JP3516104B2 - Mutanase producing microorganism and mutanase - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel mutanase-producing microorganism and a novel mutanase, and more particularly to a novel mutanase which belongs to the genus Bacillus obtained by separating from soil and produces mutanase by culturing the strain. The present invention relates to a novel mutanase which is obtained by culturing a producing microorganism and a strain of the microorganism and can be stably added to an oral composition such as toothpaste.

[0002]

2. Description of the Related Art Streptococcus mutans and Streptococcus sobrinas (Streptococcus mutans) which are bacteria present in the oral cavity
cus sobrinus) and dental plaque composed of insoluble glucan produced by these bacteria are presumed to be one of the factors for cariogenic formation. Caries is caused by the production of acid from bacteria at the plaque site, which in turn decalcifies teeth.

As one of the methods for removing plaque, it is effective to decompose insoluble glucan, which is a polysaccharide forming plaque, with an enzyme. In the insoluble glucan, glucose is mainly composed of α-1,6-glucoside bonds and α-1,3-
Glucans having glucosidic bonds are complexly entangled and formed, but conventionally, dextranase that cleaves α-1,6-glucosidic bonds and mutanase that cleaves α-1,3 bonds are each It is known to have an effect of suppressing plaque formation, and oral compositions containing these compounds alone have been proposed (Patent No. 782154, No. 1055365, etc.).

It is also known that the combined use of dextranase and mutanase, which have different points of action on plaque, provides a higher plaque formation-inhibiting effect than the use of each of them alone (Japanese Patent Laid-Open Publication No. Sho. 57-165312).

However, actually, the mutanases which have been put on the market so far are limited to a very small part, and the mutanase-containing composition which is put on the market as a product of oral composition such as toothpaste containing mutanase. Nor. The reason is that the currently marketed mutanases have insufficient stability required for incorporation into oral compositions such as toothpaste or mouthwash, such as insufficient heat resistance and stability in the composition. , Because it has not yet reached a stable supply of such products.

[0006] The present invention has been made in view of the above circumstances, and a novel mutanase and a soil which are effective in preventing plaque formation and removing plaque, and which can be stably incorporated into oral compositions such as toothpaste. It is an object of the present invention to provide a novel mutanase-producing microorganism belonging to the genus Bacillus obtained by isolation and culturing the strain to produce the above-mentioned mutanase.

[0007]

Means and Actions for Solving the Problems The present inventors have conducted diligent studies to achieve the above-mentioned object, and have excellent stability in the coexistence of the blending components of the oral composition such as toothpaste and mouthwash. As a result of broadly searching for microorganisms that produce mutanase, which has high activity and is highly effective for plaque decomposition, from the natural world, a new mutanase having a higher plaque removing ability than known mutanases and excellent thermal stability is obtained. The following microorganisms produced in the medium have been found, and the present invention has been completed.

That is, the present invention relates to Bacillus sp.
acillus sp. ) RM1 strain
No. 836), which provides a mutanase-producing microorganism having the following mycological properties. (I) Gram stainability is negative. (II) Has periflagellates and is motile. (III) Protease production is negative. (IV) Growth pH is 5-8.5, 25-60 degreeC. (V) On an agar medium, it grows in the presence of 2% sodium chloride and does not grow in the presence of 5% sodium chloride. (VI) Generates an acid from arabinose, xylose, fructose and mannose. (VII) Produces a mutanase having the following physicochemical properties. (1) Action: It has a property of decomposing the α-1,3-glucoside bond of mutan. (2) Substrate specificity: Decomposes mutan well. (3) Optimum action pH: 10 at 35 ° C with mutan as a substrate
When it is allowed to act for a minute, the action is optimum at pH 4, and high activity is exhibited in the range of pH 4 to 6. (4) Stable pH range: When kept at 25 ° C for 24 hours,
It is stable in the pH range of 4 to 10. (5) Optimum temperature: When mutan is used as a substrate and reacted at pH 5, the action is optimum at a temperature of 60 ° C. (6) Temperature stability: When heat-treated at pH 5 for 10 minutes,
The activity is stable at 60 ° C or lower, and deactivates at 75 ° C. (7) Effect of metal ion: When mutan is used as a substrate, the action is inhibited by mercury, silver or trivalent iron. (8) Effect of inhibitor: When mutan is used as a substrate, it is inhibited by p-chloromercury benzoic acid (PCMB). (9) Molecular weight: The molecular weight by SDS-polyacrylamide gel electrophoresis is about 150,000. Also provided is a mutanase having the above-mentioned physicochemical properties, and a method for producing mutanase, which comprises collecting mutanase from a culture obtained by culturing a strain of the mutanase-producing microorganism.

