JP5314955B2 - Novel microorganism, inulinase, inulin degrading agent, method for producing inulooligosaccharide, and method for producing inulinase - Google Patents

Description

  The present invention relates to a novel microorganism, inulinase, an inulin degrading agent containing these as active ingredients, a method for producing inulooligosaccharides using the novel microorganism or inulinase, and a method for producing inulinase using the novel microorganism.

There are various storage polysaccharides synthesized by plants, and the presence of glucans, fructans, mannans and the like is known. Among them, there are many plants that use starch, which is a kind of glucan, as a storage polysaccharide, and the second most common plant is plants that use inulin, which is a kind of fructan, as a storage polysaccharide. Examples of the plant using inulin as a storage polysaccharide include plants of the family Amaryllidaceae, Oleaceae, or Asteraceae. Inulin is a linear polysaccharide in which 30 to 40 molecules of fructose are β-2,1 linked to fructose residues of sucrose.
On the other hand, polysaccharides are useful as raw materials for producing oligosaccharides, and oligosaccharides are expected to be used in various applications including functional foods. Then, the manufacturing method of the oligosaccharide using inulin is also examined from both sides of a cyclic oligosaccharide and a linear oligosaccharide.

Cyclic oligosaccharides are known to be generated by the action of intramolecular glycosyltransferases, whereby the fructoside bond of inulin is cleaved and transferred to the terminal fructoside residue of inulin. Specifically, a method for producing difructose dianhydride III ( DFAIII ) using inulin fructotransferase produced by a microorganism belonging to Arthrobacter ureafaciens (Non-patent Documents 1 and 2) Reference), Cyclic inulooligosaccharide-producing enzyme (CFTase) produced by Bacillus circulans OKMZ31B strain, from inulin 6-8 molecules of fructose β-2,1-linked cyclic inulooligosaccharide (See Non-patent Document 3 and Patent Document 1), a method for producing cyclic inulooligosaccharides using a medium containing inulin and 0.01 to 1.0% organic nitrogen source (see Patent Document 2) Is disclosed There.

On the other hand, linear oligosaccharides are obtained by degrading inulin with inulinase. There are two types of inulinase, endo-type and exo-type, and in order to efficiently produce linear oligosaccharides, the reaction should be carried out under conditions with as much endo-type inulinase as possible and as little exo-type inulinase as possible. is important.
In response to such problems, inulinase obtained by culturing Penicillium purpurogenum var. Rubri-sclerotium (FERM P-8705) is used as an endo-inulinase in a cation exchanger. A method for obtaining endo-inulinase by separating it into exo-type inulinase (see Patent Document 3) is disclosed. However, this method has a problem in that it requires a capital investment because of the special purification method, and further requires a long time for the production of oligosaccharides, resulting in an inevitable increase in cost.
As a method that does not require such purification, a method using inulinase produced by a microorganism belonging to the genus Penicillium (see Patent Document 4) is disclosed. However, even in this method, depending on the medium composition, there are problems that the main inulinase produced becomes exo-type, and that it takes a long time for inulinase to change from exo-type to endo-type.
Also, inulin-containing plants such as Jerusalem artichoke tubers and chicory underground are used as carbon sources, maltose and ammonium phosphate are added, inulinase-producing bacteria are cultured using a medium not containing potassium phosphate, and endo-type inulinase is A method of obtaining (see Patent Document 5) and a method of using a medium in which magnesium and zinc are further added as minerals to the same medium (see Patent Document 6) have been disclosed. There was a problem that the composition was limited.
Above all, the conventional techniques mentioned so far have a problem that it is difficult to efficiently obtain an oligosaccharide having a desired specific degree of polymerization.
In contrast, Bacillus sp. ( Bacillus sp.) A method for producing inulotetraose using an enzyme produced by KK-4645 strain (see Patent Document 7) is disclosed.
Japanese Patent Laid-Open No. 02-255085 Japanese Patent Laid-Open No. 06-141856 JP 03-035881 A Japanese Patent Laid-Open No. 62-228293 Japanese Patent Laid-Open No. 03-198774 Japanese Patent Laid-Open No. 04-190789 JP 62-232380 A T. T. et al. Uchiyama, Biochem. Biophys. Acta. , 397, 153 (1975) New Food Industry 2005, Vol. 47, no. 3 M.M. Kawamura, T .; Uchiyama, T .; Kuramomoto, Y. et al. Tamura and K.K. Mizutani, Carbohydr. Res. 192, 83-90 (1989)

  However, the method described in Patent Document 7 also has a problem that the production efficiency of inulotetraose is low. In addition, when an enzyme is extracted from a bacterial cell and used, the enzyme is present in the cell wall of the bacterial cell. Therefore, it is necessary to extract and purify the enzyme from a fraction obtained by crushing the cells and collecting the cell wall. There was a problem that it took much time to obtain the enzyme.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide means for easily producing inulooligosaccharides, particularly inulotetraose, from inulin as a raw material.

