JPH03259090A - Production of difructose dianhydride iii - Google Patents

Abstract

PURPOSE:To continuously produce the subject compound having high thermal stability by reacting inulin with an inulinase originated from a specific microbial strain in an inulin-containing solution. CONSTITUTION:Arthrobacter sp. (MC12496) (FERM P-11288) (strain A) is separated from natural soil. The strain forms a swollen smooth circular colony when cultured on a heart infusion agar medium at 30 deg.C. It has bacillar form, exhibits Gram-positive property, grows at pH5-10 and is free from starch-decomposition activity, etc. The strain A is inoculated to a medium (B) containing about 5wt.% of inulin, 0.02wt.% of yeast extract, etc., and cultured by rollng-and shaking- incubation at about pH 7 and about 30 deg.C for about 30hr at a rotational speed of 160rpm. The obtained culture produce (C) is filtered and the filtrate is concentrated, extracted and purified to obtain the objective difructose dianhydride III.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to difructose dianhydride (hereinafter referred to as
The present invention relates to a method for producing rDFAI [[referred to as J].

[Prior art and problems to be solved by the invention] DFA
I is a disaccharide with a structure in which two fructose molecules are dehydrated and condensed between 1-2' and 2-3', and was isolated and identified in 1929 by Jackson et al. (Bur, 5ta).
nd, J, Res, J, 27.1929).

Since DFA, III 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[l] by acid hydrolyzing inulin, a polysaccharide mainly composed of fructose, but the yield was only a little less than 2%, and it cannot be said to be an efficient method.

In 1972, Tanaka et al. produced DFAII from inulin using an inulin-degrading enzyme produced by Arthrobacter ureafaciens (Biochi et al.
m, Biophys, Acta, 284,248,19
72).

However, this enzyme has low stability against temperature, and is severely inactivated at temperatures exceeding 60°C, so it cannot be said to be an appropriate enzyme for industrialization.

[Means for solving problems]

The present inventors conducted extensive research on species to solve these problems, and as a result, they discovered that Arthrobacter sp.
cter SP, ) (MCI 2496) The inulin-degrading enzyme derived from FERMP-11288 (FERMP-11288) efficiently (produces DFAIII,
Moreover, since it has higher thermal stability than conventional products, it was found that continuous industrial production of DFAI[I can be carried out efficiently, leading to the completion of the present invention.

That is, the gist of the present invention is to produce difructose, which is characterized by reacting inulin with an inulin-degrading enzyme derived from Arthrobacter sp. (MCI2496) FERMP-11288 in an inulin-containing solution.・It consists in the production method of dianhydride 4.

To explain the present invention below, the inulin component enzyme used in the present invention is Arthrobacter sp. (MCI249).
6) FERMP-1128 No. 11288 (FERMP-1128)
8) It is derived from