The present invention will be described in more detail below. The mutanase-producing microorganism of the present invention is isolated and collected from nature, belongs to the genus Bacillus, and has negative Gram stainability and negative protease production. And is characterized by producing a mutanase-producing microorganism having a growth pH of 5 to 8.5, and in particular, producing a mutanase having the above-mentioned characteristics (1) to (9), it is differentiated from other mutanase-producing microorganisms. The mycological properties of the strain are as follows.

The bacteriological properties and classification method are described in Bergey's Manual of S.
ystematic Bacteriology (1
984), The Genus Bacillus (The Genu
s Bacillus) (1973).

A. Morphological Properties The following morphological characteristics are observed when culturing on broth agar at 40 ° C. for 2 days. (1) Cell shape and size: It is a bacillus and has a size of 0.
6 to 1 μm × 3 to 4 μm. (2) Polymorphism: None. (3) Motility: Periflagellates and motility. (4) Spores: Spores are formed, the spores have an elliptical shape, and the site is neutral to sub-edible, and swelling of the sporangia is observed. The size of the spores is 1 μm × 1.2 to 1.7 μm. (5) Gram stainability: Negative. (6) Anti-acidity: Negative.

B. Culture property (1) Meat broth agar plate culture: Round, flat, and full-edge colonies are formed. The colony has a milky white to cream color in the central part and a translucent milky white to cream color in the peripheral part, and has light selection and viscosity. (2) Meat broth agar slant culture: grows in a wide band and forms white to cream colored colonies. (3) Broth liquid culture: Grows, but pellicle is not formed. (4) Meat broth gelatin stab culture: growing but not liquefied. (5) Litmus milk: No discoloration. Do not liquefy.

C. Physiological properties (1) Reduction of nitrate: negative (2) Denitrification reaction: negative (3) MR test: negative (4) VP test: negative (5) VP broth pH: 6.37 (6) Indole formation : Not detected. (7) Generation of hydrogen sulfide: Not detected. (8) Starch hydrolysis: positive (9) Utilization of citric acid: Not used in Simmons medium. Christensen
sen) Not used in medium. (10) Use of inorganic nitrogen source: Nitrate is used. Ammonium salt is used. (11) Pigment formation: Negative (12) Urease: Negative (13) Oxidase: Positive (14) Catalase: Positive (15) Range of growth: pH 5 to 8.5, grows at 25 to 60 ° C. (16) Attitude toward oxygen: Aerobic (17) OF test: Aerobic decomposition (18) Generation of acid and gas from sugar: As shown in Table 1 below. In addition, in Table 1, "+" indicates that an acid or gas is generated, and "-" indicates that an acid or gas is not generated.

[0014]

[Table 1]

D. Other properties (1) Tolerance of sodium chloride: 2% in agar medium
It grows in the presence of sodium chloride and does not grow in the presence of 5% sodium chloride. (2) Growth under anaerobic: Negative (3) Casein decomposition: Negative (4) Utilization of propionate: Negative (5) Growth at pH 6.8: Positive (6) Growth at pH 5.7: Positive ( 7) Crystal formation: negative (8) Protease production: negative

Next, the difference between the strain of the mutanase-producing microorganism of the present invention and a known strain will be described. Known mutanase-producing microorganisms include bacteria Pseudomonas, Flavobacterium, Bacillus, actinomycete Streptomyces, fungi Trichoderma, Aspergillus, and the like.

