JP5467251B2 - Method for quantifying β-1,3-1,6-glucan - Google Patents

Description

  The present invention relates to a method for quantifying β-1,3-1,6-glucan, a kit for quantifying β-1,3-1,6-glucan that can be used in the method, and a bacterium useful for the method The present invention relates to a microorganism that produces an in vitro enzyme.

  Cells produce structural polysaccharides as well as storage polysaccharides that serve as energy, such as glycogen and starch. Structural polysaccharides are polysaccharides that are components of cell membranes and cell walls that protect the cell surface, and various types such as glucan, mannan, glycoprotein, and chitin have been confirmed.

  In recent years, among such structural polysaccharides, those produced by basidiomycetes have attracted attention because they have excellent physiological activity.

  Basidiomycetes are higher fungi that produce basidiomycetous spores, including matsutake mushrooms, maiko, and himematsu mushrooms called agaricus. The structural polysaccharides produced by these basidiomycetes are effective as a dietary fiber, exhibiting antitumor, antibacterial, blood glucose and serum cholesterol lowering effects, and are effective in preventing lifestyle-related diseases such as diabetes and cancer. Some research results have been reported.

  Among the structural polysaccharides produced by basidiomycetes, β-1,3-1,6-glucan having a β-1,3-glucoside bond in the main chain and a β-1,6-glucoside bond in the side chain, In particular, it exhibits characteristic physiological activity (Non-patent Document 1).

  However, although structural polysaccharides have excellent physiological activity, they are very difficult to extract because they exist in cell walls and the like. In addition, even if extracted, the extract is insoluble or hardly soluble in water, has a unique color and unpleasant odor, has unstable quality and is very expensive.

  On the other hand, among Aureobasidium pullulans seeds called black yeast, those that release β-1,3-1,6-glucan outside the cells under certain conditions have been found. And the culture solution of the said microbe containing (beta) -1,3-1,6-glucan is tasteless and odorless, the quality is stable, and can be manufactured cheaply. Therefore, the way of utilizing β-1,3-1,6-glucan obtained from the bacterium as a functional food is being opened.

  However, when a culture solution containing β-1,3-1,6-glucan or the like is used as a raw material for food, it is necessary to evaluate the quality by measuring the content thereof, but an accurate quantitative method. Does not yet exist.

  For example, the β-glucan quantification method carried out by the Japan Food Analysis Center is a non-specific hydrolysis of all glucoside bonds with sulfuric acid after enzymatic treatment with amylase, amyloglucosidase, etc. It is to be quantified. However, this method cannot distinguish β-1,3-1,6-glucan from other β-glucans. Furthermore, glucose derived from heteroglucan containing monosaccharides other than glucose is decomposed and measured. Therefore, this method is not suitable as a method for quantifying β-1,3-1,6-glucan.

  In addition, since the generally used β-glucan quantification kit uses a protein factor that recognizes and binds to a continuous β-1,3-glucoside bond, β-glucan contained in the sample is used. The larger the side chain ratio in 1,3-glucan, the greater the error in the measured value.

By the way, as described in Non-Patent Documents 2 to 4, in the laboratory to which the present inventors belong, β-1,3-glucanase and β-1,6-glucanase derived from microorganisms belonging to the genus Ceriporiopsis isolated from soil are respectively β It has been found that it has high reactivity with respect to -1,3-glucoside bonds and β-1,6-glucoside bonds. Therefore, it is considered that the β-1,3-1,6-glucan content in the sample can be accurately measured using these enzymes.
Toshio Miyazaki, “The Structure and Physiological Activity of Polysaccharides” Asakura Shoten (1990) Hirohiko Noda, “Department of Agricultural Chemistry, Graduate School of Agriculture, Kochi University, Master's Thesis” (1994) Hideo Nakajima, “Master's Thesis, Department of Fermentation and Brewing Studies, Graduate School of Agriculture, Kochi University” (1993) Hironori Ikegami, “Master's thesis, Applied Microbiology Laboratory, Department of Bioresource Science, Graduate School of Agriculture, Kochi University” (2004)

  As described above, the laboratory to which the present inventors belong has found a highly reactive β-1,3-glucanase and β-1,6-glucanase-producing microorganism belonging to the genus Cerioporopsis, and uses these enzymes. Therefore, it is conceivable to accurately measure the β-1,3-1,6-glucan content in the sample.

  However, in order to completely purify the enzyme, it is necessary to repeat various chromatographies, and it takes time and cost. Therefore, the quantification method using the purified enzyme is definitely not practical for industrial implementation. hard. Therefore, it can be considered that the enzyme released outside the cells is used for quantification without being completely purified. However, since the microorganisms belonging to the genus Ceriporiopsis have a very strong α-glucosidase activity, α-glucosidase is mixed in the crudely purified enzyme. Therefore, if α-glucan is present in the sample, it is degraded to glucose. , Β-1,3-1,6-glucan content cannot be measured accurately. Therefore, in order to quantify β-1,3-1,6-glucan in a sample using a crudely purified enzyme derived from a microorganism belonging to the genus Ceriporiopsis, the α-glucan is first decomposed and removed by α-glucosidase or the like. It is necessary to decompose β-glucan with the crudely purified β-1,3-glucanase and β-1,6-glucanase and to quantify the resulting glucose. Such a method is time consuming and laborious and inefficient.

  Therefore, an object of the present invention is to provide a method capable of accurately and efficiently quantifying β-1,3-1,6-glucan without using a completely purified enzyme. Another object of the present invention is to provide a kit that can be used in the method and a microorganism that is useful for the method.

  The inventors of the present invention have made extensive studies in order to solve the above-mentioned problems. As a result, they have the ability to produce β-1,3-glucanase and have β-glucanase activity other than β-1,3-glucanase and α-glucosidase. It is derived from an inoculum of Mitsaria chitosanitabida that does not show activity, and an inoculum of Streptomyces omyaensis that has the ability to produce β-1,6-glucanase and does not show β-glucanase activity other than β-1,6-glucanase and α-glucosidase activity If an extracellular enzyme solution is used, β-1,3-1,6-glucan can be accurately and efficiently quantified regardless of the presence of α-glucan, other β-glucan and heteroglucan in the sample. By demonstrating, the present invention has been completed.

  The method for quantifying β-1,3-1,6-glucan according to the present invention has a β-1,3-glucanase producing ability in a polysaccharide-containing sample and a β other than β-1,3-glucanase. -An extracellular enzyme solution derived from an inoculum of Mitsubishi chitosanitabida that does not exhibit glucanase activity and α-glucosidase activity, and β- other than β-1,6-glucanase and having β-1,6-glucanase producing ability A step of allowing an extracellular enzyme solution derived from Streptomyces omiyaensis inoculum that does not exhibit glucanase activity and α-glucosidase activity; and a step of measuring the amount of glucose produced.

  In the method of the present invention, the above M.I. As an inoculum of chitosanitabida Chitosanitabida H1 strain (NITE AP-629) is preferable. S. omiyaensis The omiyaensis SY26 strain (NITE AP-616) is preferred. The excellent effect of the above strain has been demonstrated by experiments.

  In the above-mentioned method of the present invention, the polysaccharide-containing sample is subjected to the above M.M. an extracellular enzyme solution derived from an inoculum of chitosanitabida It is preferable to simultaneously act on an extracellular enzyme solution derived from an omiyaensis inoculum. It is preferable from the viewpoint of efficiency that the two types of extracellular enzyme solutions simultaneously act on a polysaccharide-containing sample. However, β-1,3-glucanase and β-1,6-glucanase contained in each enzyme solution are preferable. The optimum temperature is different. However, in the temperature range of 20 ° C. or higher and 45 ° C. or lower, both enzymes can exert their effects well, and β-1,3-1,6-glucan contained in the polysaccharide-containing sample is efficiently used. Can be disassembled.

