JPH03247295A - Production of difructose-dianhydride i - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to difructose dianhydride I (hereinafter referred to as
It is called rDFAI J. ).

(Prior art and problems to be solved by the invention) DFA
I is a 2W class with a structure in which two fructose molecules are dehydrated and condensed between 1-2' and 2-1', and was isolated and identified by Jackson et al. in 1929 (Bur, 5tan
d, J, Res,.

Lord, 27. 1929).

Since DFAI is a non-fermentable sugar that is not metabolized in the animal body, it is attracting attention as a low-calorie sweetener, and it is expected that it will be used in many fields such as beauty foods in the future.

Jackson et al. obtained DFAI by acid hydrolyzing inulin, a polysaccharide whose main component is fructose, but the yield was only a little less than 2%, and the method of chemically hydrolyzing inulin was not efficient. I can't say that.

Therefore, in recent years, using microbiological methods, inulin can be extracted from D.
A method of manufacturing FAI has been proposed.

For example, in 1979, China and China produced DFAI from inulin using an inulin-degrading enzyme produced by a fungus. (Carbohydr, Res,.

Yu 5, 340.1979). Atsugi et al. also produced DFAI from inulin using an inulin-degrading enzyme produced by Arthrobacter globiformis (Japanese Patent Application Laid-open No. 275693/1983).

However, the number of microbiological methods for producing DFAI is still small, and it is necessary to provide an enzyme that can be efficiently produced.

(Means for Solving the Problems) Therefore, the present inventors conducted intensive research on a method for producing DFAI from inulin by a microbiological method, and as a result, they discovered that Arthrobacter sp.
ter sp,) Microtechnical Research Institute No. 11287 (FER
The inulin-degrading enzyme derived from MP-11287) is highly efficient.
It was discovered that DFAI can be produced, and the present invention was developed.

That is, the gist of the present invention is that inulin is added to Arthrobacter sp.
The present invention relates to a method for producing difructose dianhydride I, which is characterized by reacting with inulin degrading enzyme derived from No. (FERMP-11287). To explain the present invention below, the inulin-degrading enzyme used in the present invention is Arthrobacter sp.
MP-11287).

Arthrobacter sp. (MCI2493) of the present invention
Bacterium No.) is a bacterium isolated from natural soil by the present inventors, and its mycological properties are as follows.

1. Morphological characteristics Characteristics of colonies after 11 weeks of culture at 30°C on heart infusion agar medium 1) External shape: circular 2) Size: 2-3 mm 3) Surface ridges: convex 4) Surface shape: Smooth 5) Gloss: Dull light 6) Color: Yellowish gray 7) Transparency: Translucent 8) Periphery: Gold ○ On heart infusion agar medium, 30°C 13~
Morphological properties during 48-hour culture 1) Cell morphology: Until about 6 to 12 hours after culture, cells elongate unevenly and become long rod-shaped.

After that, a septum is formed in the center, and the cell bends into a curved shape and repeats gradual segmentation. After 18 hours, most of the cells change to a uniform short culm shape.

2) Cell division mode: Bending type 3) Motility: None 4) Spore formation: None 5) Durham staining: Positive 6) Acid-fastness: Negative 2, Physiological properties 1) Growth under anaerobic conditions 2) Growth in air Growth 3) Catalase 4) Oxidase 5) 0-F Testro) Gelatin hydrolysis: Negative: Positive: Positive: Negative: No acid production: Positive 8) 9) 10) 11) 12) Reduction of nitrate Methyl Red Test VP Tests Production of indole Production of hydrogen sulfide: Negative: Negative: Negative: Negative: Negative 13) Hydrolysis of starch: Negative 15) 16) 17) 18) Utilization of inorganic nitrogen sources Hydrolysis of urease casein Production of DNase : Positive: Negative: Positive: Negative 20) Production of pigment 21) Growth temperature range 22) Growth pH: Negative 210-37°C: pH 5-10 23) Production of acids from various sugars Observation for 1-3 weeks after culturing +: Generating ability.

±: Doubtful.