First, the Bacillus circulans strain (Bac) which is the same Bacillus strain as the mutanase-producing microorganism of the present invention is used.
illus circulans) BC-8 (hereinafter,
BC-8 bacterium) (Japanese Unexamined Patent Publication No. 63-301788), the mutanase-producing microorganism of the present invention is negative in Gram stainability, whereas BC-8 bacterium is positive. In addition, the mutanase-producing microorganism of the present invention has a spore site that is neutral to sub-edge, whereas BC-8 is sub-edge. Furthermore, while the mutanase-producing microorganism of the present invention is negative in the viability of 5% NaCl, BC-8
The bacterium is positive. The mutanase-producing microorganism of the present invention is L-
Acid production from arabinose is positive, whereas BC
-8 is negative and the mutanase-producing microorganism of the present invention is positive for acid production from D-xylose, whereas
C-8 is negative. The mutanase-producing microorganism of the present invention is positive in its viability at 45 ° C., whereas BC-8 is negative.

On the other hand, Bacillus circulans (Ba) belonging to the same genus Bacillus as the mutanase-producing microorganism of the present invention.
cillus circulans) FERM-P4
The 765 strain (hereinafter referred to as the FERM-P4765 strain) (Japanese Patent Laid-Open No. 55-88693) has unknown mycological properties, but the mutanase produced by the mutanase-producing microorganism of the present invention has a molecular weight of Is about 150,000, the optimum temperature is 60 ° C, and the optimum pH is 4, while FERM
The mutanase produced by the P4765 strain has a molecular weight of 70,000 or more, an optimum temperature of 30 to 40 ° C., and an optimum pH of 6.2 to 6.
7, the mutanase-producing microorganism of the present invention is F
Differentiated from ERM-P4765 strain.

Further, the mutanase-producing microorganism described in JP-A-52-34980 belongs to the genus Streptomyces, and the mutanase-producing microorganism described in JP-A-50-145583 belongs to the genus Flavobacterium. Since the mutanase-producing microorganisms described in JP-A-47-9743 are Trichoderma harzianum, Penicillium lilacinum, Penicillium funiculosum, Penicillium melinii, and Penicillium yansinerum, the mutanase-producing microorganisms of the present invention are Differentiated from these known mutanase-producing microorganisms.

From the above points, the strain of the mutanase-producing microorganism of the present invention is different from known strains.

The present inventors have developed a novel mutanase-producing microorganism, Bacillus sp.
p. ) The RM1 strain (hereinafter referred to as the RM1 strain) was deposited at the Institute of Biotechnology, National Institute of Industrial Science and Technology as Life Research Institute No. 14836 (FERM P-14836). This R
The M1 strain has the above-mentioned mycological properties. The soil was collected from this soil in Ninomiya Town, Naka-gun, Kanagawa Prefecture, and the soil was collected in 1994.

The RM1 strain is isolated and collected from nature by the screening method described below.

One spatula of soil is suspended in 5 ml of sterilized physiological saline, and 100 μl of the suspension is applied to an agar medium containing mutan. By culturing at 45 ° C for 3 to 5 days and separating transparent colonies around the colony as mutanase-producing microorganisms, and examining the characteristics of the enzyme obtained by culturing, excellent plaque-removing ability and stability The RM1 strain having the sex was selected.

Mutanase is produced by inoculating this RM1 strain in a natural or synthetic medium and culturing under ordinary culture conditions. The medium is usually a microorganism such as carbon source, nitrogen source, inorganic salt or the like. Those containing components necessary for growth are preferable, and those generally used by microorganisms can be used as the nutrient source of the medium. Examples of such nutrient sources include glucose, inositol, fructose, and mutan. , Starch, etc., nitrogen sources such as peptone, soybean flour, yeast extract, etc., inorganic salts such as phosphoric acid,
Examples thereof include salts such as sodium, potassium and magnesium, and these may be used alone or in appropriate combination of two or more, and among these, inositol, mutan, peptone, phosphate and the like are preferably used. To be done. The pH of the culture solution is preferably 5 to 8.5, and particularly pH 7
Is suitable. The culture temperature is preferably 35 to 48 ° C, and particularly preferably 40 ° C. The culture time varies depending on the culture conditions, but is usually preferably 1 to 3 days.

Next, a supernatant obtained by removing the bacterial cells and the like from the culture solution of the RM1 strain in which mutanase is sufficiently produced by centrifugation or the like is obtained as a crude enzyme solution of mutanase of the present invention. This crude enzyme solution can be used as it is, but further known separation and purification methods such as ultrafiltration membrane concentration method, vacuum concentration method, salting out method using ammonium sulfate, fractionation method using ethanol, etc. It is preferable that the purified enzyme solution is used alone or by appropriately combining the focusing method, the column chromatography fractionation method and the like. This purification process is SD
It is desirable to carry out until it can be confirmed by S-polyacrylamide gel electrophoresis that a single band has been purified.