To solve the above problem,
The invention according to claim 1 is a microbacterium sp. ( Microbacterium sp.) YK-01 (FERM P-21593) strain.
Invention of Claim 2 is a manufacturing method of inulo-oligosaccharide which has the process of culture | cultivating microorganisms of Claim 1 in the presence of inulin.
The invention according to claim 3 is a microbacterium sp. ( Microbacterium sp.) Inulinase derived from YK-01 (FERM P-21593) strain.
The invention according to claim 4 is the inulinase according to claim 3 having the following physicochemical properties. (1) Acts on inulin to produce a linear inulooligosaccharide, and the amount of the product at this time is inurotetraose>inurotriose> inurobiose, fructose. (2) The optimum pH is 6 to 8.5. (3) The optimum temperature is 45 to 55 ° C. (4) Stable at 50 ° C. or lower .
Invention of Claim 5 is inulinase of Claim 3 or 4 which makes the production amount of inulotetraose 4 times or more (mass ratio) of the production amount of inulotriose.
The invention according to claim 6 is a method for producing inulooligosaccharides, wherein the inulinase according to any one of claims 3 to 5 is allowed to act on inulin.
The invention according to claim 7 is an inulin degrading agent comprising the microorganism according to claim 1 as an active ingredient.
The invention according to claim 8 is an inulin degrading agent comprising the inulinase according to any one of claims 3 to 5 as an active ingredient.
The invention according to claim 9 is a method for producing inulinase, comprising the steps of culturing the microorganism according to claim 1 and extracting a water-soluble component from the obtained culture.

  According to the present invention, inulooligosaccharide can be easily produced using inulin as a raw material. In particular, inulotetraose and inulotriose can be preferentially produced, and inulotetraose can be produced most efficiently. Therefore, the present invention provides a new use of inulooligosaccharides for various foods and drinks including functional foods.

Hereinafter, the present invention will be described in detail.
In the present invention, “inuro-oligosaccharide” refers to an oligosaccharide having a structure in which fructose is linked by β-1,2 bonds. Among them, disaccharide oligosaccharide is referred to as “inurobiose”, trisaccharide oligosaccharide. “Inurotriose”, a tetrasaccharide oligosaccharide is called “inurotetraose”.
In the following, “microbacterium sp. ( Microbacterium sp.) YK-01 (FERM P-21593) strain” is abbreviated as “microbacterium sp. YK-01 strain”.

<Microbacterium sp. Acquisition of YK-01 stock>
Microbacterium sp. YK-01 strain was obtained by the following method.
A suspension of 1 g of soil in Mt. Asahigaoka, Kashihara City, Osaka Prefecture, in 100 ml of sterilized water was allowed to stand. Sterile water was added to 1 ml of the supernatant to prepare a 1000-fold diluted solution. The diluted solution was sucked up with a Pasteur pipette, dropped in the center of the following plate medium, spread over the entire surface, and cultured at 30 ° C. for 2 days. The resulting colony was transplanted to the following slant medium and cultured at 30 ° C. for 2 days.
(Plate medium and slant medium)
The plate medium and slant medium are yeast extract (Becton Dickinson) 0.1 w / v%, sodium nitrate 0.2 w / v%, magnesium sulfate heptahydrate 0.05 w / v%, potassium chloride 0.05 w / V%, medium containing 0.05 w / v% potassium dihydrogen phosphate was adjusted to pH 7.0 with 2N aqueous sodium hydroxide solution, inulin (manufactured by Wako Pure Chemical Industries, Ltd.) 2 w / v%, agar ( (Wako Pure Chemical Industries, Ltd.) 3 w / v% was added, and the mixture was sterilized at 121 ° C. for 15 minutes.

