JP2901458B2 - Method for producing gentianose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for easily producing gentianose with good yield.

[0002]

BACKGROUND ART Gentianose is contained in rhizomes of Gentian plants such as gentian from southern Europe,
O-β-D-glucopyranosyl- (1 → 6) -α-D-
It is a non-reducing trisaccharide having a structure of glucopyranosyl- (1 → 2) -β-D-fructofuranoside, which exists only in a small amount in nature, but is synthesized into a nitrosourea derivative,
It has application as a pharmaceutical as an anti-leukemic agent and an anti-tumor agent (Japanese Patent Publication No. 60-41078). However, conventionally, to obtain gentianose as a raw material, extraction from a natural product or a multi-step chemical synthesis method is only available. In the former case, it is difficult to isolate gentianose because there are many impurities and the like. The latter has the disadvantage that the reaction process is complicated and multi-step, and the final yield of gentianose is low.

[0003]

The inventors of the present invention have conducted intensive studies in order to solve the above-mentioned problems by a microbiological technique, and as a result, have greatly simplified the production process, thereby contributing to the final production of gentianose. It has been found that the target yield can be increased, and the present invention has been completed.
Therefore, an object of the present invention is to provide a method for easily producing gentianose with good yield.

[0004]

The object of the present inventors is to provide a β-
It is achieved by reacting a solution containing a polysaccharide having a glucoside bond or a partially degraded product thereof and sucrose with a specific microorganism having β-glucosyltransferring activity or an enzyme derived from the microorganism.

The details of the present invention will be described below. The polysaccharide having a β-glucoside bond used in the present invention is not particularly limited as long as the β-glucosyltransfer reaction proceeds, and it is easily available and low in price. Practical, practical Pakiman, curdlan and laminarin are preferred. As the decomposed product of the polysaccharide having a β-glucoside bond, any of those obtained by appropriately decomposing the above-mentioned saccharide or the like with each decomposing enzyme to form oligosaccharides or oligosaccharides may be used. Regarding sucrose, the degree of purification is not particularly problematic, and industrial sucrose can be used sufficiently.

The microorganism used in the present invention is obtained from soil collected in Komatsushima City, Tokushima Prefecture, and is selected from those grown on an agar medium containing laminarin as a single carbon source. is there.

The microbiological properties of the microorganism used in the present invention are as follows. A. Morphological characteristics Cell shape and size: Bacillus, size 0.5μm × 1.0 ～
3.0 μm Cell polyplasia: none Motility: none Sporulation ability: none Gram stain: negative Acid resistance: none

B. Cultural properties Growth status in the following media Broth agar: good growth. The color of the colony is pale yellow at the beginning of the culture, but becomes deep yellow with the passage of days.
The colony is perfectly round, shiny, raised and sticky. The diameter of a colony after culturing on a normal agar medium for 2 days is 2 to 3 mm. It emits a fruity aroma. Gravy slant medium: Normal growth. Yellow, transparent and sticky. It is shiny and wet. Broth liquid medium: Growth is normal to good. Turbid, with precipitation.
Incomplete ring formation. Broth gelatin stab culture: liquefaction started after 2 days at 20 ° C. Layered liquefaction. Litmus milk: coagulated by acid

C. Physiological properties Nitrate reduction:-Denitrification:-MR test:-VP test:-Indole formation: + Hydrogen sulphide formation: + Starch hydrolysis: + Use of citric acid: Grow slightly in Christensen medium Does not grow in the medium of Coser. Utilization of inorganic nitrogen: Almost no ammonium salt or nitrate is used. Dye formation: Produces a water-insoluble yellow dye. Urease: + Oxidase: + Catalase: + Growth range: Growing at pH 5.0 to pH 9.0, 4 ° C to 40 ° C
Grows in. Attitude to oxygen: facultative anaerobic OF test ^* : fermentative (oxidation reaction is very slow under paraffin inclusion) * The anaerobic condition of the OF test was performed by enclosing slant with paraffin.