In addition to the RM1 strain described above, an RM1 mutant strain or a recombinant microorganism having the mutanase gene can be used for the production of the mutanase of the present invention.

Next, the novel mutanase of the present invention has the above characteristics (1) to (9), which will be described in detail below.

First, the mutanase is an insoluble glucan (α-1,3-1,6-) produced from sucrose by oral streptococci, particularly Streptococcus mutans and Streptococcus sobrinus which are caries fungi.
(Glucan) is an enzyme that uses mutan, which is an insoluble α-1,3-glucan obtained by removing the side chain of α-1,6 bond, as a substrate. The novel mutanase of the present invention has the following physicochemical properties. Is. The mutan used in the following properties as a substrate is obtained by a known method, for example, α-1,6-glucoside of insoluble glucan produced by culturing Streptococcus mutans or Streptococcus sobrinus. It is obtained by cleaving the bond with dextranase.

(1) Enzymatic action Mutanase (α-1,3-glucanase) has an exo-type which is cleaved from the end and an endo-type which is randomly cleaved according to the cleavage pattern of its glucoside bond. Is presumed to have the property of decomposing the α-1,3-glucoside bond of mutan into endo type.

That is, 50 μl of 100% 3% mutan
Of the present invention, or Novozyme 234 (trade name: manufactured by Novo Co., derived from Trichoderma harzianum) that decomposes the α-1,3-glucoside bond of mutanase or mutan of the present invention is added, and incubated at 37 ° C. for 20 hours. After reaction, the reaction product was centrifuged and the supernatant obtained was spotted on a thin layer plate and developed (1-butanol: isopropanol: water = 4: 7: 1), and concentrated sulfuric acid was applied to the thin layer plate. Is sprayed and heated at 100 ° C. for 5 minutes to analyze the saccharide produced by the above reaction by TLC. In this case, the mutanase decomposition product of the mutanase of the present invention is randomly decomposed, and a long strip-shaped spot is detected at a position having a lower mobility than glucose, whereas the mutan decomposition product of Novozyme 234 decomposes the glucose spot. From the above, it is presumed that the mutanase of the present invention is an endo-type enzyme that randomly decomposes a substrate.

(2) Substrate specificity The mutanase of the present invention decomposes mutan well. That is, 100 μl of the mutanase solution of the present invention (500 units / ml)
In contrast, 100 μl of each substrate solution shown in Table 2 (1% solution)
Is reacted for 30 minutes, and the amount of reducing sugar is detected according to the method for measuring the enzyme activity shown below. The results are also shown in Table 2.
The relative degree of water hydrolysis in Table 2 means the relative value of the reducing sugar amount of each substrate when the amount of free reduction for mutan decomposition of mutanase is 100. Method of measuring enzyme activity 100 μl of mutanase solution was added to 100 μl of 3% mutan (0.1 M acetate buffer, pH 5), and the mixture was incubated at 35 ° C. for 1 hour.
Let react for 0 minutes. During this, shake at 100 times / minute. The reaction was stopped with 0.4 ml of a DNS solution (4,6-dinitrosalicylic acid), centrifugation was performed, and the supernatant was added to 0.5
Transfer to a ml test tube, place a glass ball on it, and perform 10 at 100 ° C.
Heat for minutes. Cool in running water for 5 minutes, dilute with 4 ml of distilled water and measure the absorbance at OD ₅₃₀ . 0 to 90 μg
The titer is determined from the calibration curve of the glucose standard solution of. The amount of enzyme that produces 1 μg of reducing sugar per minute is defined as 1 unit. The enzyme activities in the following physicochemical properties are measured by this method.

