JPH01187084A - Bacteria-bound type glucosyltransferase - Google Patents

Abstract

PURPOSE:To provide the subject enzyme useful for the elucidation of the mechanism of dental caries development, development of drugs, etc., having each specific action, substrate specificity, optimum pH, appropriate temperature range for action, deactivation condition, molecular weight and immunogenicity. CONSTITUTION:The objective glucosyltransferase having the following physical- chemical characteristics: (a) action and substrate specificity...acting on sucrose to synthesize water-insoluble glucan. (b) optimum pH...6.7-7.0 (c) appropriate temperature range for action...15-50 deg.C (d) deactivation condition ... treatment at 80 deg.C for 5min. (e) molecular weight...150-165K daltons determined by the SDS-polyacrylamide gel electrophoresis (f) immunogenicity...becoming immunogen in animals to be capable of producing specified antibody to the present engyme. This glucosyltransferase can be obtained by separation and purification of e.g., a water-insoluble glucansynthesizing enzyme produced in an bacteria- bound state, from said bacteria.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a bacterial cell-bound glucosyltransferase that acts on sucrose and catalyzes the reaction of synthesizing water-insoluble glucans, a method for purifying the same, and a method for producing the same. Regarding the method.

[Prior art] From the viewpoint of caries prevention, etc., Streptococcus mutans (Streptococcus mutans), which is known as a caries pathogen, has been studied.
Much work is being done to elucidate the properties of S. occus mutans and the mechanism by which this bacterium is involved in the development of dental caries.

Currently, strains of S. mutans, which are involved in the development of caries, have been found to be serologically classified into eight serotypes from a to h, and serologically, they are classified into d-type seedlings and g-type seedlings.
The type seedlings are similar, as well as C mural, e Doga, and f pear garden.
These are classified into two groups.

An important process in the mechanism by which S. mutans is involved in caries development is the production of sticky, water-insoluble glucan from sucrose involving the glucosyltransferase (hereinafter abbreviated as GTF) possessed by S. mutans. Something is already known.

GTF produced by S. mutans can be divided into two types: one that exists in the culture supernatant and one that exists bound to the bacterial cell surface.

For example, when culturing serologically similar d-type seedlings or g-type seedlings in the absence of sucrose, most of GTF is present in the culture supernatant, and currently three types of GTF are present in the culture supernatant. , i.e. GT F-5t , -S2
and I (S is water-soluble glucan synthase, ■ is water-insoluble glucan synthase) have been separated and purified. Water-insoluble and sticky glucans are synthesized by the joint action of these three GTFs [e.g.
“Glucan synthesis and its attachment mechanism in occus mutans”, Toshihiko Koga, Journal of the Japanese Society of Bacteriology, No. 41
@, No. 4, 67g (1986)].

It is also known that when culturing d-type and g-type seedlings in the presence of sucrose, most of the above three GTFs bind to bacterial cells via glucans and become bacterial cell-bound [Haa + ada]. , S, and 5lade.

H,D, ,^rchs oral, biol,,2
4.399-402 (1979)].

On the other hand, GTF-5 and GTF-r have also been isolated from the culture supernatants of serologically similar types C, E, and f Rion [Kuramitsu, H., andl.
londrack, L,, Infect, Tt+au
nity, 42 Near 63-770 (1983)].

However, there is no report that a water-insoluble and sticky glucan is synthesized through the joint action of GTF-5 and GTF-1.

[Problem N to be solved by the invention] With the aim of establishing a more effective caries prevention technique, the present inventors discovered that the most abundant caries are distributed within the hydrocavitary cavity and play the most important role in the development of caries in humans. In the process of conducting various studies to elucidate the mechanism of their involvement in the development of touch, we focused on the C mural, which is thought to have played an active role, and the e-child's painting, F Rien, which is serologically similar to the C mural. , GT involved in the process of synthesizing water-insoluble glucan produced by these bacteria.
Research has been further advanced from the viewpoint that it is important to separate and purify F and elucidate its properties.

As a result, the present inventors found that among the GTF produced by these bacteria, the cell-bound water-insoluble glucan synthase activity, which seems to be particularly important in the sucrose-dependent attachment of the cell to the southern surface, GTF (hereinafter referred to as GA-GTF-
We have confirmed the existence of C^-GTF-
An attempt was made to separate and purify I.

