JPH0441597B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to apatite on which glucanase is immobilized and a method for immobilizing glucanase on apatite. Glucanase is a general term for enzymes that decompose glucan, and includes various enzymes, but glucanase as used in the present invention refers to levanase that decomposes levan, dextranase that decomposes dextran, and mutanase that decomposes mutan. Apatite means hydroxyapatite and fluoroapatite. However, when the enzyme is dextranase, only fluoroapatite is referred to as apatite. Dental caries is caused by polysaccharides produced by various bacteria present in the oral cavity, such as levan, dextran, mutan,
It is a well-known fact that this occurs due to the formation of dental plaque. Therefore, in order to prevent dental caries, it is considered that polysaccharides produced by these bacteria can be removed to prevent the formation of dental plaque. The present invention relates to apatite in which polysaccharide degrading enzymes related to the development of dental caries are immobilized, and a method for producing the same. (Prior Art) Various methods of removing dental plaque have been used to prevent dental caries. For example, there are methods to scrape off dental plaque with zeolite, calcium carbonate, alumina, silica, and other abrasives, and methods to use dextranase together with a stabilizing agent. Until now, there has been no method for immobilizing enzymes such as enzymes that decompose polysaccharides that cause dental caries into apatite, which is used as an abrasive for tooth brushing, and no apatite in which these enzymes are immobilized has existed. (Problems to be Solved by the Invention) Since glucanase is an enzyme, it is relatively unstable, and if it is mixed directly into toothpaste, its activity will decrease over time and it will eventually cease to exhibit any activity. Dextranase is currently used in tooth brushing, but various stabilizers have been proposed to prevent its deactivation. For example, JP-A No. 56-63915 discloses a combination of dextotanase and aluminum oxide, JP-A No. 56-110609 discloses a mixture of dextranase, carvone, and l-menthol, and JP-A No. 52-49055 The specification proposes the combination of dextranase and gelatin or pefton. However, since toothpaste is used in the oral cavity, it is necessary to consider its effect on the human body, and it is subject to various restrictions, such as the need to feel refreshed after use. Since the stabilizer is naturally subject to such restrictions, the selection of the stabilizer involves difficult problems.
When using enzymes other than dextranase, exactly the same problems arise as when using dextranase. Therefore, as a result of various studies on methods for stabilizing glucanase, we found that by immobilizing glucanase on apatite, which is used as an abrasive,
We were able to develop a method to obtain glucanase that does not require stabilizers and exhibits stable activity over a long period of time.
The present invention provides apatite with immobilized glucanase and a method for producing the same. (Means for solving the problem) It is a well-known fact that there is an enzyme immobilization method in which enzymes are physically adsorbed and immobilized on hydroxyapatite, activated carbon, kaolinite, clay, etc. It is also generally known that the enzyme is stable, exhibits less change in activity over time than the enzyme alone, and is convenient to handle. It has been known for a long time that apatite strongly adsorbs and binds certain proteins. Based on the idea that apatite can immobilize glucanase and reduce changes in its activity over time, it has been used as an abrasive agent in toothpaste. If it is possible to immobilize glucanase on apatite, which is used as an apatite, by a physical adsorption method, the immobilized enzyme can be obtained with a simple operation, and when used for tooth brushing, it has both abrasive and polysaccharide decomposing effects. In addition to having
We considered immobilizing glucanase with apatite, thinking that it would be a preferable polishing material without the need to select an enzyme stabilizer. As a result, it was found that it was impossible to directly adsorb and immobilize glucanase on apatite because the adsorption power of glucanase to apatite was extremely weak. Therefore, we were able to obtain apatite with glucanase immobilized through a protein that firmly binds to apatite. The present invention provides apatite on which glucanase is immobilized, and a method for immobilizing glucanase on apatite together with a certain type of protein that strongly adsorbs to apatite. As mentioned above, the glucanases referred to in the present invention include dextranase, levanase, mutanase,
Apatite means hadroxyapatite and fluoroapatite, but when the enzyme is dextranase, fluoroapatite is used as apatite. Apatite is suspended in an aqueous solution containing glucanases and proteins that strongly adsorb to apatite and have no adverse effects on the human body, or in a phosphate buffer solution with a 0.01 to 0.05 molar concentration, and a bifunctional aldehyde is added while stirring vigorously. Drip. The protein used is selected from albumin, casein, lysozyme, cytochrome C, protamine, etc. The titer of the immobilized enzyme produced and its binding strength to apatite depend on the type of protein, but among them, lysozyme, cytochrome C, etc. are preferred. As mentioned above, the glucanase can be arbitrarily selected from levanase, dextranase, and mutanase, and in some cases, a mixture of these enzymes can be used. The apatite is selected from hydroxyapatite and fluoroapatite. The reaction is at a pH that does not decompose apatite,
That is, it is carried out at a pH of 5.6 or higher, but a higher pH has a negative effect on adsorption, so a pH of 9.0 or higher is not preferable, and a pH of around 7.0 is preferable. It is desirable that the particle size of the apatite used is as uniform as possible, but particles commonly used as an abrasive for toothpaste are sufficient, and particles with a particle size of 2μ to 200μ are easy to use and are effective against proteins. Use 10 to 100 times the amount. To ensure efficient stirring,
It is desirable to use a large amount of water or buffer solution relative to the amount of apatite used, and the solid content of the reaction phase is from 4 to 4.
