JPS62248487A - Apatite containing immobilized glucanase or such and production thereof - Google Patents

Abstract

PURPOSE:To obtain a glucanase exhibiting stable activity over a long period without necessitating a stabilizer, by immobilizing a glucanase to apatite useful as an abrasive by the aid of a protein which can be firmly adsorbed and bonded to apatite. CONSTITUTION:(A) A glucanase such as dextranase, levanase, mutanase, etc., (B) an apatite such as hydroxyapatite, fluoroapatite, etc., and (C) a protein such as albumin, casein, protamine (preferably lysozyme or cytochrome C), etc., are used as raw materials. An immobilized enzyme is produced either by dripping glutaraldehyde to a solution containing the above materials to effect the immobilization or by adsorbing the protein to the apatite and reacting with the glucanase. The immobilized enzyme produced by this process is easily handleable, has high stability, polysaccharide-decomposing capability and abrasive power of the apatite and is useful for the hygienics of oral cavity.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to apatite on which glucanases and the like are immobilized, and a method for immobilizing glucanases and the like on apatite. Glucanase is a general term for enzymes that decompose glucan, and includes enzymes of various types, but glucanase as used in the present invention refers to levanase that decomposes levan, dextranase that decomposes dextran, and mutanase that decomposes mutan. Moreover, apatite means hydroxyapatite and fluoroapatite. However, when the enzyme is dextranase, only fluoroapatite is referred to as apatite.

Tooth decay is caused by polysaccharides produced by various bacteria that exist in the oral cavity.
For example, it is a well-known fact that levan, dextran, mutan, etc. cause plaque formation. Therefore, it is considered to prevent dental caries by removing polysaccharides produced by these bacteria and preventing the formation of dental plaque. The present invention relates to apatite immobilized with polysaccharide degrading enzymes related to the development of dental caries and its loss.

(Prior Art) Various methods of removing dental plaque have been used to prevent dental caries. For example, there are methods to scrape off #scale with zeolite, calcium carbonate, alumina, silica, and other abrasives, and methods to use dextranase together with a stabilizing agent. Apatite, which immobilizes the enzymes that cause dental caries, such as enzymes, did not exist until now.

(Problem to be solved by the invention) Since glucanase is an enzyme, it is relatively unstable, and m
If you mix it directly in your bladder, its activity will decrease over time, and it will eventually show no activity.Currently, dextranase is used for young teeth, but in order to prevent its deactivation, various virtues are added. Stabilizers have been proposed. For example, JP-A-56
JP-A-63915 discloses the combination of dextranase and aluminum oxide, JP-A-56-110609 discloses the mixture of dextranase, carvone, and t-menthol, and JP-B No. 52-49055 specifies the combination of dextranase and aluminum oxide. A combination of stranase and gelatin or beftone is proposed.

However, since it is used in the oral cavity, it is necessary to consider the effect on the human body, and it is also required to feel cool after use, so it is subject to various restrictions. Since the stabilizer is naturally subject to such restrictions, the selection of the stabilizer involves difficult problems. When using enzymes other than dextranase, the same problems arise as when using dextranase.As a result of careful consideration of methods for stabilizing glucanase, we found that glucanase was fixed to apatite, which is used as an abrasive. By doing so, we were able to develop a method for producing glucanain that exhibits stable activity over a long period of time without having to rely on stabilizers.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 since ancient times that apatite strongly adsorbs and binds the proteins of oak, so the idea of immobilizing glucanase and reducing changes in its activity over time is also thought to be the reason for polishing with toothpaste. If glucanase can be immobilized on apatite, which is used as an agent, by physical adsorption, the immobilized enzyme can be changed with a simple operation. In addition to having both functions, we thought that it would be a preferable toothpaste material without the need to select an enzyme stabilizer, so we investigated the immobilization of glucanase with apatite. Because the suction power is extremely weak,
It was recognized that it is impossible to directly adsorb and immobilize glucanase on hepatite. 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 hepatite together with a certain woodcutter protein that strongly adsorbs to apatite.

As mentioned above, glucanase in the present invention includes dextranase, levanase, and mutanase, and apatite means hydroxyapatite and fluoroapatite, but when the enzyme is dextranase, fluoroapatite is used as apatite. It means.