[0032]

[Table 2]

(3) Optimum pH of action The mutanase of the present invention was prepared by mixing 50% of the mutanase of the present invention in 3% mutan adjusted to each pH with the following buffers.
And react at 35 ° C. for 10 minutes to measure the activity, and p for each activity when the activity at the optimum pH is 100%.
When determining the relative activity at H, as shown in FIG.
4, the action is optimum, pH 4 to 7, especially pH 4
High activity is shown in the range of ~ 6. Buffer pH 3 to 7: Citric acid-Na ₂ HPO ₄ buffer pH 6 to 8: NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer pH 9 to 10: Glycine-NaOH buffer pH 10 to 11: Na ₂ HPO ₄ -NaOH buffer liquid

(4) Stable pH range For the mutanase of the present invention, the activity of the mutanase of the present invention was measured after adding the mutanase of the present invention to 300 units / ml in 20 mM of each of the above buffers and incubating at 25 ° C. for 24 hours. At 100% of the activity before incubation
As shown in FIG. 2, the relative activity at pH 4 to
Approximately 100% residual activity is shown in the range of 10 and pH
3, the residual activity rate of 90% or more is exhibited even at pH 11.

(5) Optimum temperature The mutanase of the present invention is prepared by adjusting the concentration of the mutanase enzyme of the present invention to 50 units / ml in a 20 mM acetate buffer of pH 5 containing 3% of the substrate mutan in the whole solution. Addition and reaction at each temperature for 10 minutes
When the relative activity at each temperature is determined as 0%, the action is optimum at a temperature of 60 ° C. as shown in FIG.

(6) Temperature stability The mutanase of the present invention is 20 mM acetate buffer (pH 5).
When 300 units / ml of the mutanase of the present invention dialyzed in is added, and the mixture is heat-treated at each temperature for 10 minutes and ice-cooled, the enzyme activity before heat treatment is taken as 100% to determine the residual activity rate,
As shown in FIG. 4, the residual activity rate becomes 30% at 65 ° C. and deactivates at 75 ° C., but the residual activity rate at 30 to 60 ° C. is 100%.

(7) Effect of metal ion The mutanase of the present invention is 50 mM acetate buffer (pH 5).
Was used to prepare the mutanase of the present invention at 50 units / ml, various metal salts were added to this to a final concentration of 1 mM, and the mixture was incubated at 35 ° C. for 30 minutes,
The activity of each solution after the treatment was measured, and the relative activity in the solution to which various metal salts were added with the activity without addition of metal salt as 100% was determined. As shown in Table 3, mercury, silver or trivalent iron was added. It is recognized that the addition of the enzyme inhibits the enzyme activity of the mutanase of the present invention.

[0038]

[Table 3]

(8) Effect of inhibitor The mutanase of the present invention is 50 mM acetate buffer (pH 5).
Was used to prepare the mutanase of the present invention at 50 units / ml, various inhibitors were added to this so that the final concentration was 1 mM, and the mixture was incubated at 35 ° C. for 30 minutes, and then each solution was added. The residual activity of p-chloromercury benzoic acid (PCM)
It is observed that the addition of B) inhibits the enzyme activity particularly strongly.

[0040]

[Table 4]

(9) Molecular weight The mutanase of the present invention was electrophoresed for 1 hour at 40 mA using a 5 to 20% polyacrylamide gradient gel (ready-made gel manufactured by Bio-Rad Co., Ltd.), and sigma marker. When the molecular weight of the enzyme protein purified by SDS-polyacrylamide gel electrophoresis using High Range (manufactured by Sigma) is determined, the molecular weight is calculated to be about 150,000.

The mutanase of the present invention is not limited in its production method as long as it has the above-mentioned physicochemical properties, and is not limited to that produced from the RM1 strain and the like as described above, but is produced from, for example, the RM1 strain. Mutanase is also modified into a more stable and effective enzyme by chemical modification or gene recombination.