  The kit for quantifying β-1,3-1,6-glucan according to the present invention has the ability to produce β-1,3-glucanase and has β-glucanase activity and α other than β-1,3-glucanase. -M. not exhibiting glucosidase activity. extracellular enzyme solution derived from chitosanitabida inoculum or dried product thereof; has ability to produce β-1,6-glucanase and does not exhibit β-glucanase activity and α-glucosidase activity other than β-1,6-glucanase S. an extracellular enzyme solution derived from omyaensis inoculum or a dried product thereof; and a reagent for measuring the amount of glucose.

  The present invention also relates to M.I. chitosanitabida H1 strain (NITE AP-629) and S. The present invention relates to omiyaensis SY26 strain (NITE AP-616).

  Conventionally, in order to accurately measure β-1,3-1,6-glucan in a polysaccharide-containing sample containing α-glucan, α-glucan is first decomposed and removed with amyloglucosidase or the like, and then β-glucan. The amount had to be measured, and it took time and effort. Moreover, with the conventional method, it has been difficult to accurately measure β-1,3-1,6-glucan in a sample containing other β-glucan. However, according to the method of the present invention, it is not necessary to decompose α-glucan in advance without using a completely purified enzyme, and β-1,3-1,6-glucan is accurately and efficiently quantified. be able to. Moreover, the quantification kit according to the present invention has the same effect. Further, the two strains according to the present invention are very suitable for the quantification method and quantification kit according to the present invention. Therefore, the present invention is extremely useful industrially as an index that can be used to evaluate the quality of a sample containing β-1,3-1,6-glucan, which has attracted attention for its excellent physiological activity.

  The method for quantifying β-1,3-1,6-glucan according to the present invention has a β-1,3-glucanase producing ability in a polysaccharide-containing sample and a β other than β-1,3-glucanase. -M. not exhibiting glucanase activity and α-glucosidase activity. an extracellular enzyme solution derived from an inoculum of Chitosanitabida, and S. cerevisiae having an ability to produce β-1,6-glucanase and not showing β-glucanase activity and α-glucosidase activity other than β-1,6-glucanase. a step of allowing an extracellular enzyme solution derived from Omiyaensis inoculum to act; and a step of measuring the amount of glucose produced.

  The polysaccharide-containing sample which is the measurement target of the method of the present invention contains or is considered to contain β-1,3-1,6-glucan, and particularly if the content is to be measured. The type is not limited. For example, yeast cells, culture media, dried products, extracts, purified products, suspensions, solutions; basidiomycetes and seaweeds themselves, dried products, extracts, ground products, products, suspensions A turbid liquid, a solution, etc. can be mentioned.

  In the method of the present invention, the polysaccharide-containing sample has an ability to produce β-1,3-glucanase and does not exhibit β-glucanase activity and α-glucosidase activity other than β-1,3-glucanase. an extracellular enzyme solution derived from an inoculum of Chitosanitabida, and S. cerevisiae having an ability to produce β-1,6-glucanase and not showing β-glucanase activity and α-glucosidase activity other than β-1,6-glucanase. An extracellular enzyme solution derived from omiyaensis inoculum is allowed to act. While these extracellular enzyme solutions show each β-glucanase activity, they do not degrade α-glucan or other β-glucan. Therefore, if these extracellular enzyme solutions are used, β-1,3-1,6-glucan in the polysaccharide-containing sample can be specifically decomposed to glucose without purifying the enzyme.

  The bacterium is not limited to those producing no β-glucanase other than α-glucosidase, β-1,3-glucanase and β-1,6-glucanase, and β-1,3-1,6 -Those that release these enzyme activities to the extent that they do not inhibit the accurate quantification of glucan, or those that produce these enzymes that can be easily separated and removed from β-1,3-1,6-glucanase Those that are released outside the body are also included. Specifically, α-glucosidase having a specific activity of 0.05 unit / mg or less, or β-glucanase other than β-1,3-glucanase and β-1,6-glucanase is released into the extracellular enzyme solution. Fungus is also included. However, those that do not substantially exhibit these enzyme activities are suitable as the above-mentioned bacteria. The term “substantial” as used herein means that the activity of these enzymes in the extracellular enzyme solution is below the detection limit in the measurement methods described in the Examples below.

  Examples of the extracellular enzyme solution include the above M.I. chitosanitabida inoculum and S. An enzyme solution obtained from a liquid culture solution of Omiyaensis sp., β-1,3-1, such as β-glucanase other than glucose, α-glucosidase, β-1,3-glucanase and β-1,6-glucanase It does not contain a component that inhibits accurate quantification of 6-glucan. That is, low molecular weight substances such as glucose can be removed by adding ammonium sulfate to the above liquid culture solution of the bacterium to separate the precipitated protein component or by dialysis. In addition, the extracellular enzyme solution of the above-mentioned bacterium may show α-glucosidase activity and the like, but the activity is very weak. Therefore, α-glucosidase and the like can be easily removed by adjusting the concentration of ammonium sulfate. .

  Specifically, the extracellular enzyme solution can be prepared as follows, for example. First, solid components such as bacterial cells are removed by subjecting a liquid culture solution obtained by culturing the above-mentioned bacteria to centrifugation or filtration. It is preferable to add a protease inhibitor such as PMSF (phenylmethylsulfonyl fluoride) to the obtained liquid. Next, the protein component is separated by a conventional method. For example, after concentrating the obtained liquid to some extent, a buffer solution is added, and then the precipitated protein component is separated by adding ammonium sulfate while cooling. At this time, it is efficient to preliminarily determine the ammonium sulfate concentration at which β-1,3-glucanase and β-1,6-glucanase are sufficiently precipitated and α-glucosidase is not precipitated by preliminary experiments. . The desired β-glucanase may be further purified from the obtained protein component to some extent by a simple method such as dialysis. However, the M.M. chitosanitabida inoculum and S. cerevisiae Since the omyaensis inoculum does not release α-glucosidase or the like out of the cells, or the amount released is extremely small, it is not necessary to purify the enzyme by a method that is not industrially suitable, such as chromatography.

  The extracellular enzyme solution may be once dried for storage or the like and used as a solution at the time of use. However, the β-1,3-glucanase and β-1,6-glucanase are inactivated at 70 ° C. and 50 ° C., respectively. Therefore, it is preferable not to raise the temperature to 70 ° C. or 50 ° C. or higher when the enzyme solution is concentrated or dried.

  In the present invention, the above-mentioned M.I. an extracellular enzyme solution derived from an inoculum of chitosanitabida and the aforementioned S. An extracellular enzyme solution derived from omiyaensis inoculum is allowed to act, but these enzyme solutions may be acted on simultaneously or sequentially. Considering the efficiency, it is preferable to act simultaneously.

  The concentration of β-1,3-glucanase and β-1,6-glucanase in the extracellular enzyme solution may be adjusted as appropriate according to the polysaccharide concentration in the reaction mixture. For example, the reaction mixture As the activity of each β-glucanase contained therein, when laminarin, which is β-1,3-1,6-glucan, is used as a substrate, about 1.0 unit / ml or more and about 5.0 unit / ml or less, β- When pasturan which is 1,6-glucan is used as a substrate, it is preferably about 0.1 unit / ml or more and 3.0 unit / ml or less. The unit in the present invention is defined as a unit of the amount of enzyme that purifies 1 μmol of reducing sugar per hour at 30 ° C.

  The pH of the exoenzyme solution may be adjusted as appropriate, but in the experiments by the present inventors, good results have been obtained at pH 6.0, so that the solvent is about pH 5.0 or more and 7.5 or less. These buffers are preferred.