The isolated strain was transplanted from the slant medium to 2 ml of the following liquid medium and cultured with shaking at 30 ° C. for 2 days. The supernatant obtained by centrifuging the culture solution is subjected to thin-layer chromatography by the following method, and the decomposition pattern of inulin in the culture solution is confirmed from the sugar spots generated by spray heating the coloring solution. This strain in which the production of tetraose was confirmed was obtained.
(Liquid medium)
The liquid medium is yeast extract (Becton Dickinson) 0.1 w / v%, sodium nitrate 0.2 w / v%, magnesium sulfate heptahydrate 0.05 w / v%, potassium chloride 0.05 w / v% The medium containing 0.05 w / v potassium dihydrogen phosphate was adjusted to pH 7.0 with a 2N aqueous sodium hydroxide solution, and 2 w / v% inulin (manufactured by Wako Pure Chemical Industries, Ltd.) was added. For 15 minutes.
(Thin layer chromatography)
Thin layer chromatography (TLC) was performed by the following method.
Using a thin layer chromatography plate (HP-K high performance silica gel, manufactured by Whatman), 2-propanol: 1-butanol: distilled water = 12: 3: 4 (V / V / V) was used as a developing solvent. used.
1.0 μl of the supernatant of each culture solution was spotted and developed by the ascending method, dried with a dryer, and then dried with a naphthoresorcinol sulfate reagent (M. Chebregzabher, S. Rufimi, B. Monabbi and M. Lato, J. Chromatogr. 127, 133-162 (1976)) and heated at 110 ° C. for 5 minutes to develop sugar.

<Microbacterium sp. Identification of YK-01 (FERM P-21593) Strain>
[Morphological properties]
One platinum loop of this strain was inoculated into the following agar medium sterilized, cultured at 30 ° C. for 2 days, and the morphological properties were confirmed by microscopic observation of the colonies. The results are shown in Tables 1 and 2.
(Agar medium)
The agar medium is yeast extract (Becton Dickinson) 0.1 w / v%, sodium nitrate 0.2 w / v%, magnesium sulfate heptahydrate 0.05 w / v%, potassium chloride 0.05 w / v% The medium containing 0.05 w / v% potassium dihydrogen phosphate was adjusted to pH 7.0 with 2N aqueous sodium hydroxide solution, and then agar (Wako Pure Chemical Industries, Ltd.) 1.5 w / v%, inulin ( Wako Pure Chemical Industries, Ltd.) 1.5 w / v% was added, and the mixture was sterilized at 121 ° C. for 15 minutes.

[Physiological properties]
The physiological properties of this strain were evaluated by known methods. The results are shown in Table 3.

[Base sequence]
The base sequence (partial sequence) of 16S rDNA of this strain was determined by a known method. Among the determined base sequences, the base sequence from the 5 ′ end (first) to the 1482th is shown in SEQ ID NO: 1.
This determined base sequence was subjected to homology search using MicroSeq Bacterial Full Gene Library.
As a result, the base sequence shown in SEQ ID NO: 1 of the present strain showed the highest homology to the 16S rDNA base sequence of Microbacterium trichothecenoliticum with a homology rate of 99.72%. In addition, as a result of homology search using the international nucleotide sequence database, the nucleotide sequence shown in SEQ ID NO: 1 of this strain is homologous to the 16S rDNA nucleotide sequence of DSM8608 strain, which is a reference strain of Microbacterium trichothesenolyticum. It showed a homology of 98.7%. Furthermore, as a result of searches using both databases, most of the 16S rDNA base sequence that showed high homology with the base sequence shown in SEQ ID NO: 1 of this strain was derived from strains belonging to the genus Microbacterium. It was.
As described above, this strain is a Gram-positive short gonococcus having no motility, the colony color was yellow, the catalase reaction was positive, and the oxidase reaction was negative. These properties are consistent with the general properties of Microbacterium trichothecenolyticum. On the other hand, the base sequences of 16S rDNA of both strains do not match, and this strain does not hydrolyze Tween 80 and does not produce hydrogen sulfide. It was different from the (Yokota A., Takeuchi M., Sakane T. and Weiss N.:Proposal of six new species in the genus Aureobacterium and transfer of Flavobacterium esteraromaticum Omelianski to the genus Aureobacterium as Aureobacterium esteraromaticum comb. Nov. Int. J. Syst. Bacteriol., 1993, 43, 555-564.).
From the above, it was determined that this strain belongs to the genus Microbacterium and is closely related to Microbacterium trichothesenolyticum, but is a different strain.

  The microbacterium sp. The YK-01 strain was entrusted to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, under the accession number FERM P-21593 dated June 9, 2008.

Microbacterium sp. The YK-01 strain produces inulinase. The inulinase acts on and degrades inulin to produce linear inulooligosaccharides, but the main products are inulotetraose and inulotriose, and hardly produces inulobiose and fructose. It is particularly characterized in that the production ratio of inulotetraose is high, and the ratio of inulotetraose / inurotriose (w / w, mass ratio) can be increased four times or more.
That is, the inulinase is
(A) It has physicochemical properties such that the amount of the product when acting on inulin is inulotetraose>inulotriose> inulobiose, fructose.
Thus, it has not been known that microorganisms belonging to the genus Microbacterium are known as microorganisms producing inulinase which produces linear inuro-oligosaccharides preferentially and in particular produces "inulotetraose" at a high ratio. .
Thus, the present invention provides for the first time a microorganism belonging to the genus Microbacteria in which the amount of a product when acting on inulin is inulotetraose>inulotriose> inulobiose, fructose. Such a microorganism is hereinafter referred to as a microorganism of the present invention.