D. Other physiological properties Degradation of esculin: + Utilization of malonic acid:-Degradation of arginine:-Decarboxylation of lysine:-Decarboxylation of ornithine:-Deamination of phenylalanine:-β-galactosidase: + of sodium chloride Tolerance: grows up to 2% sodium chloride concentration. Growth on MacConkey medium:-Lysozyme resistance: + Lecithinase (Egg-york reaction): + Degradation of casein: + Degradation of tyrosine: + Formation of pigment in tyrosine medium: + Formation of dioxyacetone: + Oxidation of gluconic acid :-Decomposition of cellulose:-Hemolytic (sheep blood agar): + (haemolysis on the 7th day) Decomposition of Tween 80: +

Based on the mycological properties described above, the microorganism used in the present invention was prepared using the method described in Bergey's Manual of Systematic Bacteriology, Vol.
l Description of ofsystematic Bacteriology Volume 2) and other research (Gifu University School of Medicine, Vol. 38, P380 (1990),
Microbiol. Immunol., Vol. 34, P73 (1990), Ann. Rev. Mic
robiol., Vol. 37, P233 (1983), J. Gen. Appl. micro-b
iol., Vol. 27, p. 57 (1981)), it is a bacterium belonging to the genus Flavobacterium and is presumed to be a species close to Flavobacterium indologenes. , Urease, β-galactosidase and the use of malonic acid. Indologenes is a bacterium isolated from a clinic, whereas the bacterium used in the present invention is a bacterium isolated from soil. Based on the significant differences in growth environment and physiological characteristics as described above, it was considered that Indrogens and this fungus were related but not the same species. Therefore, this bacterium was named Flavobacterium sp. SKP4001 and deposited on March 22, 1993 as "Microtechnical Laboratories No. 13537 (FERM P-13537)".

In practicing the present invention, the bacterial cell culture obtained by aeration and agitation culture of the above-mentioned microorganisms is centrifuged to remove cells from the culture supernatant, or the cells are prepared from the cells. A crude enzyme solution, a solution obtained by purifying the crude enzyme by various column chromatography, and increasing the specific activity can be used.

[0013] The enzyme used in the present invention is a microorganism SKP4001 belonging to the genus Flavobacterium or an enzyme derived from the microorganism SKP4001.

Since these enzymes are cell membrane-bound or intracellular enzymes, they are subjected to ultrasonic crushing, French press treatment,
Crush cells by a known method such as bacterial wall lysis enzyme treatment,
After removing bacterial cell residues by centrifugation, the enzyme is purified by various chromatography, for example, using a known method such as a DEAE-cellulose column or a gel filtration column to obtain an enzyme having a high specific activity.

The microorganism used in the present invention or the enzyme derived from the microorganism not only produces gentianose, but also uses its β-glucosyltransferase activity to produce other disaccharides such as maltose, cellobiose, oligosaccharides and oligosaccharides. ,
Beta-glucosyl transfer to saccharide analogs such as ascorbic acid, riboflavin, or glycosides such as stevioside can also be performed.

Next, the transfer reaction conditions of the present invention will be described. The polysaccharide having sucrose and β-glucosyl bond used for the transfer reaction in the present invention or its decomposed product is first dissolved in water, and the concentration of sucrose is 0.1 to 15 W / V%, which has β-glucosyl bond. 0.1 ~ 50W / V% for polysaccharide or decomposed product
Use as a degree. In the transfer reaction, since the behavior of the polysaccharide having a β-glucosyl bond and the decomposition product thereof are the same, the same purpose can be achieved by using either of them. In some cases, a low water-soluble decomposed product is used to facilitate the reaction operation.

In the transfer reaction, the amount of transferase added is
When cells are used, 5 ml or more, preferably 20 to 50 ml, of the culture solution is used for 1 g of the substrate. When enzymes derived from bacteria are used, the amount of protein is 0.5% relative to the substrate.
As described above, the content is preferably 2.0 to 5.0%.

The pH and temperature of the reaction solution in the transfer reaction are as follows:
Any condition can be used as long as the enzyme having β-glucosyl transfer activity can react and produce gentianose,
The pH is usually from 4 to 10, preferably from 5 to 8, and the reaction temperature is from 20 to 85 ° C, preferably from 30 to 75 ° C. The reaction time may be 10 minutes or more, preferably 2 to
48 hours.

Under the conditions under which the transferase reacts optimally, 40% of the sucrose used was converted to gentianose.

As the transfer reaction method, besides the batch method, a method of immobilizing cells or their enzymes by a known method and performing a continuous conversion reaction can be used.

The gentianose-producing solution obtained under the above-mentioned transfer reaction conditions was subjected to sucrose and remaining β-
It can be separated from glucosyl sugar compounds.