  What is necessary is just to adjust the reaction temperature in the said enzyme reaction suitably. For example, M.M. The optimum temperature of β-1,3-glucanase derived from the chitosanitabida H1 strain is about 40 ° C., and if it is about 30 ° C. or more and about 55 ° C. or less, about 60% of the activity is exhibited. S. The optimum temperature of β-1,6-glucanase derived from omiyaensis SY26 strain is about 30 ° C., and if it is about 20 ° C. or more and about 45 ° C. or less, it shows about 60% of the activity. Therefore, when each extracellular enzyme solution is allowed to act on a polysaccharide-containing sample sequentially, the reaction temperatures are adjusted to about 30 ° C. or more and about 55 ° C. or less, and about 20 ° C. or more and about 45 ° C. or less, respectively. It is preferable to do. In addition, the above M.I. an extracellular enzyme solution derived from an inoculum of chitosanitabida and the aforementioned S. In the case where an extracellular enzyme solution derived from an Omiyaensis inoculum is simultaneously applied to a polysaccharide-containing sample, the reaction temperature is preferably 20 ° C. or higher and 45 ° C. or lower in order to make both glucanases function effectively. More preferably, the reaction is performed at 35 ° C. or higher and 40 ° C. or lower.

  The reaction time of the enzyme reaction is a time during which β-1,3-1,6-glucan present in the polysaccharide-containing sample is sufficiently decomposed to glucose. That is, the reaction time includes the concentration of β-1,3-1,6-glucan contained in the reaction mixture, β-1,3-glucanase and β-1,6-glucanase contained in the extracellular enzyme solution. Specifically, the amount of glucose produced is measured over time while adding the extracellular enzyme solution several times every few hours into the reaction mixture in a preliminary experiment. The time when it can no longer be confirmed that glucose is not newly produced by the enzyme reaction may be used. Usually, it is preferable to react overnight to sufficiently degrade β-1,3-1,6-glucan. However, depending on the amount of the extracellular enzyme solution, the reaction time may be as short as 2 hours or more and 10 hours or less. It is also possible to terminate the reaction.

M. used in the present invention. As the inoculum of chitosanitabida, Chitosanitabida H1 strain (hereinafter simply referred to as “H1 strain”) is preferred. The H1 strain is deposited with the depository as follows.
(I) Name and address of the receiving organization Name: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center Address: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan
(Ii) Date of receipt: August 12, 2008 (iii) Receipt number: NITE AP-629

  When the H1 strain is grown on Wicherham agar medium, a white colony is formed at the initial stage of culture, and gradually turns from milky white to yellow as it matures. The morphological features observed using an optical microscope are as follows. That is, it is a gram-negative bacillus having a width of 0.7 to 1.0 μm and a length of 1.0 to 4.0 μm, and exhibits motility and catalase activity. The above morphological features are described in M.M. This applies to bacteria belonging to the chitosanitabida species. The H1 strain is aerobic, grows well at 30 ° C., and grows well in neutral (pH 6.8-7.0) medium. Therefore, the temperature at which the H1 strain is cultured is preferably about 25 ° C. or more and 35 ° C. or less, and is preferably cultured in the vicinity of neutrality.

  Hitherto, no bacteria have been known that release β-1,3-glucanase outside the cells but do not substantially release other β-glucanase or α-glucosidase. Therefore, the H1 strain according to the present invention is novel.

S. used in the present invention. Examples of the Omiyaensis sp. The omiyaensis SY26 strain (hereinafter simply referred to as “SY26 strain”) is preferred. The SY26 strain is deposited with the depository as follows.
(I) Name and address of the receiving organization Name: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center Address: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan
(Ii) Receipt date: July 29, 2008 (iii) Receipt number: NITE AP-616

  When the SY26 strain is grown on Wicherham agar medium, a white colony is formed at the initial stage of the culture, and gradually changes from a beige to a gray colony as it matures. Morphological features observed using an optical microscope are as follows. That is, colorless and transparent basic hyphae are observed in the medium substrate, the diameter is 0.8 to 1.5 μm, the hyphae are linearly branched, and when they mature, their tips are cut to form spores. To do. The spore surface has no wrinkles and is oval or oblong, and has a diameter of 0.5 to 1.0 μm and a length of 1 to 4 μm. Furthermore, in this bacterium grown in the medium, spore sac, motile spore and segmental spore are not confirmed, and melanin production is negative. The above morphological features apply to basidiomycetes belonging to the species of Streptomyces omiyaensis.

  The SY26 strain was aerobic and grew well at 30 ° C., and grew well in a neutral (pH 6.8 to pH 7.0) medium. Therefore, the temperature at which the SY26 strain is cultured is preferably about 25 ° C. or more and 40 ° C. or less, and culture in the vicinity of neutrality is suitable.

  Hitherto, no bacteria have been known that release β-1,6-glucanase outside the cells but do not substantially release other β-glucanase or α-glucosidase. Therefore, the SY26 strain according to the present invention is novel.

  After the reaction with the extracellular enzyme solution, the amount of glucose produced by the action of β-1,3-glucanase and β-1,6-glucanase is measured.

  The β-1,3-glucanase and β-1,6-glucanase according to the present invention specifically cleave β-1,3-glucoside bond and β-1,6-glucoside bond, respectively. Therefore, since β-1,3-1,6-glucan present in the polysaccharide-containing sample is selectively decomposed to glucose by the extracellular enzyme solution, β-1 , 3-1,6-glucan can be grasped.

Specifically, the amount of glucose produced is determined by a method for measuring free reducing sugars such as the Somogyi-Nelson method (see M. Somogyi, J. Biol. Chem., 195, p. 19 (1952)). It is possible to measure. It can also be measured by enzymatic methods or HPLC. Enzymatic methods include G6PDH-HK method (F-kit, manufactured by Roche) in which glucose-6-phosphate dehydrogenase and hexokinase are combined, and mutarotase / GOD method in which mutarotase and glucose oxidase are combined (glucose CII-Test Wako, Wako). Further, as columns for HPLC analysis, there are many columns suitable for analyzing glucose, such as TSKgel Sugar AXG (manufactured by Tosoh Corporation) and Sugar-Pak ^™ I (manufactured by Waters), which can be used.

The kit for quantifying β-1,3-1,6-glucan according to the present invention has the ability to produce β-1,3-glucanase and has β-glucanase activity and α other than β-1,3-glucanase. -M. not exhibiting glucosidase activity. extracellular enzyme solution derived from chitosanitabida inoculum or dried product thereof; has ability to produce β-1,6-glucanase and does not exhibit β-glucanase activity and α-glucosidase activity other than β-1,6-glucanase S. an extracellular enzyme solution derived from omiyaensis inoculum or a dried product thereof; and
A reagent for measuring the amount of glucose.

  M. above. an extracellular enzyme solution derived from an inoculum of chitosanitabida The extracellular enzyme solution derived from Omiyaensis species can be the same as that used in the method of the present invention. Moreover, those dry substances should just dry the said extracellular enzyme solution. However, as described above, since β-1,3-glucanase and β-1,6-glucanase are inactivated at high temperatures, the above M.I. An extracellular enzyme solution derived from an inoculum of chitosanitabida is 60 ° C. or lower, and the above S. It is preferable to dry the extracellular enzyme solution derived from the seed of omiyaensis at 45 ° C. or lower.

  As a reagent for measuring the amount of glucose, for example, when the amount of glucose produced is measured by the Somogyi-Nelson method, Somogyi reagent and Nelson reagent, or a compound for preparing them can be mentioned. When using the enzyme method, for example, when measuring the amount of glucose produced by the enzyme method, the G6PDH-HK method (F-kit, manufactured by Roche) in which glucose-6-phosphate dehydrogenase and hexokinase are combined, Examples include reagents similar to the mutarotase GOD method (glucose CII-Test Wako, manufactured by Wako) in which mutarotase and glucose oxidase are combined, or compounds for preparing them.

  The kit can be used in the β-1,3-1,6-glucan quantification method according to the present invention.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. It is also possible to implement, and they are all included in the technical scope of the present invention.