An aqueous solution containing glucanase and a protein that strongly adsorbs to apatite and has no adverse effect on the human body, or 0
Apatite is suspended in a phosphate buffer solution with a molar strength of 0.01 to 0.05 molar, and the difunctional aldehyde is added dropwise with vigorous stirring. The protein used is selected from albumin, casein, lysozyme, cytochrome C1 protamine, and the like. The titer of the immobilized enzyme produced and the binding strength to a6tite vary depending on the type of protein, but among them, lysozyme, cytochrome C, etc. are preferred. As described 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 takes place at pH 1 or pMs, which does not decompose apatite, 6
It is carried out at a pH of 9.0 or above, but a pjl of 9.0 or above is not preferable because it does not have a sudden effect on adsorption if the pit is high (,
9117, preferably around 0. It is desirable that the particle size of the apatite used is as uniform as possible, but generally 1
Particles that are used as abrasives can be used sufficiently, and particles with a particle size of 2μ to 200μ are easy to use.
Use 10 to 100 times the amount of protein/XK. For the apatite violet, use so that stirring is efficient (done).
It is desirable to use a large amount of water or buffer solution to adjust the amount of water so that the solids content of the reaction phase is between 4 and 20 percent. 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. The commonly used difunctional aldehyde is preferably glutaraldehyde. The amount used is the factor that most influences the titer of immobilized enzyme. In general, if the amount of glutaraldehyde added is too small, the binding strength of the immobilized enzyme to apatite is weak (and the deactivation is significant over time. Also, if the amount added is too large, the titer of the immobilized enzyme obtained will decrease). However, as the amount of glutaraldehyde added is increased, it finally stops showing activity.The amount of glutaraldehyde used varies somewhat depending on the quality of the enzyme and protein, but is in the range of 3 to 60 glutaraldehyde per protein used, preferably 60 glutaraldehyde.
The range is from 2011Q to 2011Q. The reaction is carried out below room temperature, preferably around 5°C.

A suspension in which protein, glucanase, and apatite coexist is cooled to below room temperature, preferably around 5°C, and an aqueous glutaraldehyde solution is gradually added dropwise to this solution while stirring vigorously, and after the addition is complete, the mixture is stirred several times at the same temperature. Terminate the reaction after a certain amount of time. After the reaction is complete, the apatite obtained by filtration is thoroughly washed with water or the buffer solution used to remove accompanying proteins and enzymes, and then either 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 a water bath solution or buffer solution in which protein has been soaked, and stirred sufficiently to saturate the apatite with adsorption of protein.After that, the adsorbed apatite is collected, and the adsorbed apatite is added to water or buffer solution in which glucanase has been dissolved. Then, while stirring vigorously, the glutaraldehyde water bath solution was gradually added dropwise, and after the addition was completed, stirring was continued.

In this case, the reaction temperature and other conditions may be the same as those described above.

(Function) It has already been revealed that apatite adsorbs certain proteins, and it is also known that glutaraldehyde is used as a crosslinking agent to immobilize enzymes. Although it is not clear what mechanism causes immobilization to apatite, it is presumed that the adsorption of proteins that are easily adsorbed to apatite to apatite and the cross-linking of proteins and glucanases occur simultaneously, resulting in immobilized enzymes. .

The present invention will be specifically described below with reference to Examples.

Example 1. Immobilization of levanase on hydroxyapatite Lysozyme 100'q, levanase 1o0111i.

was dissolved in 50 m of pure water, 2 tons of abrasive hydroxyapatite was added to the solution, and the mixture was cooled to 4°C. The solid was collected by stirring and washing with glumeraldehyde water bath solution (glutaraldehyde 28) 3 times while stirring vigorously at 4°C to obtain undried immobilized hydroxyapatite (in some cases, it may be used as is). This was freeze-dried to obtain a powder of approximately 2.059. This powder 1f was weighed and the pH was adjusted to 6.
, 8. Add 1 molar potassium phosphate buffer solution 10-, stir at room temperature for 1 hour, centrifuge to collect the solution, repeat the same operation twice for the remaining amount, combine the P solution and transfer to a lorry. - The amount of protein was measured by the method, and the remaining amount was washed with water, dried, and weighed. As a result, it was confirmed that hydroxyapatite was intact and protein 11.7aF was bound. When the levanase activity of undried immobilized hydroxyapatite was measured by the method described below, it was found that hydroxyapatite-binding protein fx? It was confirmed that 0.47 tons of leban was decomposed per unit.

