. for 1 to 15 days. In particular, as the carbon source, for example, D-galactitol, D-tagatose,
-It is desirable to aerobically culture in a liquid medium containing one or more saccharides such as sorbitol, D-galactose, D-glucose and D-fructose and other various nutrient sources.

The bacteria (live cells) obtained by the above-described culturing method are brought into contact with an aqueous solution containing a saccharide selected from D-galactitol and D-tagatose, preferably, shaking, aeration and stirring, oxygen injection, and the like. Under aerobic conditions to convert the saccharide to D-sorbose. At this time, even if relatively inexpensive D-galactitol is used as a raw material, it can be conveniently converted into D-sorbose via D-tagatose.

The bacteria used for this conversion need not be limited to living cells isolated from the culture solution as they are, for example, bacteria in which living cells are encapsulated in hollow fibers made of a semipermeable membrane, agar, gelatin, alginate. Bead-like,
As bacteria immobilized in various shapes such as sheets,
It is also advantageous to use repeatedly for conversion of a saccharide selected from D-galactitol and D-tagatose into D-sorbose.

The aqueous solution containing D-sorbose generated and accumulated by the various methods described above can be separated by a suitable separation method, for example,
The bacteria are separated and collected by a method such as centrifugation or filtration.

The resulting D-sorbose aqueous solution is purified as necessary by, for example, ammonium sulfate precipitation, protein removal by zinc hydroxide adsorption, decolorization by activated carbon adsorption, H-type, desalting by OH-type ion exchange resin, etc. The syrupy D-sorbose product can be collected by concentration. Further, column chromatography using an ion-exchange resin, for example, Dowex 50W-X4, Dowex WGR manufactured by Dow Chemical Company, Amberlite XT-1008 manufactured by Tokyo Organic Chemical Industry Co., Ltd., Amberlite IRA47 , Manufactured by Mitsubishi Kasei Kogyo Co., Ltd.
Crystallization can be achieved by fractionation, purification, and concentration by column chromatography using Diaion WA11 or the like, and a highly purified crystal sample of 99% or more can be easily obtained.

D-sorbose produced in this way is usually
It is obtained in a high yield of about 30 W / W% or more based on the raw material saccharide selected from D-galactitol and D-tagatose, and is suitable as an industrial production method for supplying a large amount at low cost.

Therefore, D-sorbose is used in the food industry, pharmaceutical industry,
It can be used freely as a raw material for chemical industry and as an intermediate.

 Hereinafter, examples will be described.

Example 1 Ammonium sulfate 0.2W / V%, monopotassium phosphate 0.24W /
V%, dipotassium phosphate 0.56W / V%, magnesium sulfate
7 100 mL of culture solution consisting of 0.01 W / V% of salt, 0.5 W / V% of yeast extract, 2 W / V% of D-sorbitol and deionized water
After placing the autoclave in 20 00 mL shake flasks at 120 ° C. for 20 minutes, Pseudomonas chicory ST-24 (FERM
BP-2736) was inoculated one loop at a time, and cultured with shaking at 30 ° C. for 2 days.

After culturing, the cells were collected by centrifugation, and about 20 live cells were obtained.
g is added to 1 L of 0.05 M phosphate buffer (pH 7.0) containing 0.5 W / V% of D-galactitol and mixed.
Each was dispensed into a 500 mL shake flask and shaken at 30 ° C. for 3 days to convert D-galactitol into D-sorbose. The bacteria were then removed by centrifugation.

The obtained supernatant was adjusted to pH 7.6 by adding 1/10 volume of 25 W / V% zinc sulfate, and centrifuged to collect the supernatant. The supernatant is decolorized using activated carbon according to a conventional method, and then Diaion SK1B (H type, trade name manufactured by Mitsubishi Chemical Industry Co., Ltd.)
And desalted using Diaion WA30 (OH type, trade name manufactured by Mitsubishi Kasei Kogyo Co., Ltd.), and concentrated under reduced pressure to obtain a transparent syrup having a concentration of about 60% containing D-tagatose as an intermediate as an impurity. .

This was purified and concentrated by column chromatography using Dowex 50W-X4 (a calcium-type cation exchange resin, trade name manufactured by Dow Chemical Company), and D-sorbose was crystallized and separated to obtain D-sorbose crystals. Collected.

The yield of D-sorbose to D-galactitol was about 40% per solid.

In order to identify the product thus obtained, the physicochemical properties were examined by experiment and the properties were compared with literature values, or compared with the values of reagent D-sorbose or reagent L-sorbose marketed by Sigma. Compared. In this experiment, the product obtained by the method of the present invention was called this substance,
The reagent D-sorbose or reagent L-sorbose from gma is referred to as standard D-sorbose or standard L-sorbose.