Adjust the water amount to 20%. It is preferable to use approximately equal amounts of protein and glucanase, and extreme differences between the two, especially a small amount of glucanase relative to the protein, should be avoided as this will lower the titer of the immobilized enzyme produced. Use 2
As the functional aldehyde, commonly used glutaraldehyde is preferred. The amount used is the factor that most influences the titer of immobilized enzyme.
Generally, when the amount of glutaraldehyde added is too small, the binding strength of the immobilized enzyme to apatite is weak, and the enzyme is significantly deactivated over time. Furthermore, if the amount added is too large, the titer of the immobilized enzyme obtained will decrease, and if the amount added is further increased, it will eventually cease to show any activity. The amount of glutaraldehyde used varies somewhat depending on the type of enzyme and protein, but is 3 to 60 mg per gram of protein used.
preferably in the range of 6 to 20 mg. The reaction is carried out below room temperature, preferably around 5°C. Cool the suspended station in which protein, glucanase, and apatite coexist to below room temperature, preferably around 5°C, and gradually drop an aqueous glutaraldehyde solution into this solution while stirring vigorously. After the dropwise addition is complete, stir at the same temperature for several hours. and complete the reaction. After the reaction is complete, the apatite obtained is thoroughly washed with water or the buffer solution used to remove accompanying proteins and enzymes, and then kept at a low temperature or freeze-dried to form a solid. Keep at room temperature. The immobilized enzyme of the present invention can also be produced as follows. First, apatite is added to an aqueous solution or a buffer solution in which a protein is dissolved, and the apatite is thoroughly stirred to saturate the apatite with protein adsorption, and then the adsorbed apatite is collected, and the adsorbed apatite is added to water or a buffer solution in which glucanase is dissolved, Gradually add the glutaraldehyde aqueous solution dropwise while stirring vigorously, and continue stirring after the drop plate is finished.
In this case, the reaction temperature and other conditions may be the same as those described above. The immobilized enzyme obtained in this way is stable and
It has little change over time and is easy to handle. (Function) It has already been revealed that apatites adsorb certain proteins well, and it is also known that glutaraldehyde is used as a crosslinking agent to immobilize enzymes. It is not clear by what mechanism glucanase is immobilized on apatite by the present method, but adsorption of proteins that are easily adsorbed to apatite and cross-linking of proteins and glucanase occur simultaneously, and the immobilization of glucanase occurs beyond the immobilized enzyme. It is estimated that there are. The present invention will be specifically described below with reference to Examples. Example 1 Immobilization of levanase on hydroxyapatite 100 mg of lysozyme, 100 mg of levanase, and 50 mg of pure water
ml, add 2 g of abrasive hydroxyapatite to this solution, and cool to 4°C. While maintaining the temperature at 4°C and stirring vigorously, 2 ml of an aqueous glutaraldehyde solution (glutaraldehyde 28 mg/100 ml) was gradually added dropwise. After dropping, maintain the temperature 2.
Stir for hours. Thereafter, the solid was collected by centrifugation and washed with 50 ml of pure water with stirring three times to collect the solid to obtain undried immobilized hydroxyapatite (which may be used as is in some cases). This was freeze-dried to obtain about 2.05 g of powder. This powder 1
Weigh out 10 ml of potassium phosphate buffer solution with pH 6.8 and 1 molar concentration. After stirring at room temperature for 1 hour, centrifuge to collect the liquid. Repeat the same operation twice with the residue to collect the liquid. In addition, the amount of protein was measured by the Lowry method, and the residue was washed with water, dried, and weighed. As a result, per gram of hydroxyapatite, protein
It was confirmed that 11.7mg was bound. The levanase activity of undried immobilized hydroxyapatite was measured by the method described below.