Furthermore, microbacterium sp. Inulinase derived from the YK-01 strain has the following physicochemical properties.
(B) The optimum pH is 6 to 8.5, and shows the highest activity at about pH 7.5.
(C) The optimum temperature is 45 to 55 ° C, and the highest activity is exhibited at about 50 ° C.
(D) Stable at 50 ° C. or lower, and highest stability at about 30 ° C.
(E) The molecular weight measured by SDS-polyacrylamide gel electrophoresis is about 128,000.
The inulinase is presumed to be an exo type from the distribution of products obtained by acting on inulin.

The inulinase can be obtained by culturing the microorganism of the present invention and extracting a water-soluble component from the obtained culture.
For example, microbacterium sp. The YK-01 strain is preferably cultured in a medium containing inulin. And it is preferable that it is 0.5-5 w / v%, and, as for content of inulin in a culture medium, it is more preferable that it is 1-3 w / v%. The medium may be either a liquid medium or a solid medium.
The culture method may be appropriately selected from stationary culture, shaking culture, agitation culture, and the like according to the type of medium.
The culture temperature is preferably 20 to 40 ° C, and more preferably 25 to 35 ° C.
The culture time may be appropriately adjusted according to the culture temperature, but it is usually preferably 24 to 72 hours, more preferably about 36 to 60 hours.

  A water-soluble component may be extracted from the obtained culture by a known method, and the method is not particularly limited. Microbacterium sp. The YK-01 strain is considered to secrete the produced inulinase outside the cells, and the inulinase can be easily taken out without disrupting the cells. For example, a crude enzyme solution containing inulinase can be obtained by centrifuging the culture together with the cells and separating the supernatant.

The crude enzyme solution containing inulinase may be appropriately purified according to the purpose to obtain a purified enzyme solution. The purification method is not particularly limited, and any known method may be used alone or in combination of a plurality of types. Examples include purification methods using various chromatographies such as ion exchange chromatography, gel filtration chromatography, and reverse phase chromatography, and various separation membranes such as microfiltration membranes and ultrafiltration membranes.
Inulinase may be used after the purified enzyme solution is further subjected to freeze-drying or the like and taken out as a powder, or may be used as it is as a purified enzyme solution.

Those having inulinase activity, such as the microorganism of the present invention, culture thereof, or inulinase having the above physicochemical properties, are suitable for preparing an inulin degrading agent containing this as an active ingredient. In addition, as long as the said culture contains inulinase, the life and death of the microorganisms of this invention are not ask | required.
The inulin degrading agent may contain other components in addition to the effective component within a range not impeding the effect.

  When inulin oligosaccharides are produced by degrading inulin, for example, the microorganism of the present invention may be cultured in the presence of inulin, or inulinase having the physicochemical properties may be allowed to act on inulin. Thereby, as mentioned above, inulooligosaccharide which hardly contains inulobiose and fructose and contains a mixture of inulotetraose and inulotriose as a main component is obtained.

A culture obtained by culturing the microorganism of the present invention in the presence of inulin, or a reaction product obtained by allowing inulinase having the above physicochemical properties to act on inulin can be used as it is depending on the purpose. It may be used after being appropriately post-processed. Furthermore, it may be used in a liquid state, or may be used after being dried and used as a solid or powder.
The method of the post-treatment is not particularly limited, and may be performed by using a known technique such as neutralization, extraction, filtration, centrifugation, concentration, various chromatographies, purification using various separation membranes alone or in combination of plural kinds. . The drying method may be appropriately selected from known methods such as blow drying, heat drying, reduced pressure drying, freeze drying and the like.

  Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples. The unit of concentration “mg%” in the following examples represents the amount of mg of solute contained in 100 ml of the solution and is synonymous with the unit “mg / 100 ml”.

