JPH0317100A - Substance capable of inhibiting glucosyltransferase - Google Patents

Abstract

NEW MATERIAL:A substance obtained by covalently bonding a protein having about 4.5 isoelectric point or a Maillard reaction product thereof to dextran having 1000-2000000 molecular weight. Molecular weight; >=30000 [the sodium dodecyl sulfate(SDS) electrophoresis method]. Isoelectric point; about pH3.5 (the isoelectric focusing electrophoresis method). Protein content; 10-90% (the Lowry method). Sugar content; 10-90% (the phenol-sulfuric acid method). Solubility; readily soluble in water and insoluble in methanol, acetone, etc. Color reaction; positive to the biuret reaction, xanthoprotein reaction, etc. Action; inhibiting reaction for synthesizing adhesive and insoluble glucan by action of glucosyltransferase on sucrose. Distinction of acidic, basic and neutral; a weakly acidic aqueous solution. Color; white or light brown, etc. USE:An active ingredient of anticarious agents. PREPARATION:A protein having about 4.5 isoelectric point (e.g. casein) or a Maillard reaction product thereof is bonded to activated dextran.

Description

[Detailed description of the invention]

(1) Industrial field in Icheon The present invention is directed to the use of Streptococcus mutans, which is a caries-causing bacterium.
>glucosyltransferase (hereinafter referred to as 'G') produced by
This invention relates to an inhibitor of Tase, a method for producing the same, and a caries prevention agent containing the same as an active ingredient. (2) Conventional technology In the oral cavity, Streptococcus mutans synthesizes adhesive and insoluble glucan from sucrose to form dental plaque, which is a bacterial flora, on the tooth surface. It has been revealed that the production of acid by bacteria within dental plaque is the cause of dental caries. Traditionally, it has been used as one of the methods of caries prevention. Streptococcus mutans G. It has been thought that plaque formation can be suppressed by inhibiting Tase. As GTase inhibitors derived from natural products,
6-10319, JP 57-98215, JP 5 7 - 9 9 5 1 8 JP 61-4
7515, JP-A-58-12i218, JP-A-60-154312, etc. In addition, the inventors of the present application have discovered that Maillard reaction products (hereinafter abbreviated as 'MRP') between specific proteins such as casein and sugars having reducing properties have GT-ase inhibitory activity, and have filed a patent application. A patent application was filed in 1981-118184. (3) Problems to be Solved by the Invention GTase may be activated by ingredients contained in foods. Therefore, under conditions where these components are present at high concentrations, it is thought that the inhibitory activity of the inhibitor is difficult to express. In the case of MRP, the raw protein itself is
Since these proteins have the property of activating GTase, in the presence of an excessive amount of these proteins, the expression of their inhibitory activity is suppressed, and the caries prevention effect is limited. The inventors believe that GTa
Even in the presence of proteins that have the property of activating se,
Research was conducted to obtain a GTase inhibitor that exhibits an effective caries prevention effect. (4) Means for solving the problem A protein-dextran conjugate with enhanced binding strength to GTase by binding dextran, which has strong binding strength to GTase, to a protein or MRP; Alternatively, an IARP-dextran conjugate is used. These conjugates can maintain G even in the presence of large amounts of protein.
We conducted research and completed the present invention with the idea that Tase inhibitory activity would be fully expressed. In order to produce the substance of the present invention, it is preferable that the protein has an isoelectric point of 4 to 5, such as casein, albumin, or MRP that is similar to these proteins. By covalently bonding dextran to L(R P derived from R
It is refined. The dextran used in the present invention has a molecular weight of 1.
000~2. OOG, 000 commercially available dextran can be used. Specifically, examples include Dextran T-10 to T-2000 manufactured by Pharmacia. In addition, the above protein and its M R P are p }
Precipitation occurs in the pH range of f 4 to 5, but by binding dextran, precipitation does not occur in this pH range, so the range of use for acidic foods is expanded. To measure GTase inhibitory activity, Streptococcus mutans (
Streptococcus mutans B-13
> from the culture iP solution using the method of Fukushima et al. (IRC3 Med.
Sci. ．． 13. 501 (1976)) using the method of Koga et al.
-fect. l+nmun. 28, 882
(1982)). MRP was performed using the method of Kobayashi et al. (Agric. Biol.
Chem. ．． 523169 (1988)) was used. (4> Examples Hereinafter, details of the substance of the present invention, its production method, the GTase inhibitory effect, and the effect of the dental caries preventive agent containing the substance of the present invention in inhibiting dental plaque formation against Bacillus attenuans will be explained using examples. Example 1> Marshall et al.'s method (J. Biol. C
helQ. , 251, 1081 (1976))
According to 2. 5g nodextran'r-10 (7y
(manufactured by Lumacia, molecular weight 10,000) at pH 10.
7 was dissolved in 250 ml of an aqueous solution of 0.7, while keeping the pH at 10.7. Add 625 g of cyanogen bromide twice,
Activated Dex1 Run. 0.25 g of casein (manufactured by Sigma) was added to this activated dextran, reacted at 4°C for 12 hours, and dialyzed.
& 2 g of glycine was added to stop the reaction. The activated dextran and casein binding substance produced as a result of the reaction was passed through the gel with Bio-Gel A0.5m.
The active fraction located in the void volume was collected, and proteins and unreacted dextran were removed. The obtained active fraction contains approximately 50× protein and approximately 50× sugar.
It contained. 0.50 to 1 g of this active fraction (0.25 μg of protein) has an adhesive property of 1 mg in 16 hours.
GTase that synthesizes insoluble glucan, 50? <Inhibition, even in the presence of high concentration casein (500 μg/ml),
The inhibitory activity remained unchanged (see Figure 3). Example-
2> In the same manner as in Example-1, α
0.25 g of MRP obtained by the Maillard reaction of 5-casein and glucose was combined. The reaction product is. B
The active fraction located at the void volume was collected by gel p-filtration using io-Gel A5@, and MRP and unreacted dextran were removed. The obtained active fraction is approximately 50X
The GTase inhibitory activity of MRP was measured in the presence and absence of casein and compared with that of MRP, as shown in FIG. In the absence of casein, MRP is 0.26 μg (0
．． 25 μg protein), the activity of GTase was increased by 50
In the presence of a high concentration of casein (500
MRP-
The active fraction of the dextran conjugate has a high concentration of casein (5
Even in the presence of 0.00 μg/ml), 0.55 μg (
0.28μg protein>, the activity of GTase was increased by 5
0〉=Inhibited. Example 3 Streptococcus muta, a caries-causing bacterium
IngbritJ GS-5, P belonging to ns
s-14, MT6265, MT8148.
6715 was added with 10 mg/ml of sucrose and the casein-dextran conjugate obtained in Example 1, or 1 mg/ml of the N+ R P-dextran conjugate obtained in Example-2. After culturing in Plain Heart Infusion Medium at 37°C for 18 hours, the bacterial cells grew to 2m young. The results are shown in Table 1 J3 and Table 2. Significant inhibition of bacterial cell adhesion was observed for all strains.