Example 1 Isolation of β-1,3-glucanase producing bacteria A soil sample was collected from within the Faculty of Agriculture, Kochi University. Sterile water (15 mL) was added to the soil sample (5 g), and the mixture was stirred using a vortex mixer. The mixture was then allowed to stand for several minutes, and the supernatant (50 μL) was collected with a micropipetter.

  Separately, curdlan (β-1,3-glucan, 2 g) was suspended in tap water (200 mL) and treated in an autoclave at 121 ° C. for 20 minutes. This was applied to a mixer and the resulting gel was ground completely. Polypeptone (0.125 g) and yeast extract (0.125 g) were dissolved in this, and tap water was added to make up to 500 mL. Further, 10 g of agar was added, sterilized in an autoclave at 121 ° C. for 20 minutes, and hardened on a petri dish to prepare a curdlan plate medium. A starch medium (0.4% starch, 0.025% polypeptone, 0.025% yeast extract, 2% agar, pH 7.0) was also prepared.

  The supernatant was spread on a curdlan plate medium, spread with a spreader, and cultured at 30 ° C. for 1 day. 480 strains were isolated, inoculated on the same plate medium, and further cultured at 30 ° C. for 2 days. This medium was stained with a 0.1% Congo red solution for 2 to 3 hours and then decolorized in running water for 2 hours to confirm the formation of halo. As a result, only one strain formed halo. The strain was applied to a starch plate medium and cultured at 30 ° C. for 1 day. After staining this medium with iodine solution for 2 to 3 hours (see Nakamura Michinori and Kakinuma Junji, “Biochemical Experimental Method 19 Starch and Related Carbohydrate Experimental Method”), and decolorized in running water for 2 hours. No halo formation was observed. FIG. 1 shows a Congo red staining photograph after culturing in a curdlan plate medium, and FIG. 2 shows an iodine staining photograph after culturing in a starch medium.

  As shown in FIGS. 1 and 2, the above strain shows β-1,3-glucan degrading activity, whereas starch (α-1,4-1,6-glucan) does not show degrading activity. It was thought that α-glucosidase was not produced while producing -1,3-glucanase. This strain was temporarily named H1 strain and used for the subsequent experiments.

Example 2 Microscopic Observation of H1 Strain The H1 strain obtained in Example 1 was magnified 1000 times with a microscope (OLYMPUS, product name “BH-2”) and observed. An enlarged photograph is shown in FIG.

  As shown in FIG. 3, the H1 strain was a koji mold having a width of 0.7 to 1.0 μm and a length of 1.0 to 4.0 μm. The H1 strain was gram-negative and showed catalase activity.

Example 3 16S rDNA base sequence analysis of H1 strain (1) Polymerase chain reaction In order to determine the genus species of H1 strain obtained in Example 1 above, the 16S rDNA base sequence was analyzed. Specifically, first, a 10 × ExTaq buffer solution (5 μL), a mixed solution containing 2.5 mM each of dNTP, dTTP, dGTP and dCTP (8 μL), a 5 pmol / μL solution (1 μL each) of each primer of SEQ ID NOS: 1-2 ) And added to sterilized water to a total volume of 49.5 μL. Separately, a genomic DNA solution of the H1 strain was obtained using an Ultra Clean Microbial DNA Isolation Kit manufactured by MO BIO. The genomic DNA solution (1 μL) or a small amount of cells obtained from a colony of the H1 strain with a sterilized toothpick was added to the above solution to obtain a reaction mixture. A DNA thermal cycler (Takara Shuzo) was used for the polymerase chain reaction. The reaction conditions are as follows.
-Heat denaturation-94 ° C, 5 minutes-Addition of Ex Taq DNA polymerase (Takara Shuzo Co., Ltd., 2.5 U)-Heat denaturation-94 ° C, 45 seconds-Primer annealing-55 ° C, 1 minute-DNA strand extension reaction -72 ° C, 2 minutes-Repeat the steps after the second heat denaturation 35 cycles-Heat treatment-72 ° C, 7 minutes

(2) Separation of 16SrDNA fragment The 16SrDNA fragment amplified by the polymerase chain reaction was separated by agarose gel electrophoresis. The agarose gel used for electrophoresis was prepared from a 1.5% solution of Sea Kem GTG agarose (FMC). As the running buffer, Howly buffer (40 mM Tris, 20 mM sodium acetate, 2 mM EDTA, pH 7.8) was used. As the molecular weight marker, λDNA treated with Hind III was used.

After electrophoresis, DNA was visualized by immersing the agarose gel in a 1 μg / mL ethidium bromide solution for 10 minutes for staining, and ultraviolet rays were irradiated to take a picture. After photographing, a band portion corresponding to the target size was cut out with a sterilized razor, placed in a microtube with a membrane filter (SUPREC ^™ -01, manufactured by Takara Shuzo Co., Ltd.), and completely frozen. After leaving to stand at 37 ° C. for 5 minutes for thawing, the mixture was centrifuged at 10,000 rpm for 10 minutes, 200 μL of TE buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 7.5) was added, and the mixture was further centrifuged for 10 minutes. separated. Two times the amount of cooled ethanol and 1/20 amount of 3M sodium acetate were added, and the mixture was allowed to stand at -20 ° C for 1 hour. Thereafter, the mixture was centrifuged at 12,000 rpm for 10 minutes, ethanol was removed, and suction drying was performed to obtain a mixture containing 16SrDNA fragments.

(3) Determination of base sequence of 16SrDNA fragment The 16SrDNA fragment was amplified by polymerase chain reaction, and the base sequence was analyzed. Specifically, template DNA (4 μL), 5 pmol / μL primer solution (1 μL), BigDye terminator ver. 3.1 5 × Sequencing Buffer (1.5 μL), BigDye Terminator ver. 3.1 (1 μL) was mixed, the total volume was made up to 10 μL with sterilized water, and this was used as the reaction solution. The mixture obtained in (2) above was added to the reaction solution, and a polymerase chain reaction was performed under the following reaction conditions.
-Heat denaturation-96 ° C, 1 minute-Heat denaturation-96 ° C, 10 seconds-Primer annealing-50 ° C, 5 seconds-DNA strand extension reaction-60 ° C, 4 minutes-Steps after the second heat denaturation Repeat 25 cycles

  Next, 16SrDNA fragment was purified from the cooled reaction mixture using Sephadex G-50. Hi-Diformamide (10 μL) was added to the obtained 16S rDNA fragment, and the base sequence was analyzed by ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems). The said base sequence is shown to sequence number 3 of a sequence table.

  The 16S rDNA base sequence obtained was The strain H1 is a strain of M. cerevisiae because it shows high homology with the 16S rDNA base sequence of Chitosanitabida inoculum. It was judged to belong to the species Chitosanitabida.

Example 4 Substrate Specificity of Crude Enzyme Solution from H1 Strain (1) Preparation of Crude Enzyme Solution from H1 Strain In a test tube, 7 mL of Wicherham medium (0.5% peptone, 0.3% yeast extract, 0. 3% malt extract, 1.0% glucose, pH 7.0). The medium was inoculated with the H1 strain with a platinum loop and cultured with shaking at 30 ° C. for 1 day. Separately, a fresh baker's yeast medium (4.0% fresh baker's yeast (manufactured by Kyowa Hakko Co., Ltd., Dia Yeast)) was prepared, and the pre-cultured H1 strain was transferred to the medium and cultured at 30 ° C. for 2 days. The culture broth was centrifuged (18,000 rpm, 20 minutes, 4 ° C.), and a supernatant was obtained so that precipitation did not enter. Phenylmethylsulfonyl fluoride (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter abbreviated as “PMSF”) was added to this supernatant to a final concentration of 0.1 mM, and the ammonium sulfate pulverized in a mortar was saturated with stirring and ice cooling. It was added so that it might become 80%. The mixture was allowed to stand overnight with stirring and ice cooling, and then centrifuged (18,000 rpm, 20 minutes, 4 ° C.) to obtain a precipitate. The obtained precipitate was dissolved in a small amount of 20 mM sodium acetate buffer (pH 6.0) containing 0.1 mM PMSF. This was put into a dialysis membrane (manufactured by Viscose Companies, Inc.), and dialyzed with 2 L of the same buffer to obtain a crude enzyme solution.