In addition, in the conventional technology, the bacterial cell-bound G T F (-1) in c, e, or f Rien has not been isolated as a purified enzyme protein and its properties have not been sufficiently elucidated. It is.

For example, regarding the bacterial cell-bound GTF-I produced by C murals, there is a report by Niuramitsu [Niura@1ts
u, h.

In,, Infect, Immunity, 10
:227-235 (1974)]. That is,
Kuramitsu et al. obtained an extract having water-insoluble glucan synthase activity from bacterial cells using IMNaCl,
Testing its properties as GTF, adding GTF to the extract
It has been reported that the optimum pH for TF activity is 6.0, and the optimum temperature is 37°C.

However, this extract obtained by Uramitsu becomes insoluble in water when subjected to the saline treatment necessary for its purification, so purified proteins with GTF activity were extracted from the extract. I haven't reached the point where I can get it yet. Therefore, the data on bacterial cell-bound GTF reported by Urasitsu were not obtained using purified enzyme protein, and were therefore extremely insufficient for identifying enzymes with GTF activity. In particular, nothing has been shown about the molecular weight, isoelectric point, and other properties of the enzyme contained in the extract extracted from bacterial cells as an enzyme protein.

On the other hand, it has been shown by Kenny et al. and Urami Tsu et al. that GTF-1 exists in the culture supernatant of type C seedlings.

Kenney et al. found that the molecular weight was 153 in the culture supernatant of type C seedlings.
GT has the effect of synthesizing Dalton's water-insoluble polysaccharide.
showed the existence of F-1c [A, C, Kenn
eyand J, AJl:ole, FEMS Mic
robiol, Lett, 16:159-162
(1983)].

However, Kenny et al. only demonstrated the presence of GTF-1c in the culture supernatant, and the isolation and
No data were provided regarding purification or to determine the protein nature of the enzyme.

On the other hand, uraa+1tsu et al.
, GTF-5c), and mutansynthetase (which has the action of synthesizing water-insoluble glucan).
A fraction containing MS%GTF-1c) was separated from the culture supernatant of type C seedlings, and various analyzes were performed on GTF activity in these fractions, and it was shown that DS and MS are different enzymes. . The MS obtained here has different immunogenicity than DS, and also has a molecular weight of 155 k Daltons and a molecular weight of 4
It had an isoelectric point (pI) of ~5 [Kura
Mitsu, H.,.

and Ifondrack, L., Infect.
Immunity, 42 Near 63-770 (198
3) ].

Under these circumstances, the present inventors developed GA-GTF-1
In order to clarify the existence of CA-GTF-I, as a result of intensive studies on isolation and purification methods for this enzyme, we succeeded for the first time in isolating and purifying CA-GTF-I from the cells of type C seedlings, and discovered new information about its properties. Having obtained this knowledge, we have completed the present invention.

The purpose of the present invention is to provide GA-GTF-1 as a water-insoluble glucan synthase that is useful for elucidating the detailed mechanism of caries development and developing more effective techniques and various drugs necessary for caries prevention. It's about doing.

In particular, since the present inventors have revealed that the (:A-GTF-I) of the present invention has immunogenicity to animals, it is possible to use a specific antibody against the enzyme prepared using the enzyme. , it becomes possible to establish the technology necessary for caries prevention, and to develop caries preventive agents and various caries preventive agents containing the antibody as an active ingredient. It can be effectively used for the production of glucan from.

(:A-GTF-1 of the present invention) is an enzyme protein with a molecular weight of 150 to 165 Daltons as measured by 5DS-polyacrylamide gel electrophoresis (SDS-PAGE), and is an enzyme protein that converts water-insoluble glucan from sucrose into functions as an enzyme that catalyzes the reaction in which

The optimal pn of the enzymatic activity of CA-GTF-1 of the present invention is p
H6.7 to 7.0.

In addition, this optimal pH is almost the same as the pH in the oral cavity, and from this point as well, CA-GTF-1 has a
, it can be easily inferred that it plays an important role in the behavior of mutans.

Note that the temperature at which CA-GTF-1 of the present invention exhibits enzymatic activity is in the range of 15 to 50°C, and the optimum temperature is in the range of 27 to 37°C.
in the range of ℃.