Arthrobacter sp. (MCI2496) 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 grown on heart infusion agar medium at 30°C for 11 weeks Work) External shape: circular 2) Size: 2-311I113) Surface ridges: convex 4) Surface shape: smooth 5) Gloss / Dull light 6) Color tone / Yellowish gray 7) Transparency / Opaque 8) Periphery Gold-rimmed O heart infusion agar medium, 30'C13~
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) Sporulation: None 5) Durham staining: Positive and negative 6) Acid-fast 26 Physiological properties]) Growth under anaerobic conditions 2) Growth in air 3) Catalase 4) Oxidase 5) O -F testro) Hydrolysis of gelatin 7) Lidomus milk 8) Reduction of nitrates 9) Methyl red test 10) VP test 11) Formation of indole 12) Formation of hydrogen sulfide 13) Hydrolysis of starch 14) Utilization of citric acid (On Chris Lansen medium) 15) Utilization of inorganic nitrogen source 16) Negative for urease, positive, positive, negative, no acid formation, positive, no discoloration, peptonization, negative, negative, negative, negative, negative, positive: Positive: Negative 17) 18) 19) 20) 21 ) 22) 23) 24) 25) Hydrolysis of casein: Production of DNase: Growth in 5% sodium chloride: Generation of pigment Growth temperature range Growth pH 7'ween 80 Decomposition: Tyrosine degradable From various types Ii Production of acid Positive, negative, negative, negative, negative 10-37°C pH 5-10 Positive, positive (Continued) (Continued) O Observation for 1-3 weeks after incubation O: Possible to produce, ±: Doubtful, -: Not capable of producing 26) Assimilation of organic acids ○ Observation for 1 to 3 weeks after culture O 10: Assimilation ability, -: Assimilation ability 3, Chemotaxonomic properties 1) CC content in DNA 67% 2) Cell wall Amino acid composition molar ratio Lysine 1 Alanine 3 Threonine L Glutamic acid 1 3) Peptidoglycan cross-linked structure Lys-Ala-Thr-AI or Lys-Thr-Alaz 4) Cell wall sugar composition Rhamnose galactose 5) Glycolate testacetyl type 6) Main menaquinone MK 9 (Hz) 4. Taxonomic considerations ○ Identification of genus This strain (MCI No. 2496) has a rod-shaped to cell cycle.
It has short rod-shaped (Rods-coccus) polymorphism.

2) It is an obligate aerobic bacterium.

3) No acids are generated from Ws such as glucose.

4) CC content in DNA is 67%, showing high GC.

5) Cell wall diamino-amino acids have lysine.

6) Main menaquinone exhibits characteristics such as having MK-9 (H, ).

Based on these characteristics, the amount of ice is
of Systematic Bacteriology (Berg
ey's Manual of Systemati
c Bacteri.

Pleomorphic, non-spore-forming, Durham stain-positive bacilli (Irregular, No.
nsporing, Gram-Positive Ro
ds) group of Arthrobacter
r) It was found that it belongs to the genus Bacteria.

Currently, about 15 species of Arthrobacter genus bacteria are identified at the level of 0 species. These species are distinguished by various physical and chemical taxonomic properties, but differences in the cross-linked peptide structure of the cell wall, sugar composition, and menaquinone composition are considered to be important classification criteria at the species level. There is. (K, H, 5chl
eiber & 0. Kandler, Bacteri
ol, Rev, Vol, 36:407-477.197
2. Bergey's Manual of Syst
ematic Bacteriology %1o12
) This strain (MCI2496) contains MK 9 CHz) as a menaquinone required for generation.

2) The cross-linked peptide structure of the cell wall is Lys-Ala-Th
r-Ala or Lys-Thr-Alaz.

3) Contains rhamnose and galactose as sugars in the cell wall.

4) It has the characteristic of not exhibiting motility. These properties and Berge
y's Manual of Systematic
Bacteriology Volume 2 and M. Take
uchi & A., Yokota, J., Gen., App.
l, old crobio1. vo1.35:233-252
．． As a result of comparison with the characteristics of species of the genus Arthlobacter described in 1989, this strain was found to be similar to Arth-robacter auresc.
It was suggested that it is a species closely related to S. ens). but,
Differences were observed in physiological properties such as organic acid assimilation and starch hydrolysis. The official species assignment will be determined after comparing Arthrobacter ararecens or similar species with the amount of ice at the nucleic acid level. Therefore, at this stage, this bacterial strain (MCI No. 2496) was identified as Bacterium sp.

Furthermore, DFAII [Arthrobacter globiformis, which is known as a producing bacterium]
rIobiformis) and Arthrobacter ureafaciens (Arthrobacter urea
When we compared the mycological properties of C. faciens) with this strain, they were clearly differentiated in major points such as the structure of the crosslinked 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, molasses, animal/vegetable oils, etc. can be used. In addition, soybean flour,
Wheat germ, cornstarch liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used. In addition, it is effective to add, if necessary, inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions.