: No ability to produce 24) Assimilation of organic acids / Observation for 1 to 3 weeks after culture 10: Assimilation ability, -: No assimilation ability 3, Chemical taxonomic properties 1) CC content in DNA 2) Cell wall amino acid composition Lysine 66% Alanine Threonine Glutamic acid 3) Peptidoglycan cross-linked structure Lys-Ala-Thr-AI or Lys-Thr-Alaz 4) Cell wall sugar composition Rhamnose galactose 5) Glycolate test Acetyl type 6) Major menaquinone MK9 (H2) 4. Taxonomic considerations Identification of O belonging to Bell This bacterial strain (MC12493) has a polymorphism of rod-shaped to short culm-shaped (Rods-coccus) in the cell cycle. ) It is an obligate aerobic bacterium 3) It does not produce acid from sugars such as glucose 4) It has DNA
The GC content in the cell wall is as high as 66%; 5) diamino-amino acids in the cell wall contain lysine; 6) the main menaquinone has MK9 (Hz).

Due to these characteristics, the home country uses Purge Aids Manual.
of Systematic Bacteriology (Berg
ey s Manual of Systema
tic Bacteriology) Volume 2, pleomorphic, non-spore-forming, Durham stain-positive bacilli (
Irregular.

Nonsporing, Gram-Positive
It was found that the bacteria belong to the genus Arthrobacter of the Rods group.

Currently, about 15 species of Arthrobacter genus bacteria are identified at the level of 0 species. These species are distinguished by various physiological properties and chemical taxonomic properties, but differences in the cross-linked peptide structure of the cell wall, I1 composition, and menaquinone composition are considered to be important classification criteria at the species level. . (K, H, 5c
Hleiber & O, Kandler, Bacte
riol.

Rev., Vol. 36:407-477.1972,
Bergey's Manual of Sys
tematic Bacteriology, V
2) This strain (MCI2493) contains MK9 (H2) as a menaquinone required for generation.2) The crosslinked peptide structure of the cell wall is Lys-Ala-Th.
r-Ala or Lys-Thr-Ala, 3) It contains rhamnose and galactose as sugars in the cell wall, and 4) It has the characteristics of not exhibiting motility. These properties and Berge
y's Manual of System
atic Bacteriology Volume 2 and M.
Takeuchi & A, Y.

kota, J., Gen., Appl., Microbiol
, vol. 35:233-252. 198
As a result of comparing the characteristics of species of the genus Arthrobacter described in 9, the present strain was determined to be Arthrobacter au. However, the physiological properties such as assimilation of organic acids and hydrolysis of starch Differences were observed in the species.The official species assignment will be determined after comparing Arthrobacter ararecens or similar species with that of the home country at the nucleic acid level.Therefore, at this stage, the edited strain (MCI2493) will be determined. No. 1 bacterium)
・Identified as SP.

Furthermore, Arthrobacter globiformis (A1th r bac
When we compared the mycological properties of C. ter. lobiformis with this strain, they were clearly differentiated in major points, such as the structure of the cross-linked peptide and the sugar composition of the cell wall, as shown in the table below.

In the present invention, the above bacteria can be easily grown by culturing them in a medium containing nutrients that can be used by ordinary microorganisms. As a nutritional source, glucose,
Starch syrup, dextrin, sucrose, starch, nectar, animal/vegetable oils, etc. can be used. In addition, soybean flour,
Wheat germ, corn staple liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate.

Urea etc. can be used. Other sodium, as necessary.
It is useful to add inorganic salts that produce potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions.

In the present invention, inulin or Jerusalem artichoke.

In a solution containing an extract of a plant with high inulin content such as burdock as the sole carbon source, the above-mentioned Arthrobacter sp.
87) to act on the inulin-degrading enzyme derived from the plant. that time,
The bacterium itself may be used, or the enzyme may be extracted from the bacterium and used.

