JP2833081B2 - Method for producing inulotriose and / or inulotetraose - Google Patents

Description

The present invention relates to a microorganism belonging to the genus Aureobacterium, which specifically hydrolyzes fructan every 3 to 4 saccharide units from the fructose terminal side. The present invention relates to a method for producing inulotriose and / or inulotetraose using a novel exo-type hydrolase (exo-inuro-trioandtetrao-hydrolase (tentative name)) produced.

(Problems to be Solved by the Prior Art and the Invention) Inulotriose and inulotetraose to which 3 to 4 fructoses are bound are inulooligosaccharides having high functions (bifidus factor, anti-cariogenicity, low calorie, etc.). It is effective in the food and pharmaceutical fields.

Conventionally, as a method for producing inulotriose and inulotetraose from fructan, a method of hydrolyzing fructan, for example, inulin, with an acid is known.

However, according to this method, most of the product is fructose (F ₁ ), and the production yields of inulooligosaccharides, inurotriose and inulotetraose are extremely poor. By-products such as hydrides have been produced, making purification of inulotriose and inulotetraose difficult.

On the other hand, a method for producing inulotriose and inulotetraose by decomposing fructan using an enzyme has also been proposed. However, conventionally known enzymes are only endo-inulinases that hydrolyze fructans randomly.Therefore, for example, when acted on inulin, inurotriose, inulotetraose, and inurobiose are known. (F ₂ ) Inulooligosaccharides to which various fructoses such as inulopentaose (F ₅ ) and inulohexaose (F ₆ ) are bound are formed, and the yield of production is low, such as by-product nructose. It was not suitable for the production of inulotriose and ifrotetraose.

In addition, some of the present inventors have found that an exo-type hydrolase produced by a microorganism belonging to the genus Streptomyces hydrolyzes fructan to produce inulotriose and / or inulotetraose. And / or a method for producing inulotetraose (Japanese Patent Application No. 1-47361, Abstracts of 1989 Annual Meeting of the Japanese Society of Agricultural Chemistry, p. 286).

(Means for Solving the Problem) The present inventors have further studied the method for producing inulotriose and / or inulotetraose from fructan, and as a result, the microorganism belonging to the genus Aureobacterium acts on fructan. The present inventors have found that an exo-type inulinase that hydrolyzes with 3 to 4 sugar units from the fructose terminal side has been produced, thereby completing the present invention.

That is, the gist of the present invention is an enzyme produced by a microorganism belonging to the genus Aureobacterium, which hydrolyzes every three to four saccharide units from the fructose terminal side of fructan to inulotriose and / or inulotetratetrahydrofuran. Inulotriose by reacting an exo-type hydrolase having the property of generating aose with fructan to generate inulotriose and / or inulotetraose, and obtaining the inulotriose and / or inulotetraose. And / or a method for producing inulotetraose.

 Hereinafter, the present invention will be described in detail.

The microorganism belonging to the genus Aureobacterium used in the present invention has a property of hydrolyzing every 3 to 4 sugar units from the fructose terminal side of the above-described fructan to produce inulotriose and / or inulotetraose. What is necessary is just to have the ability to produce the exo-type hydrolase, and is not limited to the genus Aureobacterium and its variants and mutants.

Specific examples of the microorganism belonging to such Aureobacterium genus, for example, Aureobacterium Corynebacterium sp (Au
reobacterium sp.) MCI2494.

Such a strain is described in Microtechnological Research Institute No. 2679 (FERM BP-2679).
Has been deposited as

Aureobacterium sp.
p.) MCI2494 (hereinafter referred to as "the present strain" or "MCI2494") is a bacterium isolated from natural soil by the present inventors, and its bacteriological properties are as follows.

1. Morphological characteristics ○ Characteristics of colonies on a heart infusion agar medium at 30 ° C for 1 week 1) Outer shape: circular 2) Size: 2 to 4mm 3) Surface bulge: convex 4) Surface 5) Hikarizawa: Dull light 6) Color tone: Grayish white at first, yellow later 7) Transparency: Translucent 8) Peripheral: Whole ○ On heart infusion agar medium, 30 ° C, 3-48
Morphological properties during time culturing 1) Cell morphology: About 7 to 24 hours after culturing, cells elongate and branch in a hyphae-like manner. After that, a septum is inserted in places of the mycelial cells, and a gradual segmentation occurs at the septum, and the cells change into short rod-shaped cells.