[Left below]

(5) Effects of the Invention The present invention provides a novel GT-se inhibitor in which dexI/lan is covalently bonded to a protein or its Lf RP, and also provides a method for producing the same, and a method for producing the same. It has now become possible to provide a caries preventive agent that contains as an active ingredient.

[Brief explanation of the drawing]

Figure 1a shows the ultraviolet absorption spectrum of the casein-dextran conjugate. In addition, FIG. 1b shows the ultraviolet absorption spectrum of the casein-derived MRP/dextran conjugate. In both a and b, the vertical axis is the absorbance, and V! The axis is wavelength (nm).6 Figure 2a shows the infrared absorption spectrum of the casein-dextran bond. Also. Figure 2b shows the infrared absorption spectrum of the casein-derived MRP/dextran conjugate. In both a and b, the vertical axis is the absorbance, and the horizontal axis is the wave number (cm
Figure 3 shows the relationship between the amount added and the amount of soluble/insoluble glucan synthesized in the casein/dextran conjugate. In the figure, curve 1 indicates that only the casein/dextran conjugate exists. Curve 2 is the case when casein coexists.The vertical axis is the rate of adherent/insoluble glucan synthesis (5=.), and the horizontal axis is the rate of addition (μg/protein/ml). Figure 4 shows the relationship between the amount added and the adhesive/insoluble glucan combination ti in casein-derived MRP/dextran conjugate.In the figure, curve 1 is the case in which only casein-derived MRP/dextran conjugate is present. , Curve 2 is the case where casein coexists with this. Curve 3 is the case where only casein-derived MRP itself is present, and curve 4 is the case where casein coexists with it. The vertical axis is the adhesive property. - Amount of insoluble glucan synthesized (X>, the horizontal axis is the amount added (μg/glycoprotein/ml).

Claims (3)

[Claims] (1) A protein with an isoelectric point of around 4.5 or its Maillard reaction product has a molecular weight of 1,000 to 2,000.
0,000 dextrans are covalently bonded,
A substance that inhibits glucosyltransferase with the following physicochemical properties. a. Molecular weight: approximately 30,000 or more (SDS electrophoresis method) b. Isoelectric point: around pH 3.5 (isoelectric focusing method) c. Protein and sugar content Protein: 10-90% (Lowry method) Sugar: 10-90% (phenol-sulfuric acid method) d. Solubility: Easily soluble in water, insoluble in methanol, ethanol, acetone, chloroform, and ethyl acetate. e. Color reaction: Positive for all of the Buret reaction, xanthoprotein reaction, Folin reaction, and phenol-sulfuric acid reaction. f. Action: Inhibits the reaction of glucosyltransferase to synthesize adherent/insoluble glucans using sucrose as a substrate. g, pH stability: pH 2-10, stable at 30°C for 30 days. h. Thermal stability: Stable against heating at 100°C for 4 hours (pH 2-8). i. Stability against chemical treatments: Stable against oxidation by oxidizing agents (ozone, hypochlorous acid, etc.) and reduction by reducing agents (sulfite, sodium borate, etc.). j. Melting point: No clear melting point is shown. k, Distinction between acidic, basic, and neutral: Aqueous solutions exhibit weak acidity. l. Color of substance: white to light brown. m, the ultraviolet absorption spectrum is as shown in Figure 1 (a or b). n, the infrared absorption spectrum is as shown in Figure 2 (a or b). (2) A substance that inhibits glucosyltransferase having the physicochemical properties described in claim 1 by covalently bonding dextran to a protein with an isoelectric point of around 4.5 or its Maillard reaction product. 1. A method for producing a glucosyltransferase inhibitor, which comprises producing a glucosyltransferase inhibitor and separating the substance from a reaction solution. (3) A glucosyltransferase inhibitor produced by covalently bonding dextran to a protein with an isoelectric point of around 4.5 or its Maillard reaction product and having the physicochemical properties described in claim 1. An anti-caries agent containing as an active ingredient.