In the present invention, inulin or extracts of plants with high inulin content such as Jerusalem artichoke and burdock are used as the sole carbon source, and in a solution containing the above-mentioned Arthrobacter sp. No. (FER
Inulin-degrading enzyme derived from MP-11288) is activated. At this time, the bacteria themselves may be used to act on the enzyme, or the enzyme may be extracted from the bacteria and used to act on the enzyme.

When the bacteria themselves are used, it contains about 1 to 10% inulin as a carbon source, and other nitrogen sources such as soybean flour, wheat germ, cornstay 7' IJ car,
Cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. In addition, sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions can be generated as required. Inoculate the native culture into a medium supplemented with inorganic salts, etc., and perform shaking culture. In this case, the culture temperature is preferably 20 to 37°C, and the culture time is preferably 12 to 40 hours. The resulting culture solution is sterilized by centrifugation, and the supernatant is heated to deactivate the enzyme. Then, it is concentrated and adsorbed onto an activated carbon column by, for example, activated carbon column chromatography. After eluting fructose with distilled water,
Elution is performed with a 5% ethanol aqueous solution. In this fraction, D
Since FAm is obtained, the desired D is obtained by concentrating it to dryness.
FA[r can be obtained.

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 filtrate, salting out is performed, 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 adjusted to 7.0.
II is obtained.

This crude enzyme solution can be obtained from, for example, DEA E-Toyope.
arl650 M, S P-Toyopearl
By performing purification by ion exchange chromatography using a 650 M column (manufactured by Tosoh), an enzyme preparation that shows a single band electrophoretically can be obtained. The optimum pH of this enzyme preparation was 5.0, and maximum activity was exhibited at 60°C. The pH was stable over a wide range of 4.5 to 11.0, and after heat treatment for 20 minutes, it was stable up to 60°C, indicating high temperature stability.

〔Example〕

The method of the present invention will be explained in more detail with reference to Examples below, but the invention is not limited thereto unless it exceeds the gist thereof.

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 Medium 150s+2 containing .001% pH 1,
0 and steam sterilized at 120°C for 20 minutes. Into this sterilized solution, MCI No. 2496 was inoculated with a platinum blast and cultured at 160r, p, vx at 30°C for 130 hours.

After completion of the culture, the bacterial cells were removed by centrifugation to obtain a culture filtrate. The obtained culture filtrate is heated for 10 minutes to inactivate the enzyme, resulting in approximately 10 intestines! It was concentrated under reduced pressure to . This solution was prepared using an activated carbon column (30 g of activated carbon and Celite Nα53).
5 60g of the mixture is adsorbed in (filled with distilled water),
After flowing distilled water 12 times, elution was carried out with a 5% ethanol aqueous solution.

The eluted peaks were collected and concentrated to dryness under reduced pressure to obtain DFAm. The obtained DFAIII was 1% relative to the raw material inulin.
It was 0%. According to thin layer chromatography [silica gel plate (Merck); developing solvent n-butanol: ethanol: water = 2: 1: 1 (V/V/V)], the standard (7 )D
FAII[ and Rf value (0,67) matched.

Example 2 Culture filtrate 1 rml obtained in Example 1! was added to 4 ml of 0.05M phosphate buffer containing 5% inulin and reacted at 30°C for 3 hours.

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 DFAIII.

The obtained DFAII [was 83% with respect to the raw material inulin.
It was 4%.

[Effect of the invention] Arthrobacter sp. of the present invention (MCI2496
) FERMP-11288
) derived inulin-degrading enzyme can be successfully converted from inulin to DF.
Since it produces AI[[ and has higher stability against heat than conventional products, it is considered to be very effective in continuous production of DFAIII.

Claims (1)

[Claims] (1) Inulin was added to Arthrobacter sp. (MCI2496) in an inulin-containing solution.
A method for producing difructose dianhydride III, which comprises reacting with inulin degrading enzyme derived from No. 8 (FERMP-11288).