2) Cell division mode: Segmental 3) Motility: None 4) Sporulation: None 5) Gram staining: positive 6) Acid-fast: negative 2. Physiological properties 1) Growth under anaerobic conditions: 2) Growth in air: positive 3) Catalase: positive 4) Oxidase: negative 5) OF test: F 6) Hydrolysis of gelatin: negative 7) Litmus milk: change None, no peptone formation 8) Reduction of nitrate: positive 9) Denitrification reaction: negative 10) Methyl red test: negative 11) VP test: negative 12) Formation of indole: negative 13) Formation of hydrogen sulfide : Negative 14) Starch hydrolysis: Positive 15) Use of citric acid: Positive (on Christensen medium) 16) Use of inorganic nitrogen source: Positive 17) Urease: Negative 18) Casein hydrolysis: Yin Sex 19) DNase production: negative 20) 5% sodium chloride Growth at medium: Negative 21) Production of pigment: yellow, water-insoluble chloroform - 22 is extracted from the cells with methanol and the like) Growth temperature range: 15 to 37 ° C. 23) Growth pH: pH 5 to 9 3. Scientific taxonomic properties 1) GC content in DNA 69% 2) Amino acid composition of cell wall ornithine glycine homoserine glutamic acid (partially hydroxyglutamic acid) alanine 3) Peptidoglycan cross-linked structure B type D-Glu-Gly-D- Orn 4) Sugar composition of cell wall Rhamnose galactose Unidentified hexamonosaccharide 5) Glycolate test Glycolyl type 6) Major menaquinone MK-13 4. Taxonomic considerations ○ Higher taxonomic position ○ This strain (MCI2494) Has the following features.

1) The cell cycle has a mycelial to rod-shaped to short rod-shaped stage.

2) The GC content in the DNA shows a high GC of 69%.

3) The cell wall diamino-amino acids have ornithine.

4) Major menaquinone has MK-13.

Based on these characteristics, this strain (MCI2494 strain) can be used in Bergey's Manual of Systematic Bacteology.
riology, Vol. 2, pluripotency, non-spore formation, gram staining positive, bacilli (Irregular, Nonsporing, G
ram-Positive Rods) group.

As shown in the above table, this strain (MCI2494) has 1) a peptidoglycan type B type 2) a sugar chain structure of peptidoglycan 3) a glycolyl type 3) peptidoglycan contains major menaquinone MK-13. Systematic Bacteriology (Bergey's Mannual of Sy
The characteristics of the genus Aureobacterium described in Stematic Bacteriology, Vol. 2, p. 1323, were well matched. However, this strain (MCI2494
S. cerevisiae differs from Aureobacterium in that it grows like a mycelium at the beginning of culture and grows weakly under anaerobic conditions. Bergey's Manual of Systematic Bacteriolog
y) According to Volume 2, the genus level of coryneform bacteria is classified by chemical composition such as amino acid composition of cell wall, sugar composition, peptidoglycan cross-linked structure, sugar chain structure, major menaquinone composition, and GC content of DNA. It is regarded as a standard and is widely accepted by many taxonomies. Considering these circumstances, it is considered appropriate to treat the difference in physiological properties as a feature at the species level in terms of morphology of the strain. Therefore, this strain (MCI2494) is Aureobacterium ( Aur
eobacterium ). Barjay's Manual of Systematic Bacteriology (Be
According to rgey's Manual of Systematic Bacteriology, Vol. 2, six species of the genus Aureobacterium are known. This strain (MCI2494)
In that it contains a major menaquinone MK-13, Aureobacterium Corynebacterium Furabasensu (A.flavescens) and, similar to Aureobacterium Corynebacterium Teregensu (A.terregens). However, this strain (MCI2494) is clearly distinguished from the latter two in that the sugar composition of the cell wall and the terregens factor (described above) are not required. Furthermore, in terms of morphological properties, the present strain (MCI2494) has these two features in that it has mycelial elongated cells and branched cells.
It is distinguished from species. Therefore, this strain (MCI2494) is presumed to be a new species of the genus Aureobacterium ( Aureobacterium ). SP ( Aureobacterium sp.) Was identified.

Culturing of the present strain for producing the exo-type hydrolase of the present invention can be performed in a medium containing nutrients that can be used by ordinary microorganisms.

Specifically, fructans such as fructo-oligosaccharides, inulo-oligosaccharides and inulins containing β-2-1 ′ fructoside bonds are used as substrates, and other components necessary for the growth of bacteria, for example, soybean flour, wheat germ, corn Uses steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. to generate sodium, potassium, calcium, magnesium, cobalt, chlorine, sulfuric acid and other ions as required Culture in a medium containing inorganic salts, vitamins, etc. In this case, the amount of fructan added to the medium is, for example, in the case of inulin, in the range of 0.5 to 20%.