[Example 1]
<Microbacterium sp. Production of oligosaccharides by fermentation using YK-01 strain>
(Preparation of culture solution)
Yeast extract (Becton Dickinson) 0.1 w / v%, sodium nitrate 0.2 w / v%, magnesium sulfate heptahydrate 0.05 w / v%, potassium chloride 0.05 w / v%, diphosphate A medium containing 0.05 w / v% potassium hydrogen is adjusted to pH 7.0 with 2N aqueous sodium hydroxide solution, inulin (Wako Pure Chemical Industries, Ltd.) 2 w / v% is added, and sterilized at 121 ° C. for 15 minutes. Processed.
Next, this sterilized medium was inoculated with one platinum loop of this strain, and cultured with shaking at 30 ° C. for 2 days. The resulting culture solution was used as a preculture solution.
Furthermore, 10 μl of the preculture solution was added to 10 mL of the sterilized medium, and cultured with shaking at 30 ° C. for 24 hours to prepare a culture solution.
Subsequently, this culture solution was filtered using a DISMIC Cellulose acetate filter 0.45 μm (manufactured by Advantec) to obtain an analysis sample.
The obtained analytical sample was analyzed by high performance liquid chromatography under the following conditions.

Analysis conditions;
Column: Two OHpak SB-802.5 HQ (300 × 7.8 mm) (manufactured by Shodex) connected in series Column temperature: 40 ° C.
Moving layer: 0.1 M NaNO ₃ aqueous solution Detector: Differential refractometer (RID-10A, manufactured by Shimadzu Corporation)

(result of analysis)
As a result of analysis, the analysis sample contained fructose at a concentration of 13.89 mg%, inulobiose at 41.20 mg%, inurotriose at 220.70 mg%, and inulotetraose at 993.33 mg%. That is, the ratio of inulotetraose / inurotriose (w / w, mass ratio) was 4.5. Moreover, the production | generation rate of the saccharide | sugar of 4 or less saccharide | sugar from inulin calculated by following formula (1) was 58.7%.

[Production rate of sugars having 4 or less sugars (%)] = {[Fructose concentration (mg%)] + [Inulobiose concentration (mg%)] + [Inulotriose oligosaccharide concentration (mg%)] + [Inurotetra Aus-oligosaccharide concentration (mg%)]} / 2000 (mg%) × 100
... (1)

[Example 2]
<Microbacterium sp. Extraction of enzyme from YK-01 strain>
(Preparation of medium)
Yeast extract (Becton Dickinson) 0.1 w / v%, sodium nitrate 0.2 w / v%, magnesium sulfate heptahydrate 0.05 w / v%, potassium chloride 0.05 w / v%, diphosphate A medium containing 0.05 w / v% potassium hydrogen is adjusted to pH 7.0 with 2N aqueous sodium hydroxide solution, inulin (Wako Pure Chemical Industries, Ltd.) 2 w / v% is added, and sterilized at 121 ° C. for 15 minutes. Processed.

(Preparation of crude enzyme solution)
One platinum loop of this strain was ingested into the sterilized medium, and cultured with shaking at 30 ° C. for 2 days.
After completion of the shaking culture, 300 ml of the culture solution was centrifuged at 8000 rpm and 4 ° C. for 20 minutes using a centrifuge (himac CF15D, manufactured by Hitachi Koki Co., Ltd.), and the resulting supernatant was dialyzed into cellulose for dialysis. Using a tube (manufactured by Viscose Companies, Inc.), dialysis treatment was performed overnight against 0.01 M phosphate buffer (pH 7.0). A sample obtained by this dialysis treatment was used as a crude enzyme solution. And the enzyme activity was measured by the below-mentioned method about the obtained crude enzyme liquid.

(Preparation of purified enzyme solution (I))
The crude enzyme solution was poured from the upper end of an ion exchange chromatography column (SuperQ-Toyopearl, φ16 mm × 240 mm, manufactured by Tosoh Corporation) equilibrated with 0.01 M phosphate buffer (pH 7.0) to adsorb the components. I let you.
The enzyme was then eluted by a concentration gradient method using 0.01 M phosphate buffer (pH 7.0) that linearly changes the sodium chloride concentration from 0 M to 0.5 M. At this time, the eluted protein was detected by measuring the absorbance at 280 nm of the eluate using a digital beam spectrophotometer UV-1200 type (manufactured by Shimadzu Corporation). The measurement results of the absorbance of each fraction (fraction) are shown in FIG. 1 together with the concentration of sodium chloride.
Furthermore, the enzyme activity of each of the obtained fractions was measured by the method described later to obtain a purified enzyme solution (I) that was an active fraction. The measurement results of absorbance at 500 nm at this time are also shown in FIG. From the measurement results of absorbance at 500 nm in FIG. 1, the active fraction showing sufficient enzyme activity was fraction No. 28 to 32, and these were designated as purified enzyme solution (I).