(2) Activity measurement of crude enzyme solution The glucanase activity was measured by reacting the crude enzyme solution obtained in the previous section with various glucan substrates and detecting the produced reducing sugar. Specifically, 0.2M sodium acetate buffer (pH 6.0), black yeast β-glucan (derived from Aureobasidium sp., Provided by Sophie Co., Ltd.), pasturan (Calbiochem), Rikenan (Sigma) , Laminarin (derived from Eisenia araborea, manufactured by Hanai Chemicals Co., Ltd.), laminaran (derived from Laminaria, manufactured by Tokyo Chemical Industry Co., Ltd.), curdlan (manufactured by Wako Pure Chemical Industries, Ltd.), cellulose (manufactured by NIHON BASHHI), luteose (applied microorganism, Faculty of Agriculture, Kochi University) (Provided by Academic Laboratory), starch (manufactured by Wako Pure Chemical Industries, Ltd.), dextran (manufactured by Wako Pure Chemical Industries, Ltd.), and pullulan (manufactured by Hanai Chemical Co., Ltd.) were dissolved at 10 g / L. In order to dissolve completely, the lichenan was boiled in hot water, and the pasturan was heated at 121 ° C. for 5 minutes in an autoclave for 5 minutes. All substrate concentrations were adjusted to 0.5%, and the crude enzyme solution was added and reacted at 30 ° C. for 1 hour. After the reaction, it was boiled for 5 minutes, and the amount of reducing sugar produced was measured by the Somogyi-Nelson method (see M. Somogyi, J. Biol. Chem., 195, p. 19 (1952)).

Specifically, KNaC ₄ H ₄ O ₆ .4H ₂ O (4.0 g), Na ₂ HPO ₄ .12H ₂ O (7.1 g), CuSO ₄ .5H ₂ O (0.8 g), 1M NaOH (10 mL) And Na ₂ SO ₄ .5H ₂ O (18.0 g) were dissolved in distilled water (100 mL) and incubated at 37 ° C. for 24 hours. Subsequently, the solution was filtered, and the obtained filtrate was used as a Somogyi reagent. Further, (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O (5.0 g), H ₂ SO ₄ (4.2 g) and Na ₂ HAsO ₄ · 7H ₂ O (0.6 g) were distilled water (100 mL). And incubated for 24 hours. The solution was then filtered, and the resulting filtrate was used as the Nelson reagent. Somogyi reagent (1 mL) was added to the enzyme reaction solution (1 mL), and the mixture was allowed to react accurately in a hot water pan for 10 minutes, and then immediately cooled in running water for 5 minutes. Nelson reagent (1 mL) and distilled water (2 mL) were added to the solution, allowed to stand at room temperature for about 10 minutes, and then the absorbance at 520 nm was measured using a spectrophotometer (SHIMADZU, UV-1200). Note that glucose was used as the standard substance. Further, the amount of enzyme that produces 1 μmol of reducing sugar per hour at 30 ° C. was defined as 1 unit. The results are shown in Table 1.

  As the above results, the crude enzyme solution obtained from the H1 strain showed high activity against black yeast β-glucan, laminarin, laminaran, lichenan and curdlan containing β-1,3-glycoside bonds. On the other hand, there was no activity against pasturan and luteose consisting only of β-1,6-glycoside bonds. Furthermore, although activity against pullulan, an α-glucan, was observed, the specific activity was low.

  From the above results, the crude enzyme solution derived from the H1 strain contains β-1,3-glucanase that specifically cleaves β-1,3-glycoside bonds, but does not contain other β-glucanases, Although it showed a slight α-glucosidase activity, it was revealed that the activity was extremely low because starch and dextran could not be degraded.

Example 5 Preparation of Extracellular Enzyme Solution Derived from H1 Strain (1) Preparation of Crude Enzyme Solution Derived from H1 Strain Mitsuaria chitosanitabida H1 strain was pre-cultured in Wicherham medium (70 mL) in the same manner as in Example 4 (1) above. After that, the main culture was performed in a raw bread yeast medium (1 L). The culture solution was centrifuged (9,100 rpm, 20 minutes, 4 ° C.), and a supernatant was obtained so that precipitation did not enter. To this supernatant, 1/1000 amount of 0.1M PMSF and 1 / 5,000 amount of antifoaming agent were added, and concentrated under reduced pressure to about 1/10 of the total amount with an evaporator. Subsequently, potassium phosphate buffer (pH 7.4) is added so that the final concentration is 20 mM, and ammonium sulfate pulverized in a mortar is added while stirring and cooling with ice, and the precipitate is separated. % Fraction, 20-40% fraction, 40-60% fraction, 60-80% fraction were obtained. Each fraction was dialyzed with 20 mM sodium acetate buffer (pH 6.0), β-1,3-glucanase activity was measured, and total protein was quantified. For more details The enzyme solution was added to araborea-derived laminarin and reacted at 30 ° C. for 1 hour, and then the total amount and specific activity were measured by quantifying the amount of reducing sugar produced by the Somogyi-Nelson method. Proteins were quantified by the Lowry method (see Lowry, OH, et al., J. Biol. Chem., 193, pp. 265-275 (1951)). The results are shown in Table 2.

  As described above, the total activity was highest in the 60-80% saturated fraction, and the specific activity was highest in the 20-40% saturated fraction. In contrast, the 0-20% saturated fraction had low total activity and specific activity. Therefore, 20-80% saturated fraction was used in subsequent experiments.

(2) Preparation of extracellular enzyme solution derived from H1 strain Using 20 mM sodium acetate buffer solution (pH 5.0) containing 0.1 mM PMSF, the 20-80% saturated fraction of the crude enzyme solution obtained above was obtained. After dialysis, the mixture was centrifuged (18,000 rpm, 20 minutes, 4 ° C.), and a gel-like precipitate was formed. In the same manner as in Example 4 (2) above, E.I. When the glucanase activities of the resulting gel-like precipitate and the supernatant were measured using araborea-derived laminarin as a substrate, very high activity was observed in the gel-like precipitate. The gel precipitate was washed by suspending in 20 mM sodium acetate buffer (pH 5.0) and then repeating the centrifugation (18,000 rpm, 20 minutes, 4 ° C.). In order to examine whether the substrate specificity of the washed crude enzyme solution was changed, the same experiment as in Example 4 (2) was performed again. The results are shown in Table 3.

  As shown in Table 3, the obtained extracellular enzyme solution acts on a substrate having a β-1,3-glycoside bond, while an α-1,4-glycoside bond, α-1, It does not act on 6-glycoside bonds, β-1,4-glycoside bonds, and β-1,6-glycoside bonds. Therefore, it was demonstrated that the extracellular enzyme solution derived from the H1 strain does not have α-glucosidase activity or other β-glucanase activity while specifically cleaving β-1,3-glycoside bonds. . Initially, further purification was planned by column chromatography to investigate its enzymatic properties, but such purification could not be performed because the purified enzyme solution was in the form of a gel.

Example 6 Optimum temperature of β-1,3-glucanase derived from H1 strain The above-described procedure was carried out except that the extracellular enzyme solution obtained in Example 5 (2) was used and the reaction temperature was changed to 20 to 70 ° C. The specific activity was measured in the same manner as in Example 5 (1), and the optimum temperature of β-1,3-glucanase derived from the H1 strain was examined. The results are shown in FIG.

  As shown in FIG. 4, the optimum temperature of β-1,3-glucanase derived from the H1 strain was 40 ° C., and the activity began to gradually decrease from 50 ° C., and it was found that the activity was almost lost at 70 ° C.