The mutanase of the present invention has the above-mentioned physicochemical properties, and in comparison with known mutanases, the mutanase of the present invention and the above-mentioned novozyme 234 have a α-1,3-glucoside bond of mutan. The cutting patterns are different. The mutanase of the present invention has an optimum pH of pH 4 and an optimum temperature of 60 ° C., while the above BC-
Mutanase derived from 8 bacteria has a pH of 4.5 and an optimum temperature of 45 to
It is 50 ° C. Further, as described above, the mutanase of the present invention has a molecular weight of about 150,000, an optimum temperature of 60 ° C. and an optimum pH of 4, whereas the FERM-P4765 described above.
The strain-derived mutanase has a molecular weight of 70,000 or more and an optimum temperature of 30.
-40 degreeC and optimal pH are 6.2-6.7. Furthermore, the mutanases described in JP-A Nos. 52-34980, 50-145583, and 47-9743 are microorganisms belonging to the genus Streptomyces or the genus Flavobacterium, or Trichoderma harzianum, Penicillium lilacinum, It is produced by Penicillium funiculosum, Penicillium melinii, and Penicillium yansinerum. For example, the mutanase of the present invention has an optimum pH of 4 and a stable pH range of 4 to 10, whereas JP-A-52-34980. The mutanase disclosed in Japanese Patent Laid-Open No. 50-55 has an optimum pH of 5.5 and a stable pH range of 5 to 7,
The optimum pH of the mutanase disclosed in Japanese Patent No. 145583 is 6.0,
The stable pH range is 5.0 to 7.0, and their physicochemical properties differ in many respects. Incidentally, JP-A-47-9743
The physicochemical properties of the mutanase described in the publication are unknown.

The mutanase of the present invention is a toothpaste (toothpaste, toothpaste,
Powder toothpaste, liquid toothpaste), mouthwash, denture cleaner, mouthwash tablet, gingival massage cream, troche, oral pasta and other oral compositions can be compounded and applied.
In contrast, when it is blended alone or in combination with dextranase so as to be 1 to 50,000 units, excellent plaque formation inhibitory action and plaque removing ability are exhibited.

[0045]

EXAMPLES The present invention will be specifically described below by showing Examples and Comparative Examples, but the present invention is not limited to the following Examples. (1) Preparation of Mutan First, the mutan used in this example was prepared as follows. Streptococcus sobrinus (St
reptococcus sobrinus) 6715
The strain was added to 200 ml of BHI medium (Brain Heart Infusion).
n) manufactured by Oxoid Co., Ltd., and cultured at 37 ° C. for 1 day. 5% of the obtained culture is prepared in a volume of 4 liters.
Inoculate BHI medium containing sucrose and incubate at 37 ° C for 5
Culture was carried out for one day. Harvest this precipitate, 500 ml
Was dissolved in 1N aqueous sodium hydroxide solution and left at room temperature for 1 hour. The centrifugation supernatant was neutralized with hydrochloric acid, and the deposited precipitate was collected by filtration. Further, dextranase was allowed to act on this recovered product to cleave the α-1,6-glucoside bond.

(2) Preparation of Mutanase Next, the mutanase of this example was prepared as follows. First, a medium was prepared with the following culture composition. Culture composition Inositol 10 g / liter endless 1 g / l peptone 5 g / l yeast extract 3 g / l KH ₂ PO ₄ 2g / l NH ₄ NO ₃ 2g / l MgSO ₄ · 7H ₂ O 0.3g / l (pH 7.0)

The RM1 strain obtained by the above-mentioned screening method was inoculated into the above medium and cultured at 40 ° C. for 2 days, and then the culture solution was centrifuged at 8000 rpm for 15 minutes to obtain a supernatant, which was ultrafiltered. Concentrated in. Next, ammonium sulfate was added to this supernatant so as to be 90% saturated, the mixture was stirred well, and allowed to stand in ice for 1 hour or more, and then
Salting out was performed by centrifuging and collecting the precipitate.
Then, the cells were suspended in 10 mM Tris-HCl buffer (pH 8) and dialyzed with the same buffer. 8000 samples after dialysis
Centrifugation was carried out for 10 minutes at rpm, and the resulting supernatant was designated as the mutanase of this Example (hereinafter referred to as RM1 enzyme),
The purification was performed as follows. First, the anion exchange chromatography DE52 (trade name: manufactured by Whatman) was used to obtain a flow-through fraction at pH 8 and then pH 8.5.
The column operation was performed twice by adsorbing with. Anion exchange chromatography DE52 The flow-through fraction was dialyzed against 10 mM Tris-HCl buffer (pH 8.5) and equilibrated with the same buffer. Anion exchange chromatography D
I went to E52. The adsorbed enzyme was eluted with a 0 to 0.2 M sodium chloride solution to obtain a purified enzyme.

When the RM1 enzyme thus purified was used to confirm the above physicochemical properties, it was confirmed that the mutanase of the present invention has the above physicochemical properties. This purified RM1 enzyme was used for the following enzymatic characterization.