(Preparation of purified enzyme solution (II))
The purified enzyme solution (I) thus obtained was poured from the upper end of a gel filtration chromatography column (HiTrapQ, 5 ml, manufactured by Pharmacia) equilibrated with 0.01 M phosphate buffer (pH 7.0) to adsorb the components. I let you.
The enzyme was then eluted by a concentration gradient method using 0.01 M phosphate buffer (pH 7.0) that linearly changes the sodium chloride concentration from 0.3 M to 0.5 M. At this time, the eluted protein was detected by the same method as in the preparation of the purified enzyme solution (I). The measurement results of the absorbance of each fraction (fraction) are shown in FIG. 2 together with the concentration of sodium chloride.
Furthermore, the enzyme activity of each of the obtained fractions was measured by the same method as in the case of the purified enzyme solution (I) to obtain a purified enzyme solution (II) that was an active fraction. The measurement results of absorbance at 500 nm at this time are also shown in FIG. From the measurement results of absorbance at 500 nm in FIG. 2, the active fraction showing sufficient enzyme activity was fraction No. 14 to 17, and these were designated as purified enzyme solution (II).

(Preparation of purified enzyme solution (III))
The obtained purified enzyme solution (II) was further concentrated to 5 ml using an ultrafiltration unit (Molkat L, manufactured by Millipore), and 0.5 g of sucrose was added.
This was poured from the upper end of a gel filtration chromatography column (Sephacryl S-200HR, φ26 mm × 550 mm, manufactured by Pharmacia) equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 0.2 M sodium chloride. The components were adsorbed.
Subsequently, the enzyme was eluted with 0.01 M phosphate buffer (pH 7.0). At this time, the eluted protein was detected by the same method as in the preparation of the purified enzyme solution (I). The measurement results of the absorbance of each fraction (fraction) are shown in FIG.
Furthermore, the enzyme activity of each of the obtained fractions was measured in the same manner as in the case of the purified enzyme solution (I) to obtain a purified enzyme solution (III) as an active fraction. The measurement results of absorbance at 500 nm at this time are also shown in FIG. From the measurement result of absorbance at 500 nm in FIG. 3, the active fraction showing sufficient enzyme activity was fraction No. 50 to 57, and these were designated as purified enzyme solution (III).

(Quantification of total protein)
By measuring the absorbance at 595 nm of the crude enzyme solution, purified enzyme solution (I), purified enzyme solution (II) and purified enzyme solution (III) by Bradford method using bovine serum γ-globulin as a standard protein. The total protein amount (mg) in each enzyme solution was quantified. The results are shown in Table 4.

(Measurement of enzyme activity, total activity, specific activity and yield)
For each fraction (enzyme solution) in the preparation step of the crude enzyme solution and the purified enzyme solution (I), purified enzyme solution (II) and purified enzyme solution (III), reducing sugars produced by the enzyme reaction are 3, Enzyme activity was measured by measuring by 5-Dinitrosalic acid method (DNS method). Specifically, it is as follows.
After adding 0.4 ml of 0.025M phosphate buffer to 0.1 ml of each enzyme solution to be measured and incubating at 30 ° C. for 2 minutes, 0.5 ml of 2.0 w / v% inulin solution is added at 30 ° C. The reaction was allowed for 30 minutes.
Next, 1.0 ml of DNS reagent was added to stop the enzyme reaction, and the reaction solution was colored by boiling for 5 minutes.
Then, after cooling the colored reaction liquid with water, 8.0 ml of distilled water is added and diluted to prepare a measurement sample, and a digital beam spectrophotometer UV-1200 type (manufactured by Shimadzu Corporation) is used. Then, the absorbance at 500 nm of the measurement sample was measured.
A standard curve was prepared using a fructose solution having a concentration of 0 to 2.7 μmol / ml as a standard sample. From the measured absorbance value and the following formula (2), 1 mL of each enzyme solution The enzyme activity was calculated. The unit “unit” of enzyme activity shown below indicates the enzyme activity corresponding to the reducing ability to reduce 1 μmol of fructose per minute.
And from the measured enzyme activity, the total activity was calculated by the following formula (3), the specific activity was calculated by the following formula (4), and the yield was calculated by the following formula (5). The results are shown in Table 4.