Example 7 Thermostability of β-1,3-glucanase derived from H1 strain The extracellular enzyme solution obtained in Example 5 (2) above was treated at 30 to 70 ° C. for 15 minutes and then rapidly cooled on ice. Using this solution, the specific activity was measured in the same manner as in Example 5 (1) above, and the thermal stability of β-1,3-glucanase derived from the H1 strain was examined. The results are shown in FIG.

  As shown in FIG. 5, β-1,3-glucanase derived from the H1 strain showed high stability up to 50 ° C., but was found to be completely inactivated at 70 ° C.

Example 8 Identification of β-1,3-glucanase reaction product derived from H1 strain The extracellular enzyme solution obtained in Example 5 (2) above was added to the curdlan which was β-1,3-glucan. The reaction mixture was allowed to act under the same reaction conditions as in Example 4 (2), the reaction mixture was fractionated over time, and the reaction product was analyzed by thin layer chromatography. Specifically, after 0, 1, 2, 4, and 24 hours from the start of the reaction, the reaction mixture was collected, heated for 10 minutes with boiling water to stop the enzyme reaction, and then HPTLC Fertigplatten Kieselgel 60 (Merck) Manufactured, 10 cm × 10 cm). 1% glucose was used as a standard substance, and a mixed solvent of n-butanol: ethanol: water = 5: 2: 2 was used as a developing solvent, and the ascending method was used. After development, diphenylamine-aniline-phosphoric acid (2 g diphenylamine, 2 mL aniline, 100 mL acetone, 15 mL 80% phosphoric acid) is sprayed on the gel plate as a color developing solution, and heated at 100 ° C. for 10 minutes in a dry heat oven. The spot that appeared was observed. The results are shown in FIG. In FIG. 6, “G3” indicates curdlan, “G1” is gentiobiose in which two glucose molecules are β-1,6 linked, and “G0” is glucose.

  As shown in FIG. 6, the final product of the reaction by β-1,3-glucanase derived from the H1 strain was glucose, and the amount of production increased with time. Therefore, it was confirmed that β-1,3-glucanase derived from the H1 strain is an S-type enzyme capable of degrading β-1,3-glucan to glucose, which is a constituent monosaccharide.

Example 9 Isolation of β-1,6-glucanase producing bacteria A soil sample was collected from within the Faculty of Agriculture, Kochi University. Sterile water (15 mL) was added to the soil sample (5 g), and the mixture was stirred using a vortex mixer. The mixture was then allowed to stand for several minutes, and the supernatant (50 μL) was collected with a micropipetter. The supernatant was spread on a pasteurized plate medium (0.1% pasturan (manufactured by Calbiochem, 0.025% polypeptone, 0.025% yeast extract, 2% agar, pH 7.0) and spread with a spreader at 30 ° C. 180 strains were isolated, inoculated into the same plate medium and further cultured for 4 days at 30 ° C. This medium was stained with 0.1% Congo red solution for 2-3 hours (Andres Soler Et al., Journal of microbiological methods, 35, pp. 245-251 (1999)), decolorized in running water for 2 hours, and further submerged in Congo Red by immersion in 20 mM sodium acetate buffer (pH 5.5) overnight. Then, the formation of halo was confirmed, and a photograph of the medium is shown in FIG.

  As shown in the result of FIG. 7, halo-like material was observed around the colony of 1 of 180 strains. That is, this strain is considered to be producing β-1,6-glucanase because it exhibits a degrading activity against β-1,6-glucan, which is a pasteuran. This strain was named SY26 and used for the subsequent experiments.

Example 10 Microscopic Observation of SY26 Strain The SY26 strain obtained in Example 9 was magnified 1000 times with a microscope (OLYMPUS, product name “BH-2”) and observed. An enlarged photograph is shown in FIG.

  As shown in FIG. 8, the SY26 strain forms a colorless and transparent base mycelium. The diameter of the mycelium was 0.8 to 1.5 μm and was branched in a straight line. Further, when the mycelium matured, its tip was cut to form spores. The spore surface was free of wrinkles and was oval or oblong, and had a diameter of 0.5 to 1.0 μm and a length of 1 to 4 μm. Furthermore, in the SY26 strain, no sporangia, motile spores and segmental spores are confirmed, and melanin production is negative.

Example 11 16S rDNA base sequence analysis of SY26 strain The 16S rDNA base sequence of SY26 strain was analyzed in the same manner as in Example 3 above. The said base sequence is shown to sequence number 4 of a sequence table.

  The obtained 16S rDNA base sequence is S. cerevisiae. Since SY26 strain was highly homologous to the 16S rDNA base sequence of Omiyaensis sp. It was judged to belong to the omiyaensis species.

Example 12 Substrate Specificity of Crude Enzyme Solution Derived from SY26 Strain A crude enzyme solution was obtained from the SY26 strain culture solution in the same manner as in Example 4 (1) except that the main culture period was 6 days. Further, the activity of the crude enzyme solution was measured in the same manner as in Example 4 (2). The results are shown in Table 4.

  As the above result, the crude enzyme solution obtained from the SY26 strain showed high activity against black yeast β-glucan, laminarin, pasturan and luteose containing β-1,6-glycoside bond. In contrast, no activity was observed for lichenan and curdlan which did not contain β-1,6-glycosidic bonds and contained β-1,3-glycosidic bonds. Furthermore, α-glucosidase activity was not observed.

  From the above results, it was revealed that the crude enzyme solution of SY26 strain can specifically cleave the β-1,6-glycoside bond, but does not have α-glucosidase activity and other β-glucanase activities. .

Example 13 Preparation of extracellular enzyme solution derived from SY26 strain (1) Preparation of crude enzyme solution derived from SY26 strain As in Example 4 (1) above, S. The omyaensis SY26 strain was pre-cultured in Wicherham medium (14 mL) and then main-cultured in raw bread yeast medium (200 mL). From the culture solution, a 0-20% fraction, a 20-40% fraction, a 40-60% fraction, and a 60-80% fraction were obtained in the same manner as in Example 5 (1). Further, β-1,6-glucanase activity was measured and total protein was quantified in the same manner as in Example 5 (1) except that pasturan was used instead of laminarin as a substrate. The results are shown in Table 5.

  As described above, the total activity was highest in the 20-40% saturated fraction, and the specific activity was highest in the 40-60% saturated fraction. The 60-80% saturated fraction also showed a slight activity, but since both the total activity and specific activity values were low, 20-60% saturated fraction was used in the subsequent experiments.

(2) Purification of β-1,6-glucanase The culture solution was the same as in Example 13 (1) except that the SY26 strain was precultured in 35 mL of Wicherham medium and then main cultured in 500 mL of raw bread yeast medium. 20 to 60% of a saturated fraction was obtained.

  Using a 20 mM sodium acetate buffer solution (pH 5.0) containing 0.1 mM PMSF, the 20-60% saturated fraction of the crude enzyme solution obtained above was dialyzed, and then the precipitate formed during dialysis was centrifuged. What was removed at (18,000 rpm, 20 minutes, 4 ° C.) was used as an extracellular enzyme solution, and the activity was measured in the same manner as in Example 13 (1) above. Further, the total protein amount was quantified with a protein assay CBB solution (manufactured by Nacalai Tesque). The extracellular enzyme solution had a total activity of 40.9 units, a total protein amount of 3.24 mg, and a specific activity of 3.89 units / mg.