0.47g/g of hydroxyapatite binding protein
admitted to disassemble Levan. Example 2 Immobilization of levanase on fluoroapatite The procedure was carried out under the same conditions as in Example 1, except that fluoroapatite was used instead of hydroxyapatite in Example 1, and 2.05 g of a freeze-dried product was obtained. The bound protein was measured in the same manner as in Example 1, and it was confirmed that 13.4 mg of protein was bound per gram of fluoroapatite. As in Example 1, the levanase activity measurement results showed that 0.54 g of levan was degraded per g of bound protein. Example 3 Immobilization of mutanase on hydroxyapatite An experiment was carried out under the same conditions as in Example 1, except that mutanase was used instead of levanase in Example 1, and immobilized hydroxyapatite was obtained. As a result of the same test as in Example 1, 15.0 mg of protein per g of hydroxyapatite
It was admitted that they were combined. Furthermore, as a result of measuring mutanase activity, it was confirmed that 0.48 g of mutan was degraded per g of hydroxyapatite-binding protein. Example 4 Immobilization of mutanase on fluoroapatite Immobilized fluoroapatite was obtained by operating under the same conditions as in Example 3, except that fluoroapatite was used instead of hydroxyapatite in Example 3. As a result of analysis and titer measurement in the same manner as in Example 3, it was found that 17 mg of protein was bound per g of fluoroapatite and 0.45 g of mutan was decomposed per g of bound protein. Example 5 Immobilization of dextranase on fluoroapatite 50 mg of lysozyme and 50 mg of dextranase were mixed, and to this was added 5 g of fluoroapatite and 50 ml of a potassium phosphate buffer solution with a 0.05 molar concentration and a pH of 6.8.
Cool to 4°C and stir vigorously. While maintaining this temperature, add glutaraldehyde 0.2% aqueous solution.
125μ was added dropwise while stirring, and stirring was continued for 5 hours after the addition. Take the reaction product and add 100ml of the above buffer solution.
After washing three times with water, undried immobilized fluoroapatite was obtained. 5.08 g of dextranase-immobilized fluoroapatite was obtained by freeze-drying. To the precipitate obtained by centrifuging 1 ml of undried immobilized fluoroapatite, add 2 ml of potassium phosphate buffer solution with a 1 molar concentration of PH6.8, stir for 3 hours to desorb bound proteins, centrifuge, and the precipitate is the same. The operation was repeated, the liquids were combined, and the protein was measured by the Lowry method, and the precipitate was washed with water, dried, and weighed. As a result, 15.24 mg of protein was bound per gram of fluoroapatite. Dextranase activity measurement results showed that 0.43 g of dextran was degraded per g of bound protein. Various experimental results are shown in Table 1. The processing conditions are the same as in Examples. Measurement of each enzyme titer 1 ml of the undried immobilized enzyme obtained in the experiment was added to 10 ml of each 1% substrate solution, and after stirring at 37°C for 2 hours, the monomers produced in the solution were quantified. The amount of protein bound to 1 ml of immobilized enzyme was measured by the method described above, and the amount of monomer decomposed per gram of protein bound to apatite was determined as the titer of each immobilized enzyme. Dextranase uses dextran as a substrate,
Mutanase uses mutan, levanase uses levane, dextranase and mutanase decompose the mainly produced glucose using the glucose oxidase method, and levanase decomposes the resulting fructose using a conventional method using high-sensitivity liquid chromatography (column: Sugar Pack). 1, eluent: water). Time-dependent activity of immobilized enzymes The time-dependent changes in the activity of several types of immobilized enzymes obtained by this method were examined. All the samples used were undried immobilized enzymes, and their respective activities were measured by the method described above. It was found that the activity of the obtained immobilized enzyme increases with time, and after a certain period of time, it shows the highest activity and then gradually decreases in activity. (Effects of the Invention) According to the method of the present invention, glucanase can be immobilized on apatite with extremely simple operations, and the immobilized enzyme obtained is easy to handle, is stable, has little change over time, and has the ability to decompose polysaccharides. Since it has the abrasive properties of apatite, its use in tooth brushing is preferable for preventing cavities and is extremely useful for oral hygiene.

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[Table] Both toranase are aqueous solutions

Claims (1)

[Scope of Claims] 1. Glucanase-immobilized apatite, characterized in that glucanase and protein are immobilized on apatite with glutaraldehyde. Here, glucanase is an enzyme selected from levanase, mutanase, and dextranase; protein is albumin, casein, lysozyme,
Protein selected from cytochrome C and protamine; Apatite means apatite selected from hydroxyapatite and fluoroapatite. However, when the glucanase is dextranase, the apatite is fluoroapatite. 2. A method for producing glucanase-immobilized apatite, which comprises dropping glutaraldehyde into an aqueous solution containing a mixture of glucanase, protein, and apatite, and collecting the resulting precipitate. Here, glucanase is an enzyme selected from levanase, mutanase, and dextranase; protein is albumin, casein, lysozyme,
Protein selected from cytochrome C and protamine; Apatite means apatite selected from hydroxyapatite and fluoroapatite. However, when the glucanase is dextranase, the apatite is fluoroapatite. 3. The production method according to claim 2, wherein the protein is lysozyme.