[Enzyme activity per ml of enzyme solution (unit / ml)] = {[Absorbance of measurement sample] − [Absorbance of reaction solution at 0 min reaction time]} / [Slope of calibration curve] × 10/30
... (2)
[Total activity (unit)] = [Enzyme activity per ml of enzyme solution (unit / ml)] × [Enzyme solution volume (ml)]
... (3)
[Specific activity (unit / mg)] = [total activity (unit)] / [protein amount (mg)]
... (4)
[Yield (%)] = [Total activity of purified enzyme solution] / [Total activity of crude enzyme solution] × 100
... (5)

(Measurement of molecular weight of purified enzyme)
The molecular weight of the enzyme (inulinase) contained in the purified enzyme solution (III) was measured by the polyacrylamide gel electrophoresis method shown below.
That is, 0.01 ml of SDS electrophoresis sample buffer solution was added to 0.01 ml of purified enzyme solution (III) and treated at 100 ° C. for 2 minutes, and subjected to electrophoresis at a constant current of 20 mA. Electrophoresis was performed using a gel having an acrylamide concentration of 10% (polyacrylamide precast gel PAGEL, manufactured by ATTO Corporation) according to the method of Laemmli et al. (See UK Laemmli, Nature, 227, 680-685 (1970)). I went there.
As a result, the enzyme contained in the purified enzyme solution (III) showed a single band. When the molecular weight was determined by comparing the mobility with that of the molecular weight marker protein, the estimated molecular weight was 128,000. The electrophoresis image at this time is shown in FIG. In FIG. 4, the band in the left lane is the molecular weight marker protein, and the band in the right lane indicated by the arrow is the enzyme contained in the purified enzyme solution (III).

[Example 3]
<Microbacterium sp. Production of oligosaccharides using inulinase derived from YK-01 strain>
(Production of oligosaccharide by purified enzyme solution (III))
Add 0.1 ml of 5.0 w / v% inulin solution to 0.4 ml of purified enzyme solution (III), and at 30 ° C., 0 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes After each reaction, the reaction solution was boiled for 2 minutes to stop the reaction.

(Analysis of oligosaccharides)
0.3 ml of acetonitrile was added to 0.2 ml of each reaction solution after the reaction was stopped to precipitate unreacted inulin. Subsequently, this reaction solution was centrifuged at 10,000 rpm for 10 minutes using a centrifugal separator (himac CF15D, manufactured by Hitachi Koki Co., Ltd.), and 5 μl of the obtained supernatant was used as an analysis sample. The obtained analytical sample was analyzed by high performance liquid chromatography (HPLC) under the following conditions. The analysis results are shown in Table 5. In Table 5, the “concentration” of inulotriose and inulotetraose was quantified using a calibration curve prepared with a standard sample. Further, the “production rate” of inulotriose and inulotetraose was calculated by the following formula (6).

HPLC analysis conditions;
Column: CAPCELL PAK NH2 SG80 S-5 μm (φ4.6 mm × 250 mm) (manufactured by Shiseido Co., Ltd.)
Column temperature: normal temperature Moving bed: acetonitrile / water = 65/35 (v / v)
Detector: differential refractometer (RID-6A, manufactured by Shimadzu Corporation)

[Production rate (%)] = {[Inurotriose concentration (mg%)] + [Inurotetraose concentration (mg%)]} / 400 (mg%) × 100
... (6)

  Moreover, each said reaction liquid was analyzed by the thin layer chromatography (TLC) on the following conditions. A TLC development image is shown in FIG. In FIG. 5, “F” is a spot corresponding to fructose, “F2” is inulobiose, “F3” is inurotriose, and “F4” is inrotetraose. “St” is a standard sample containing fructose, inulobiose, inulotriose and inulotetraose. Furthermore, “0” to “120” indicate analysis samples of the corresponding reaction time (minutes).

TLC analysis conditions;
On a silica gel plate (HP-K high performance silica gel, manufactured by Whatman Japan Co., Ltd.), 1.0 μl of each reaction solution was spotted, and 2-propanol / 1-butanol / water = 12/3/4 as a developing solvent. (V / v / v) was developed by the ascending method, dried with a dryer, sprayed with a naphthoresorcinol sulfate reagent, and heated at 110 ° C. for 5 minutes to develop sugars.

(result of analysis)
As a result of the HPLC analysis, as is clear from Table 5, it was confirmed that the analysis sample contained inulotriose and inulotetraose as main products and did not contain fructose and inulobiose. The amount of inulotetraose was almost constant after the reaction time of 60 minutes. In addition, for example, at a reaction time of 60 minutes, it was confirmed that the ratio of inulotetraose was extremely high among the products of the enzyme reaction, such as inurotetraose was about 6 times the amount of inulotriose.

  Further, as is apparent from FIG. 5 as a result of TLC analysis, spots corresponding to fructose and inulobiose were not detected in the above reaction solutions except for a reaction time of 0 minutes, and spots corresponding to inulotriose and inulotetraose were detected. was detected. Furthermore, the concentration of the spot corresponding to inulotetraose was significantly higher than that of the spot corresponding to inulotriose. That is, each of the reaction liquids except for the reaction time of 0 minutes contains inulotriose and inulotetraose as main products, does not contain fructose and inulobiose, and has a much higher ratio of inulotriose than inulotriose. Was obtained, and the results supporting the HPLC analysis results were obtained.