(3) Comparison with commercially available enzyme Optimal p of the above-mentioned Novozyme 234 (comparative example), which is a mixed enzyme containing RM1 enzyme and currently marketed mutanase.
H, pH stability, optimum temperature and temperature stability were measured in the same manner as above. The results are shown in FIGS. According to FIGS. 5-8, it is recognized that the RM1 enzyme is clearly more stable than Novozyme 234.

(4) Enzyme action characteristics An enzyme sample prepared by repeatedly making a hole having a diameter of 3 mm and a depth of 1 mm in an agar plate containing mutan and diluting an initial value of 100 units of RM1 enzyme or Novozyme 234 by 2 times, that is, 5 μl of each 1, 2, 4, 8-fold diluted each enzyme sample was injected, and after incubating at 30 ° C. for 24 hours, a transparent decomposition zone by each enzyme sample was examined. The results are shown in Table 5.

[0051]

[Table 5]

According to Table 5, the RM1 enzyme shows a higher mutan degrading ability than Novozyme 234 of the same enzyme unit,
It is considered that mutanase, which decomposes mutan into endo type, has higher decomposition efficiency than exo type.

(5) Effectiveness evaluation of plaque removing ability Streptococcus mutans (Streptoc)
occus mutans) 10449 strain to BHI (B
in the Rain Heart Infusion medium 3
The mixture was left to stand overnight at 7 ° C to prepare a preculture liquid. 100 μl of this preculture liquid was added to 3 m of BHI medium containing 1% sucrose.
1 (M2 test tube), the test tube was tilted, and static culture was performed overnight at 37 ° C. to form plaque on the wall of the test tube. Then, discard the medium, wash the inside of the test tube twice with distilled water, and
l of artificial saliva (0.85 mM CaCl ₂ /6.22 m
_{M KH 2 PO 4 / 50mM NaCl} / 0.15mM
After adding MgCl ₂ (pH 5.94), 1 ml of each enzyme shown in Table 6 was added, and the mixture was kept at 37 ° C. for 3 minutes. After that, the enzyme solution was discarded, the inside of the tester was washed twice with distilled water, and then 4 ml of distilled water was added, and the turbidity of each solution after ultrasonic treatment was measured and no enzyme was added. Using the one as a control, the plaque removal ability of each enzyme solution was calculated. In addition, in the study of the combined use system with dextranase, an enzyme solution prepared by adding artificial saliva and adjusting RM1 enzyme or Novozyme 234 to 10 units per 2000 units of dextranase was used. The results are shown in Table 6.

[0054]

[Table 6]

From the results shown in Table 6, it was confirmed that mutanase exhibits synergistic plaque removal ability when used in combination with dextranase, and that the effect of RM1 enzyme is superior to that of Novozyme 234. Endo type mutanase is considered to be suitable as a plaque removing enzyme.

(6) Stability in the formulation 10 g of a commercially available toothpaste was added to RM1 enzyme or Novozyme 2
200 units of 34 are added respectively, and each toothpaste is mixed for 10 minutes, and 0.8 g of each toothpaste is sampled daily at 37 ° C. for about 1 month, and a phosphate buffer solution of pH 8 2 4 ml, centrifuged and centrifuged, and the supernatant was used as a sample, and the enzyme activity of each sample was measured in the same manner as in the above measuring method to determine the residual activity. The results are shown in Table 7.

[0057]

[Table 7]

The RM1 enzyme was added to powdered toothpaste, liquid toothpaste, mouthwash, denture cleaner, gargle tablet, gingival massage cream, troche, oral pasta having a general composition in an amount of 200 per 10 g of each composition. Excellent stability was also exhibited in the preparations formulated as a unit alone or in combination with dextranase.

[0059]

EFFECTS OF THE INVENTION According to the mutanase-producing microorganism of the present invention, not only it has an excellent effect of inhibiting plaque formation, but it can be added in a sufficiently stable manner as a product of an oral composition such as toothpaste. Mutanase can be obtained. The mutanase of the present invention, in combination with dextranase,
It shows a synergistic plaque removal effect.

[Brief description of drawings]

FIG. 1 is a graph showing the optimum pH of mutanase of the present invention.

FIG. 2 is a graph showing pH stability of the mutanase of the present invention.