  The extracellular enzyme solution was further purified by gel filtration chromatography. Specifically, the crude enzyme solution was applied to an SP-Sephadex C-50 column (Pharmacia, 3.0 × 30 cm) equilibrated with 20 mM sodium acetate buffer (pH 5.0) containing 0.1 mM PMSF. Provided. Non-adsorbed protein was eluted with the same buffer, and adsorbed protein was eluted with a linear gradient with the same buffer containing 0 to 0.5 M sodium chloride, and fractions were separated every 5 mL. The specific activity of each fraction was measured, and the highly active fractions were collected and concentrated to 6 mL with an Amicon membrane filter. After dialysis with 20 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM PMSF, the precipitate formed during dialysis was removed by centrifugation (18,000 rpm, 20 minutes, 4 ° C.). When activity measurement and protein quantification were performed in the same manner as described above, the total activity was 29.8 units, the total protein amount was 2.23 mg, and the specific activity was 13.3 units / mg.

  The enzyme solution was further purified by ion exchange chromatography. Specifically, the enzyme solution was applied to a DEAE-Toyopearl 650M column (Tosoh Corporation, 3.0 × 30 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM PMSF. The protein was eluted with the same buffer, and fractions were collected every 5 mL to collect highly active fractions. The total activity of the enzyme solution was 18.3 units, the total protein amount was 0.31 mg, and the specific activity was 58.2 units / mg.

  The enzyme solution was concentrated to about 1 mL with Centriprep, further concentrated to 50 μL using Microcon, and then purified by FPLC using BioLogic Duo Flow (manufactured by Bio-Rad). Specifically, the concentrated enzyme solution was applied to a Superose 12 HR 10/30 column equilibrated with 20 mM potassium phosphate buffer (pH 7.4) containing 0.15 M sodium chloride. The protein was eluted by continuing to pass through the same buffer and fractionated every 0.5 mL, and fractions with high activity were collected and used as a purified enzyme solution. When the activity measurement and protein quantification of the purified enzyme solution were performed as described above, the total activity was 3.65 units, the total protein amount was 0.01 mg, and the specific activity was 386 units / mg.

  Table 6 summarizes data such as the total amount of protein in each of the above purification steps.

  In addition, the enzyme solution of each purification step was analyzed according to the Laemmli method (see U.K.Laemmli, Nature, 277, pp. 680-685 (1970)). The current was 10 mA for the concentrated gel and 20 mA for the separated gel. As markers for molecular mass measurement, myosin (200 kD), β-galactosidase (116 kD), bovine serum albumin (66 kD), ovalbumin (45 kD), carbonic anhydrase (31 kD), trypsin inhibitor (21.5 kD) ), Lysozyme (14.4 kD), and aprotinin (6.5 kD) protein marker (manufactured by Nacalai Tesque) was used. After completion of the electrophoresis, the gel was immersed in a mixed solution of methanol: acetic acid: water = 5: 1: 88 containing 0.25% Coomassie Brilliant Blue R-250, and stained at 50 ° C. for 30 minutes while shaking. Then, it was immersed in a mixed solution of methanol: acetic acid: water = 5: 7: 88 and decolorized at 50 ° C. while shaking. The decolorized gel was stored with an image using a digital camera and dried with a gel dryer. The results are shown in FIG.

  As shown in FIG. 9, it can be seen that the purified enzyme solution finally obtained contains only a protein having a molecular mass of 10 kD.

Example 14 Optimum temperature of β-1,6-glucanase derived from SY26 strain Using the purified enzyme solution which is the Superose 12 HR 10/30 column elution fraction obtained in Example 13 (2) above, the reaction temperature was 10 The specific activity was measured in the same manner as in Example 13 (1) except that the temperature was changed to ˜60 ° C., and the optimum temperature of β-1,6-glucanase derived from the SY26 strain was examined. The results are shown in FIG.

  As shown in FIG. 10, the optimum temperature of β-1,6-glucanase derived from the SY26 strain was 30 ° C., and at 40 ° C., 83% of the activity at 30 ° C. was exhibited. It turns out that it is lost suddenly.

Example 15 Thermostability of β-1,6-glucanase derived from SY26 strain A purified enzyme solution, which is the Superose 12 HR 10/30 column elution fraction obtained in Example 13 (2) above, was obtained at 20-60 ° C. After treating for minutes, it was quenched on ice. Using this solution, the specific activity was measured in the same manner as in Example 13 (1) above, and the thermal stability of β-1,6-glucanase derived from the SY26 strain was examined. The results are shown in FIG.

  As shown in FIG. 11, β-1,6-glucanase derived from the SY26 strain showed high stability up to 30 ° C., and showed about 80% activity at 30 ° C. at 40 ° C., but was inactivated at 50 ° C. I found out that

Example 16 Identification of β-1,6-glucanase Reaction Product Derived from SY26 Strain The β-1,6-glucanase-containing solution purified in Example 13 above was added to the pasturan that was β-1,6-glucan. The reaction mixture was allowed to act under the same reaction conditions as in Example 13 (1), the reaction mixture was fractionated over time, and the reaction product was analyzed by thin layer chromatography. The results are shown in FIG. In FIG. 12, “G3” indicates pasturan, “G1” is gentiobiose in which two glucose molecules are linked by β-1,6, and “G0” is glucose.

  As shown in FIG. 12, the final product of the reaction with β-1,6-glucanase derived from the SY26 strain was glucose, and the amount of production increased with time. Therefore, it was confirmed that β-1,6-glucanase derived from SY26 strain is an S-type enzyme capable of degrading β-1,6-glucan to glucose, which is a constituent monosaccharide.

Example 17 Quantification of β-1,3-1,6-glucan Naturally-derived glucans include α-glucan and β-glucan, which are homoglucans consisting only of glucose, and heteroglucans linked to other sugars. Exists. In the conventional method, among these glucans, all remaining glucans from which α-glucan has been removed are decomposed into constituent monosaccharides and glucose is quantified. Therefore, useful β-1,3-1,6 -Glucan alone could not be quantified.

  On the other hand, it has been demonstrated by the above experiment that the extracellular enzyme solution derived from the H1 strain and the SY26 strain can specifically cleave β-1,3-glycoside bond and β-1,6-glycoside bond, respectively. Therefore, if the amount of glucose is measured after completely degrading glucan to glucose with these enzyme solutions, β-1,3-1,6-glucan can be accurately quantified without degrading and removing α-glucan in advance. I can expect it.

  Thus, first, β-1,3-1,6-glucan in naturally derived glucan is quantified using the two extracellular enzyme solutions according to the present invention, and then α-glucan is determined from the glucan. The same operation was performed after removing and the results were compared to evaluate the effect of the method of the present invention.

(1) Preparation of polysaccharide preparation Distilled water (27 mL) was added to a culture solution of Aureobasidium pullulans AFO202 strain (provided by Sophie, 3 g) to prepare a 10-fold diluted culture solution. This was centrifuged (18,000 rpm, 20 minutes, 4 ° C.), and twice the amount of ethanol was added to the resulting supernatant (7 mL), followed by stirring well to precipitate the polysaccharide. Further, after centrifugation (18,000 rpm, 20 minutes, 4 ° C.), the supernatant was discarded, and the precipitate was air-dried until it did not smell of ethanol. After air drying, the precipitate was dissolved in 14 mL of 20 mM sodium acetate buffer (pH 4.5), which was used as a polysaccharide preparation.

  Using the phenol-sulfuric acid method (see Dubois, M., et al., Anal. Chem., 28, p. 350 (1956)), the total amount of polysaccharide contained in the polysaccharide preparation was measured. Specifically, the above polysaccharide preparation (500 μL) and distilled water (500 μL) were mixed, and 5% phenol (1 mL) was further added. Sulfuric acid (5 mL) was added here, and it cooled at room temperature for 10 minutes. After the mixture was cooled with running water for 20 minutes, the amount of polysaccharide was quantified by measuring the absorbance at 490 nm, and the total amount of polysaccharide was 3.80 g / L.