[Example 4]
<Confirmation of pH dependence of enzyme activity>
To 0.1 ml of purified enzyme solution (III), 0.3 ml of distilled water and 0.1 ml of glycine-Tris-citrate buffer adjusted at 0.5 intervals between pH 4.0 and 10.0. Thirteen kinds of added samples were prepared. After incubating the samples at 30 ° C. for 2 minutes, 0.5 ml of 2.0 w / v% inulin solution was added and reacted at 30 ° C. for 30 minutes. Subsequently, the enzyme activity was measured by the DNS method. The results are shown in FIG.
As is clear from FIG. 6, it was confirmed that the purified enzyme solution (III) had the highest activity at about pH 7.5.

[Example 5]
<Confirmation of temperature dependence of enzyme activity>
Nine samples were prepared by adding 0.4 ml of 0.025 M phosphate buffer (pH 7.0) to 0.1 ml of purified enzyme solution (III), and these samples were set at 5 ° C. intervals between 20-60 ° C. After incubation for 2 minutes at each of the 9 temperatures, 0.5 ml of a 2.0 w / v% inulin solution was added and reacted at the same temperature of 20-60 ° C. for 30 minutes. Subsequently, the enzyme activity was measured by the DNS method. The results are shown in FIG. In FIG. 7, “◯” is data of this embodiment.
As is clear from FIG. 7, it was confirmed that the purified enzyme solution (III) had the highest activity at about 50 ° C.

[Example 6]
<Confirmation of temperature stability of enzyme activity>
Nine samples were prepared by adding 0.4 ml of 0.025 M phosphate buffer (pH 7.0) to 0.1 ml of purified enzyme solution (III), and these samples were set at 5 ° C. intervals between 20-60 ° C. Each of the nine temperatures was incubated for 10 minutes and then ice-cooled. Next, 0.5 ml of 2.0 w / v% inulin solution was added thereto, and reacted at 30 ° C. for 30 minutes. Subsequently, the enzyme activity was measured by the DNS method. The results are shown in FIG. In FIG. 7, “●” is the data of this embodiment.
As is clear from FIG. 7, it was confirmed that the purified enzyme solution (III) was stable up to about 50 ° C.

  The present invention can be used for manufacturing various foods and drinks including functional foods.

4 is a graph showing the results of measuring the absorbance of each fraction by ion exchange column chromatography in the purified enzyme solution (I) preparation step of Example 2. It is a graph which shows the light absorbency measurement result of each fraction by the gel filtration column chromatography in the refinement | purification enzyme liquid (II) preparation process of Example 2. FIG. It is a graph which shows the light absorbency measurement result of each fraction by the gel filtration column chromatography in the refinement | purification enzyme liquid (III) preparation process of Example 2. FIG. 2 is an electrophoresis image of inulinase in the purified enzyme solution (III) in Example 2. 4 is a TLC developed image at the time of analysis of inulooligosaccharide of Example 3. FIG. It is a graph which shows the data of the pH dependence of the enzyme activity of the refinement | purification enzyme liquid (III) in Example 4. FIG. It is a graph which respectively shows the temperature dependence data of the enzyme activity of the purified enzyme liquid (III) in Example 5, and the temperature stability data of the enzyme activity of the purified enzyme liquid (III) in Example 6.

Claims (9)

Microbacterium sp. ( Microbacterium sp.) YK-01 (FERM P-21593) strain.   A method for producing inulooligosaccharide, comprising a step of culturing the microorganism according to claim 1 in the presence of inulin. Microbacterium sp. ( Microbacterium sp.) Inulinase derived from YK-01 (FERM P-21593) strain. The inulinase according to claim 3, which has the following physicochemical properties.
(1) Acts on inulin to produce a linear inulooligosaccharide, and the amount of the product at this time is inurotetraose>inurotriose> inurobiose, fructose.
(2) The optimum pH is 6 to 8.5.
(3) The optimum temperature is 45 to 55 ° C.
(4) Stable at 50 ° C. or lower .   The inulinase according to claim 3 or 4, wherein the amount of inulotetraose produced is 4 times or more (mass ratio) of the amount of inulotriose produced.   The manufacturing method of inolo-oligosaccharide which makes the inulinase as described in any one of Claims 3-5 act on inulin.   An inulin degrading agent comprising the microorganism according to claim 1 as an active ingredient.   An inulin degrading agent comprising the inulinase according to any one of claims 3 to 5 as an active ingredient.   The manufacturing method of inulinase which has the process of culturing the microorganisms of Claim 1, and extracting a water-soluble component from the obtained culture.

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