FIG. 3 is a graph showing the optimum temperature of the mutanase of the present invention.

FIG. 4 is a graph showing the temperature stability of the mutanase of the present invention.

FIG. 5 is a graph comparing the optimum pHs of the mutanase of the present example and the mutanase of the comparative example.

FIG. 6 is a graph comparing the pH stability of the mutanase of this Example with that of the mutanase of Comparative Example.

FIG. 7 is a graph comparing optimum temperatures of the mutanase of this example and the mutanase of a comparative example.

FIG. 8 is a graph comparing the temperature stability of the mutanase of the present example and the mutanase of the comparative example.

Continuation of front page (51) Int.Cl. ⁷ identification code FI C12R 1:07) (72) Inventor Yuu Shimotsuura 1-3-7 Main Office, Sumida-ku, Tokyo Within Lion Corporation (56) References JP 63-301788 (JP, A) US Pat. No. 5,290,916 (US, A) Journal of General Microbiology (1980), Vol. 114, No. 1, p. 197-208 Journal of Ferment Bioeng. (1997), Vol. 83, No. 6, p. 593-595 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 1/00 C12N 9/00 C12P 21/00 CA (STN) BIOSIS / MEDLINE / WPID S (STN) JSTPlus (JOIS)

Claims (3)

(57) [Claims] 1. Bacillus sp.
sp. ) Mutantase-producing microorganism having the following mycological properties, which is an RM1 strain (Life Science Research Institute No. 14836). (I) Gram stainability is negative. (II) Has periflagellates and is motile. (III) Protease production is negative. (IV) Growth pH is 5-8.5, 25-60 degreeC. (V) On an agar medium, it grows in the presence of 2% sodium chloride and does not grow in the presence of 5% sodium chloride. (VI) Generates an acid from arabinose, xylose, fructose and mannose. (VII) Produces a mutanase having the following physicochemical properties. (1) Action: It has a property of decomposing the α-1,3-glucoside bond of mutan. (2) Substrate specificity: Decomposes mutan well. (3) Optimum action pH: 10 at 35 ° C with mutan as a substrate
When it is allowed to act for a minute, the action is optimum at pH 4, and high activity is exhibited in the range of pH 4 to 6. (4) Stable pH range: When kept at 25 ° C for 24 hours,
It is stable in the pH range of 4 to 10. (5) Optimum temperature: When mutan is used as a substrate and reacted at pH 5, the action is optimum at a temperature of 60 ° C. (6) Temperature stability: When heat-treated at pH 5 for 10 minutes,
The activity is stable at 60 ° C or lower, and deactivates at 75 ° C. (7) Effect of metal ion: When mutan is used as a substrate, the action is inhibited by mercury, silver or trivalent iron. (8) Effect of inhibitor: When mutan is used as a substrate, it is inhibited by p-chloromercurybenzoic acid. (9) Molecular weight: The molecular weight by SDS-polyacrylamide gel electrophoresis is about 150,000. 2. A mutanase obtained from a culture obtained by culturing a strain of the mutanase-producing microorganism according to claim 1, and having the following physicochemical properties. (1) Action: It has a property of decomposing the α-1,3-glucoside bond of mutan. (2) Substrate specificity: Decomposes mutan well. (3) Optimum action pH: 10 at 35 ° C with mutan as a substrate
When it is allowed to act for a minute, the action is optimum at pH 4, and high activity is exhibited in the range of pH 4 to 6. (4) Stable pH range: When kept at 25 ° C for 24 hours,
It is stable in the pH range of 4 to 10. (5) Optimum temperature: When mutan is used as a substrate and reacted at pH 5, the action is optimum at a temperature of 60 ° C. (6) Temperature stability: When heat-treated at pH 5 for 10 minutes,
The activity is stable at 60 ° C or lower, and deactivates at 75 ° C. (7) Effect of metal ion: When mutan is used as a substrate, the action is inhibited by mercury, silver or trivalent iron. (8) Effect of inhibitor: When mutan is used as a substrate, it is inhibited by p-chloromercurybenzoic acid. (9) Molecular weight: The molecular weight by SDS-polyacrylamide gel electrophoresis is about 150,000. 3. A method for producing mutanase, which comprises collecting mutanase from a culture obtained by culturing a strain of the mutanase-producing microorganism according to claim 1.