When using bacteria itself, it contains about 1-10% inulin as a carbon source, and other nitrogen sources such as soybean flour, wheat germ, cornstarch liquor, cottonseed meal, meat extract, peptone, and yeast extract. , ammonium sulfate, sodium nitrate, urea, etc., and if necessary, sodium,
A medium containing inorganic salts capable of producing potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions is inoculated with a sieve and cultured with shaking.

In this case, the culture temperature is preferably 20-37°C, and the culture time is preferably 12-40 hours. The obtained culture solution is sterilized by centrifugation, and the enzyme in the supernatant is inactivated by heat treatment. Then, it is concentrated and adsorbed onto an activated carbon column by, for example, activated carbon column chromatography. After fructose is eluted with distilled water, elution is performed with a 5% ethanol aqueous solution. Since DFAI is obtained in this fraction, the desired DFAI can be obtained by concentrating it to dryness.

In addition, when applying an enzyme, first, the culture solution cultured by the above method is sterilized by centrifugation, ammonium sulfate (65% saturation) is added to the obtained filter paper to perform salting out, and the precipitated precipitate is centrifuged. After suspending in a small amount of water, dialysis is performed to obtain a crude enzyme solution. This crude enzyme solution is adjusted to pH
The desired DFAI can also be obtained by acting on inulin in a 0.01-0.1M phosphate buffer prepared at 7.0.

This crude enzyme is, for example, DFAE-Toyope
arl HW 55F, SP-Toyopear1
By performing purification by ion exchange chromatography using a 650M column (manufactured by Tosoh), an enzyme preparation that shows a single band electrophoretically can be obtained. The optimum pH of this enzyme preparation was 6.0, and maximum activity was exhibited at 50°C. , the pH was stable in the range of 5-9, it was stable up to 60°C after heat treatment for 30 minutes, and the molecular weight by 5DS-polyacrylamide gel electrophoresis was about 40,000.

(Examples) The method of the present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these unless the gist thereof is exceeded.

Example 1 Commercially available inulin 5%, yeast extract 0.02%, sodium nitrate 0.2%, magnesium sulfate 0.05%, potassium chloride 0.05%, potassium phosphate 0.05%, ferric chloride 0 150d of medium containing .001%, pH 7.0
and steam sterilized at 120°C for 20 minutes. One loopful of MCI 2493 bacteria was inoculated into this sterilized solution, and the mixture was incubated at 160 r.

Cultured at 30°C for 130 hours at p, m.

After the cultivation was completed, the bacterial cells were removed by centrifugation to obtain a culture solution. The obtained culture solution was heated for 10 minutes to inactivate the enzyme and concentrated under reduced pressure to about 10 d. This solution was prepared using an activated carbon column (30 g of activated carbon and Celite Nα535).
．． 60 g of the mixture was adsorbed in a 5% ethanol aqueous solution (filled with distilled water), and after flowing distilled water 11, it was eluted with a 5% ethanol aqueous solution.

The eluted peaks were collected and concentrated to dryness under reduced pressure to obtain DFAl. The obtained DFAI was 10% relative to the raw material inulin.
%Met. According to thin layer chromatography (silica gel play) (MERCK); developing solvent n-butanol:ethanol:water-2=1:1 (v/v/v))
Standard DFAI and Rf obtained by acid digestion of inulin
The values (0, 6) matched.

Example 2 The culture solution 11d obtained in Example 1 was added to 4d of 0.05M phosphate buffer containing 10% inulin and heated at 30°C.
I let it react overnight.

After heating the reaction solution to inactivate the enzyme, activated carbon column chromatography was performed and eluted with 5% ethanol.
The eluate was concentrated to dryness under reduced pressure to obtain DFAI.

The obtained DFAI was 30% based on the raw material inulin.

(Effect of the invention) Arthrobacter sp.
Since the inulin-degrading enzyme derived from No. 287 (FERMP-11287) successfully produces DFAI from inulin, it is considered to be very effective in continuous production of DFAI.

Claims (1)

[Claims] (1) Inulin was added to Arthrobacter sp. (MCI2493) in an inulin-containing solution.
A method for producing difructose dianhydride I, which comprises reacting with inulin degrading enzyme derived from No. 7 (FERMP-11287).