(1) Melting point 164 to 165 ° C D-sorbose literature value 165 ° C L-sorbose literature value 165 ° C Reference: Chapman and Hall
Hall), “Dictionary of Organic.
Compounds (Dictionary of Organic Compound)
s) ", pp. 5020 and 5021 (1982) (2) Measurement of specific rotation [-α] ²⁰ _D ▼ = + 45.0 (C = 10%, H ₂ O) Reference of D-sorbose Value [▲ α] ²⁰ _D ▼ = + 42.9 (C
_{= 10%, H 2 O)} L- sorbose literature values _{^{[▲ α] 20 D ▼ =}} -43.2 (C
(10%, H ₂ O) References: (1) Same as for melting point (3) Infrared absorption spectrum The infrared absorption spectrum of this substance measured by the KBr tablet method is shown in the drawing. This spectrum was in good agreement with the infrared absorption spectrum of standard D-sorbose or standard L-sorbose measured separately.

(4) Measurement by ¹³ C-NMR The measurement of this substance by ¹³ C-NMR was carried out according to Biochemistry, Vol. 13, pages 146 to 153 (1974).
When 1,4-dioxane was used as an internal standard according to the method described in 1., the chemical shift was found to be standard D-sorbose or standard L for all signals.
-Good agreement with that of Sorbose. Also, as shown in the following table, the chemical shift was in good agreement with the values in the literature.

(5) Analysis by high performance liquid chromatography This substance was analyzed by high performance liquid chromatography (880-PU, manufactured by JASCO Corporation; column, GL-C611, Hitachi, Ltd .; eluent, 10
^-4 M sodium hydroxide, 60 ° C .; flow rate, 1 mL / min; detection, analyzed by Shimadzu RID-6A), the elution position was found to be keto such as standard D-psicose, D-fructose, D-tagatose. Unlike hexose, it was 16.8 minutes, the same as standard D-sorbose or standard L-sorbose.

As is evident from the above results, the melting point, infrared absorption spectrum, ¹³ C-NMR, and high performance liquid chromatography were in good agreement with the literature values of D-sorbose and L-sorbose or the values of these standard substances, The product obtained by the method of the present invention was judged to be D-sorbose, since the value obtained from the method of the present invention was + and-opposite to that of L-sorbose, although it was in good agreement with the literature value of D-sorbose. Is done.

This product can be advantageously used not only as a reagent as a rare saccharide, but also as a raw material, an intermediate and the like in the food industry, the pharmaceutical industry, the chemical industry, and the like.

Example 2 15 L of a culture solution having the same composition as in Example 1 was used in two 30-L jar fermenters except that D-sorbitol was replaced with D-galactitol in the culture solution of Example 1.
After sterilizing at 0 ° C. for 20 minutes, the mixture was cooled to 30 ° C., and a seed culture of Pseudomonas chicory ST-24 (FERM BP-2736) cultured in a culture of the same composition at 30 ° C. for 1 day with shaking was added to the flask for 1 W. / V%
The cells were inoculated and cultured with aeration and agitation at 30 ° C. for 5 days to convert D-galactitol into D-sorbose, and then centrifuged to separate cells and supernatant. According to the method of 1, after the treatment with activated carbon, desalting was performed using an ion-exchange resin, and concentrated, and then purified and concentrated by column chromatography to collect D-sorbose crystals. The yield of D-sorbose to D-galactitol was about 30% per solid.

This product showed the physicochemical properties of D-sorbose as in Example 1.

This product can be advantageously used not only as a reagent as a rare saccharide, but also as a raw material, an intermediate and the like in the food industry, the pharmaceutical industry, the chemical industry, and the like.

Example 3 About 20 g of the viable cells obtained by the method of Example 1 were mixed with 1 L of 0.05 M phosphate buffer (pH 7.0) containing 0.5 W / V% of D-tagatose.
, And then converted to D-sorbose in the same manner as in Example 1, purified and concentrated to obtain D-sorbose crystals in a yield of about 50% based on D-tagatose, based on solids. Obtained.

This product can be advantageously used as a raw material, an intermediate, and the like in the food industry, the pharmaceutical industry, the chemical industry, and the like.

[Effects of the Invention] As is apparent from the above description, the present invention establishes a method for easily producing D-sorbose, which has been extremely difficult to obtain conventionally, by a biochemical method. In particular, using microorganisms belonging to the genus Pseudomonas,
The fact that D-sorbose can be produced via D-tagatose even using an inexpensive sugar alcohol called D-galactitol as a raw material is extremely advantageous for a method for producing D-sorbose.

Therefore, the method of the present invention is suitable as an industrial production method of D-sorbose, facilitates large-scale, inexpensive supply, and is not only used as a rare saccharide as a reagent, but also in the food industry, which could not be expected even before. It opens the way for use in raw materials and intermediates in the pharmaceutical and chemical industries.

[Brief description of the drawings]

The drawing shows the infrared absorption spectrum of D-sorbose obtained by the method of the present invention.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:38)

Claims (3)

(57) [Claims] 1. A bacterium belonging to the genus Pseudomonas which has a D-sorbose-producing ability from a saccharide selected from D-galactitol and D-tagatose, and a saccharide selected from D-galactitol and D-tagatose. A method for producing D-sorbose, which comprises contacting an aqueous solution to produce D-sorbose and collecting the D-sorbose. 2. The method for producing D-sorbose according to claim 1, wherein the bacterium belonging to the genus Pseudomonas is Pseudomonas chicory ST-24 (FERM BP-2736). 3. Pseudomonas chicory ST-24 (FERM)
BP-2736).