[0022]

【Example】

(1) The adjustment Flavobacterium sp SKP4001 of bacteria laminarin _{0.5W / V%, KH 2 PO} 4 0.5W / V%, (NH 4) 2 S
_{O 4 0.3W / V%, MgSO} 4 · 7H 2 O0.05W / V%, polypeptone 0.1 W / V%, metal solutions (FeSO ₄ · 7
_{H 2 O0.01g, MnCl 2 · 4H} 2 O 0.01g, ZnS
The O ₄ · 7H ₂ O 0.01g dissolved in deionized water, 100 ml
The solution was inoculated into 1 liter of a medium containing 0.5 V / V%, and cultured with aeration and agitation at 30 ° C. for 48 hours. The obtained cell culture solution was centrifuged, and the cells from which the culture supernatant was removed were used as cells.

(2) Transfer reaction 5 g of sucrose (commercially available granulated sugar, purity 98%), 20 g of laminarin, and the above-prepared cells were added to a 50 mM phosphate buffer (p
H7.0) dissolved in 100 ml and reacted at 70 ° C for 24 hours.
The enzyme was inactivated by heating at 10 ° C for 10 minutes.

(3) Separation of Gentianose The obtained reaction solution was subjected to gel filtration column chromatography (Sephadex LH-20, manufactured by Pharmacia Co.), eluted with water, and a mixture of laminarin and sucrose / reaction product was obtained. separated. The sucrose / reaction product mixture is concentrated under reduced pressure, and further silica gel (silica gel 60 manufactured by MERCK)
The residue was subjected to column chromatography (solvent; chloroform: methanol: water = 6: 4: 1) to obtain 1.7 g of a reaction product.

(4) Confirmation of Gentianose The reaction product was measured by high performance liquid chromatography (LC-10A, manufactured by Shimadzu Corporation) under the following conditions, and compared with a control in which the enzyme was inactivated and reacted. Then, new peaks were confirmed at the retention times of 17 minutes and 29 minutes. (Figures 1 and 2) (Analysis conditions by HPLC) Column: Normal phase silica gel column (Ami manufactured by Tosoh Corporation)
do-80) Mobile phase: acetonitrile: water = 70:30 Flow rate: 1.0 ml / min Temperature: 30 ° C Detection: RI detector

Next, for the product having a retention time of 17 minutes,
Nuclear magnetic resonance apparatus (GX-27 manufactured by JEOL Ltd.)
0 type) and a nuclear magnetic resonance apparatus (GX-
The structure was confirmed by the following conditions under the following conditions. As a result, it was found to be gentianose. (FIGS. 3 and 4) (Analysis conditions by H-NMR) Measurement solvent: heavy water Standard substance: Tsp-d6 270 KHz, 20 ° C. (Analysis conditions by C-NMR) Measurement solvent: heavy water Standard substance: Tsp-d6 68 KHz, 20 ° C

From the results of the above analysis by H-NMR and C-NMR, it was confirmed that the product obtained by the present invention was gentianose. The other holding time 29
The minute peak is presumed to be a substance obtained by adding one glucose to gentianose.

[0028]

Industrial Applicability According to the present invention, gentianose can be easily produced at a high yield and is industrially superior to those produced by conventional extraction from natural or chemical synthesis methods. It is.

[Brief description of the drawings]

FIG. 1 is a diagram showing chromatography of a reaction product obtained in an example.

FIG. 2 is a diagram showing chromatography of a blank of a transfer reaction of an example.

FIG. 3 is an H-NMR spectrum diagram of a reaction product obtained in an example.

FIG. 4 is a C-NMR spectrum diagram of a reaction product obtained in an example.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 19/00 C12P 19/18 CA (STN)

Claims (3)

(57) [Claims] 1. A beta-glucoside polysaccharide having a coupling or in a solution containing a degradation product thereof and sucrose, beta-glucosyltransferase activity Flavobacterium genus microorganism belonging SKP4001 or reaction of the enzyme derived from the microorganism SKP4001 having A method for producing gentianose. 2. A polysaccharide having a β-glucoside bond or a decomposition product thereof and a microorganism SK belonging to the genus Flavobacterium.
A method of performing β-glucosyl transfer to sucrose using P4001 or an enzyme derived from the microorganism SKP4001. 3. The method according to claim 1, wherein the polysaccharide having a β-glucoside bond is pakiman, curdlan, or laminarin.