Furthermore, the culture temperature is 20-37 ° C, and the culture time is 24-
96 hours is appropriate.

After completion of the culture, the exo-type hydrolase of the present invention can be purified from the culture supernatant by appropriately combining various known purification methods. One example of the method is as follows.

Bacteria are removed from the culture by an appropriate purification means such as filtration centrifugation to obtain a supernatant, which is then dialyzed against distilled water. Ammonium sulfate was added to the obtained dialysate so as to make it 40% saturated, and adsorbed on Toyopearl HW65F (manufactured by TOOH) equilibrated with a phosphate buffer (0.02M, pH 7.0) containing 40% saturated ammonium sulfate. And column chromatography. At this time, elution is performed with a linear concentration gradient of a 40-0% saturated aqueous ammonium sulfate solution. Next, fractions having inulinase activity are collected and dialyzed against a 20 mM phosphate buffer (pH 7.0). When the obtained crude enzyme solution was passed through a DEAE-Toyopearl column (manufactured by Tosoh Corporation), inulinase activity was observed in the non-adsorbed fraction, and the enzyme solution was further separated by molecular sieving using Toyopearl HW55F (manufactured by Tosoh Corporation). Then, it is adsorbed to QAE-Toyopearl (manufactured by Tosoh Corporation) equilibrated with 10 mM Tris-HCl buffer (pH 8.7). Elution is performed with a linear concentration gradient of 0-0.5 M saline, and the active fraction is collected and dialyzed to obtain a purified product of a novel exo-type hydrolase.

For example, the purified enzyme of the present invention obtained as in Example 1 described below has the following properties.

(A) Fructose is hydrolyzed every 3 to 4 units from the fructose terminal side of fructan to produce inulotriose and / or inulotetraose.

(B) The optimum pH is 6 to 7, and the pH is stable at 5.0 to 10.0.

(C) When the optimum temperature at pH 6 to 7 is about 50 ° C,
It shows good activity at 0 ° C.

(D) Stable up to 50 ° C under heat treatment conditions of pH 6 to 7 for 30 minutes.

(E) The molecular weight by SDS-polyacrylamide gel electrophoresis is about 150,000.

The method for producing inulotriose and / or inulotetraose using the enzyme of the present invention comprises using a fructan containing at least four fructoses, particularly inulin as a substrate,
By acting an enzyme, inulotriose and / or inulotetraose can be produced. As the enzyme to be acted on, a purified enzyme purified from a culture supernatant may be used, or a culture supernatant obtained by culturing cells in a medium containing the above substrate may be used. Alternatively, an enzyme or a cell obtained by adsorbing, binding, and entrapping an immobilized carrier by a known method may be used.

For example, when a purified enzyme is used, inulin 0.1
Enzyme 0.01-10% in 0.1M phosphate buffer (pH 6.5) containing ~ 10%
When acted on a unit basis at 20 to 50 ° C. for 5 minutes to 100 hours, inulotriose and / or inulotetraose are specifically produced from the initial stage of the reaction.

When cells are cultured in the substrate-containing medium, inulotriose and / or inulotetraose are produced in the culture solution. This culture is sterilized, and the obtained culture supernatant is
Activated carbon treatment and desalination treatment removes impurity salts and the like. Next, the obtained product is subjected to activated carbon column chromatography. When a concentration gradient elution of ethanol of 0 to 10% is performed, inulotriose and / or inulotetraose are eluted in a fraction portion of 5 to 10%, and inulotriose and / or inurotetraose in the culture supernatant is removed by removing ethanol. Aose can be refined.

(Examples) Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following unless it exceeds the gist.

Example 1 Aureobacterium sp. MCI2494 was prepared by inulin 1.5%, sodium nitrate 0.2%, magnesium nitrate 0.
05%, potassium chloride 0.05%, potassium phosphate 0.05%,
A liquid medium containing 0.02% yeast extract and a trace amount of iron chloride was inoculated, cultured at 30 ° C. for 1 day, a portion was taken, transferred to a liquid medium having the same composition, and cultured at 30 ° C. for 1 day with shaking.

After completion of the culture, the mixture was centrifuged, and the obtained supernatant was added with ammonium sulfate so as to be 80% saturated, salted out, and the precipitated precipitate was obtained by filtration and suspended in a small amount of water.
Dialysis was performed to obtain a crude enzyme solution.