Example 2. Immobilization of levanase on fluoroapatite A freeze-dried product 2.05F was obtained by operating under the same conditions as in Example 1, except that fluoroapatite was used instead of hydroxyapatite in Example 1. The bound protein was measured in the same manner as in Example 1, and it was confirmed that 13.4 WI of protein was bound per 2 fluoroapatites. As in Example 1, the levanase activity measurement result was 0.54 f per bound protein.
I learned that the Leban is disassembled.

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 in place of levanase in Example 1, and immobilized hydroxyapatite was obtained. As a result of testing with Example 1 and colleagues, it was found that protein J15.0 was bound per hydroxyapatite f. Furthermore, as a result of measuring the mutanase activity, it was confirmed that the hydroxyabatite binding protein 5Itts degraded 0,489 mutan.

Example 4. Fluoroapatite Henoimmobilization of Mutanase Immobilized 70 oloabatite was obtained by operating under exactly 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 the protein 1711q was bound to the fluoroapatite, and the binding protein f was 0.4
Approved to disassemble 5F Mutan.

Example 5. Immobilization of dextranase on fluoroapatite Lysozyme 50wy and dextranase 504 were mixed, and fluoroapatite 5f was added to the mixture.

Add 50° of a 0.05 molar, pi 6.8 potassium phosphate buffer solution, cool to 4°C and stir vigorously.

While maintaining this temperature, add 0.0% glutaraldehyde.
Add 125 μL of 2% water '#I solution dropwise while stirring, and after dropping
Stirring was continued for an hour. The reaction product was deposited and added to the above buffer bath solution 1.
After washing three times with 00- and water, undried fixed fluoroapatite was obtained. Dextranase-immobilized fluoroapatite s, ost was obtained by freeze-drying. Potassium phosphate buffer bath solution 2- with a 1 molar concentration of 9116.8 was added to the precipitate obtained by centrifuging the undried immobilized fluoroapatite 1-3.
The bound protein was desorbed by stirring for a period of time, and the mixture was centrifuged. The same procedure was repeated for precipitation. The F solution was combined and the protein was measured by the Lowry method. The precipitate was washed with water, dried, and weighed. As a result, 15.24 W of protein was bound to the fluoroapatite. Dextranase activity measurement result is 0.43 for binding protein? Decomposed dextran.

Each 8 [Experimental results are shown in Table 1. The processing conditions are the same as in Examples.

Measurement of each enzyme titer 1 td of the undried immobilized enzyme obtained in the 10 rd K experiment was added to each substrate solution, and after stirring at 37C for 2 hours, the monomers produced in the solution were fixed and filtered, while the undried Dry immobilized enzyme 1
Measuring the dK-bound protein by the method described above,
The titer of each solid-sludge-forming enzyme was determined from monomeric violet in which proteins bound to apatite were decomposed.

Dextranase uses dextran, mutanase uses mutan, and lebanase uses levano as substrates. Dextranase and mutanase decompose the glucose produced by the glucose oxidase method, and levanase decomposed the produced fructose using high-sensitivity liquid chromatography using a conventional method. (Column: Sugar Pack 1.114HIF: Water)
It was quantified by

Activity of immobilized enzyme over time Changes in the activity over time of the number of immobilized fermented confections 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.

Claims (1)

[Claims] 1. Apatite with immobilized glucanase, etc. Here, glucanase means levanase, mutanase, and dextranase, and apatite means hydroxyapatite and fluoroapatite. However, dextranase is immobilized only on fluoroapatite. 2. The method for producing immobilized apatite according to claim 1, which comprises dropping glutaraldehyde into a solution containing a mixture of glucanase, protein, and apatite. 3. The production method according to claim 2, which uses lysozyme as the protein.