(2) When α-glucan is contained in the sample The polysaccharide preparation (500 μL) obtained in the above (1) is added to the 1M sodium acetate buffer (pH 6.0, 50 μL), the extracellular enzyme solution derived from the H1 strain. (225 μL) and an extracellular enzyme solution (225 μL) derived from the SY26 strain were mixed and incubated at 40 ° C. for 6 hours. The activity of the extracellular enzyme solution derived from the H1 strain used was 2.2 units against laminarin (derived from E. araborea), and the activity of the extracellular enzyme solution derived from the SY26 strain was 1. 4 units is sufficient to completely degrade the glucan in the polysaccharide preparation.

  About the reaction liquid mixture after the said enzyme reaction, the amount of reducing sugars was measured by the Somogyi-Nelson method. The results are shown in Table 7.

(3) When α-glucan is not contained in the sample: Amyloglucosidase (derived from Rhizopus sp, manufactured by Sigma) is dissolved in 0.2 M sodium acetate aqueous solution (pH 4.5) at a concentration of 1 μg / mL to obtain an amyloglucosidase solution. Got. The polysaccharide preparation (3.6 mL) and the amyloglucosidase solution (0.4 mL) were placed in a falcon tube and incubated overnight at 55 ° C. Thereafter, it was boiled for 5 minutes to complete the enzyme reaction. The reducing sugar produced by this amyloglucosidase treatment was quantified by the Somogyi-Nelson method to obtain the amount of α-glucan. To this reaction solution (500 μL), an extracellular enzyme solution (225 μL) derived from the H1 strain and an extracellular enzyme solution (225 μL) derived from the SY26 strain were added and reacted at 40 ° C. for 6 hours. For the reaction mixture after the enzyme reaction, the amount of reducing sugar was measured by the Somogyi-Nelson method, and the amount of α-glucan was subtracted from the measured value to obtain the amount of β-1,3-1,6-glucan. . The results are shown in Table 7.

  When α-glucan is not removed, the amount of β-1,3-1,6-glucan is 3.09 g / L, and when α-glucan is removed, it is 3.18 g / L. There was no big difference. Therefore, it was found that the method of the present invention can accurately quantify β-1,3-1,6-glucan even when α-glucan is present in the preparation.

Example 18 Quantification of β-1,3-1,6-glucan As in Example 17 above, according to the method of the present invention, β-1,3-1 was accurately detected even when α-glucan was present in the preparation. , 6-glucan can be quantified, but if the amount of α-glucan in the reaction solution is larger than this, the amount of β-1,3-1,6-glucan cannot be accurately quantified without removing α-glucan. There is a possibility. Therefore, A. which produces more α-glucan. The same experiment was performed using the culture medium of pullulans 132 strain as a sample.

(1) Preparation of polysaccharide preparation The pullulans 132 strain (Keisuke Terao (Department of Bioresource Sciences, Faculty of Agriculture, Kochi University), see “Applied Microbiology Laboratory Graduation Thesis” (2006)) is scraped with a platinum ear and 5 mL of a liquid medium containing rice bran (0.2 % Rice bran, 0.2% ascorbic acid, 1% sucrose, pH 5.2) and precultured by shaking at 25 ° C. and 125 rpm for 48 hours. Next, 100 mL of rice bran-containing liquid medium was prepared in a 30 mL Erlenmeyer flask, and the above preculture solution (1 mL) was added thereto, followed by main culture by shaking at 25 ° C. and 125 rpm for 72 hours. It should be noted that the rice bran-containing liquid medium promotes polysaccharide production.

  A. A polysaccharide preparation was obtained in the same manner as in Example 17 (1) except that the culture solution (3 g) of the main culture was used instead of the culture solution of pullulans AFO202 strain. It was 3.04 g / L when the amount of polysaccharide contained in the said polysaccharide sample was quantified by the method similar to the said Example 17 (1).

(2) Quantification of β-1,3-1,6-glucan For the above-mentioned polysaccharide preparation, the amount of β-1,3-1,6-glucan was determined when the α-glucan was not removed and when it was removed. It measured similarly to Example 17 (2)-(3). The results are shown in Table 8.

  As described above, A. α-glucan contained in the culture solution of pullulans strain 132 is A. glucan. More than α-glucan contained in the culture solution of pullulans AFO202 strain. However, the amount of β-1,3-1,6-glucan measured by the method of the present invention is 0.92 g / L when α-glucan is not removed, and 1. It was 10 g / L, and there was no big difference between these two.

  From the results of Examples 17 to 18 above, according to the method of the present invention using β-1,3-glucanase and β-1,6-glucanase derived from H1 strain and SY26 strain, α-glucan in the sample was obtained. It was demonstrated that β-1,3-1,6-glucan can be accurately quantified regardless of the presence or absence of and the presence of mixed α-glucan.

In Example 1, it is the photograph which carried out Congo red dyeing | staining after culture | cultivating the strain isolated from the soil sample on the curdlan flat plate culture medium. In Example 1, it is the photograph which carried out the iodine dyeing | staining after culture | cultivating the strain isolated from the soil sample in the starch culture medium. 2 is a photomicrograph of H1 strain obtained in Example 1. It is a graph which shows the relationship between reaction temperature and the activity of the β-1,3-glucanase derived from H1 strain. It is a graph which shows the relationship between the activity of β-1,3-glucanase derived from H1 strain and the heat treatment temperature. It is the result of the thin layer chromatography which shows the time-dependent change of the decomposition reaction product of curdlan by the β-1,3-glucanase derived from the H1 strain. In Example 9, it is the photograph which carried out Congo red dyeing | staining after culture | cultivating the strain isolate | separated from the soil sample with the pasturan flat plate culture medium. 4 is a photomicrograph of SY26 strain obtained in Example 9. It is a result of SDS-PAGE of each purification step of β-1,6-glucanase derived from SY26 strain. In the figure, 1 is a molecular weight marker lane, 2 is a 20-60% saturated ammonium sulfate precipitation lane, 3 is a lane after purification by gel filtration chromatography, 4 is a lane after purification by ion exchange chromatography, 5 shows the lane after purification by FPLC. It is a graph which shows the relationship between reaction temperature and the activity of (beta) -1,6-glucanase derived from SY26 strain. It is a graph which shows the relationship between the activity of (beta) -1,6-glucanase derived from SY26 strain, and heat processing temperature. It is the result of the thin layer chromatography which shows the time-dependent change of the decomposition reaction product of the pasturan by the β-1,6-glucanase derived from the SY26 strain.

Claims (5)

Mitsaria chitosanitabida H1 strain that has the ability to produce β-1,3-glucanase and does not exhibit β-glucanase activity or α-glucosidase activity other than β-1,3-glucanase (Mitaria chitosanitabida) An extracellular enzyme solution derived from NITE AP-629) and β-1,6-glucanase producing ability and no β-glucanase activity and α-glucosidase activity other than β-1,6-glucanase A step of allowing an extracellular enzyme solution derived from Streptomyces omiyaensis SY26 strain (NITE AP-616) to act; and a step of measuring the amount of glucose produced;
A method for quantifying β-1,3-1,6-glucan. In the polysaccharide-containing sample, an extracellular enzyme solution derived from the H1 strain (NITE AP-629) and an extracellular enzyme solution derived from the SY26 strain (NITE AP-616) at 20 ° C. or higher and 45 ° C. or lower. The method for quantitative determination according to claim 1, wherein Mitsaria chitosanitabida H1 strain (NITE AP-629) has the ability to produce β-1,3-glucanase and does not exhibit β-glucanase activity or α-glucosidase activity other than β-1,3-glucanase. Derived extracellular enzyme solution or dried product thereof;
Streptomyces omiyaensis SY26 strain (NITE AP-616) that has the ability to produce β-1,6-glucanase and does not exhibit β-glucanase activity or α-glucosidase activity other than β-1,6-glucanase ) Extracellular enzyme solution derived from or dried product thereof; and a reagent for measuring glucose amount;
A kit for quantitative determination of β-1,3-1,6-glucan. Mitsuaria chitosanitabida H1 strain (NITE AP-629). Streptomyces omiyaensis SY26 strain (NITE AP-616).