The crude enzyme solution was adsorbed on DEAE-Toyopearl 650M (manufactured by Tosoh Corporation) and eluted with a linear concentration gradient of 0-0.3M saline. Fractions having inulinase activity were collected and separated by molecular sieve using Toyopearl HW55 (manufactured by Tosoh Corporation) to obtain an enzyme preparation which electrophoretically showed a single band. Table 1 shows the purification results in each step.

The measurement of the titer was performed as follows. That is, 0.
0.25 ml of 2% inulin aqueous solution and pH 6.5, 0.1 M phosphate buffer
0.15 ml of the enzyme solution was added to 0.1 ml of the mixed substrate, reacted at 37 ° C. for 1 hour, and purified inulotriose and / or inulotetraose were quantified by HPLC (high performance liquid chromatography). The amount of the enzyme that produces 1 μmol of inulooligosaccharide per minute was defined as 1 unit (u).

Example 2 A solution containing 0.1 unit of the purified hydrolase obtained in Example 1 was added to 1 ml of a 0.05 M phosphate buffer (pH 6.5) containing 0.1% (w / v) inulin as a substrate, and the solution was added at 30 ° C. For 10 minutes.

After heating the reaction mixture to deactivate the enzyme, the inulooligosaccharide production rate in the reaction mixture was determined using HPLC.Inurotriose was 5.1 mg and inulotetraose was produced at 24.8 mg, respectively. No sugar was found. The amount of unreacted inulin was 70.1 mg.

As described above, the use of the enzyme of the present invention enables the specific production of inulotriose and inulotetraose.

Example 3 The reaction was carried out for 2 hours in the same manner as in Example 2, and
As a result of analysis by the same method as in the above, the production rate of inulotriose and inulotetraose (inulotriose: inulotetraose = 1: 5) was 80% in total.

Example 4 20 g of inulin was added to 80 ml of the enzyme solution that had been cultured in the same manner as in Example 1, and was sterilized after the culture, and reacted at 30 ° C. for 48 hours.

After the reaction, the reaction solution was analyzed by liquid chromatography. As a result, the production rate of inulotriose and inulotetraose (inurotriose: inulotetraose = 1: 5) in the reaction solution was about 75%.

Example 5 Five cultures were carried out in the same manner as in Example 1, and after the culture, the bacteria were removed by centrifugation, and the supernatant was used as a crude enzyme solution. 5 80 ml (wet state) of a crude enzyme solution buffered with a 67 mM KH ₂ PO ₄ -NaHPO ₄ (pH 6.92) phosphate buffer of HPA75 (manufactured by Mitsubishi Chemical) with a strong porous anion exchange resin ) Add 3 ℃ at 30 ℃
The mixture was shaken and stirred for hours to adsorb and immobilize the enzyme. After washing the immobilized enzyme suspension three times (300 ml in total) with 100 ml of the above phosphate buffer,
After suction, a wet immobilized enzyme was obtained. This immobilized enzyme was packed in a column having an inner diameter of 20 mm to a height of 180 mm and kept at 40 ° C. After flowing 100 ml of water at a flow rate of 0.1 ml / min to stabilize the packed column, a 3% aqueous solution of inulin 1 was flowed at a flow rate of min / min.
The reaction was allowed to flow at 0.1 ml. After the reaction, the mixture was decolorized with activated carbon, desalted and concentrated under reduced pressure.

As a result of analysis in the same manner as in Example 2, inulotriose 6.4 g and inulotetraose 16.9 g were produced.

(Effect of the Invention) The enzyme of the present invention is a novel exo-type hydrolase that specifically hydrolyzes fructan for each sugar unit to which 3 to 4 fructoses are bound from the fructose terminal side, and comprises inulotriose and // Inulotetraose can be advantageously produced.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Reiko Sada, 1000 Kamoshita-cho, Midori-ku, Yokohama-shi, Kanagawa Prefecture, Mitsubishi Chemical Research Institute (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 19 / 14 CA (STN)

Claims (1)

(57) [Claims] 1. An enzyme produced by a microorganism belonging to the genus Aureobacterium, which hydrolyzes every three to four saccharide units from the fructose terminal side of fructan to produce inulotriose and / or inulotetraose. Reacting an exo-type hydrolase having the property of causing the reaction with fructan to produce inulotriose and / or inulotetraose, and obtaining the inulotriose and / or inulotetraose, wherein inulotriose and / or inulotriose are obtained. A method for producing inulotetraose.