Note that the GTF activity of the enzyme is inactivated by treatment at 80° C. for 5 minutes.

On the other hand, CA-GTF-I of the present invention can act as an immunogen in animals, and specific antibodies against the enzyme can be generated by administering the enzyme to animals. The antibodies thus obtained are useful in the field of caries prevention, etc., as described above.

The enzymatic activity of GA-GTF-1 of the present invention can be achieved by, for example, adding [14C-glucose] sucrose ([14C-glucose]
It can be measured by measuring the amount of radioactivity incorporated into glucan produced when reacting with lucoselsucrose) as a substrate.

In the present invention, the activity of transferring 1 μmol of glucose to glucan per minute is defined as 1 unit (U).

GA-GTF-1 of the present invention can be obtained, for example, by separating and purifying GTF-1 produced in a state bound to bacterial cells from bacterial cells.

GA-GTF-1 of the present invention is GTF-5 (GTF-5), which has the effect of synthesizing water-soluble glucan present in the culture medium of type C seedlings, as will be clarified in the examples below.
CF-GTF-5).

CA-GTF-I of the present invention can be separated and purified from c, e, or f doga as follows.

First, C, e, or f Doga is cultured in an appropriate medium, and the resulting bacterial cells are collected and washed as necessary.

The type C seedlings used here include S, mutans 1
78148 strain, Ingbritt strain, 10449 strain, etc. can be used.

Note that these bacteria are known and easily available.

For example, S. mutans strain 178148, Ing.
The britt strain is from the Osaka University School of Dentistry, and the 10449 strain is from the National Collection of Types and Cultures [
National Co11ection of Ty
pe (:ultures(NCTfl:))]. Also, for e-murals and f-children's paintings, known strains may be obtained and used.

Further, as a medium, a medium containing at least glucose can be used, for example, TTY medium (Trypticas
e%Tryptose%Yeast extract complex medium), BHI (Brain Heart
Infusion medium, FMC medium, etc. can be used.

In addition, the culture temperature is such that bacterial cell growth is obtained and CA-GT
The temperature may be within a range suitable for production of F-1, but from the viewpoint of good cell proliferation and production of GA-GTF-I, it is usually recommended to set the temperature to about 37°C.

In addition, the culture time varies depending on culture conditions such as culture temperature and type of medium, but it can be determined by selecting the time when the optimum yield of GA-GTF-1 is reached, and it is usually about 18 to 20 hours. .

In addition, other culture conditions may be appropriately selected from the above viewpoint.

Next, CA-GTF-1 is extracted from the bacterial cells.

Extraction of CA-GTF-I from bacterial cells can be carried out by a method of bringing the bacterial cells into contact with an extraction solution such as a urea solution or a guanidine hydrochloric acid solution.

The extraction conditions, such as the concentration of suspended bacterial cells in the extraction solution, the concentration of the extraction solution, and the temperature and time of extraction, are determined by the type of extraction solution used, etc., so that the desired enzyme can be sufficiently extracted. It is sufficient to select and set the settings as appropriate.

In addition, from the point of obtaining an extract with high specific activity at a high recovery rate, preferably a urea solution of 6 to IOM, more preferably 8M is used, preferably at 20 to 30 °C, more preferably at 25 °C, It is recommended that the treatment be carried out for about 15 minutes to 2 hours.

When the extraction operation is completed, solid substances such as bacterial cells are removed from the extract using a method such as centrifugation, and then this is treated with dialysis, ultrafiltration, gel filtration, etc. to remove urea and low molecular weight substances. Impurities etc. are removed from the extract and CA-GTF-
1 crude extraction sample can be obtained.

In addition, if a precipitate is generated during the treatment such as dialysis, it may be removed by means such as centrifugation.

Furthermore, a crude extracted sample can be purified to obtain a purified CA-GTF-1 sample.

For this purification, a method combining an adsorption method using an anion exchanger and an adsorption method using hydroxyapatite can be suitably applied.

The anion exchanger used for purifying the crude extraction sample is diethylaminoethyl [diethyl, aminoet].
Anion exchangers having a functional group such as hyl (DEAE)] can be used, and specifically, DEAE-cephacel (D
EAE-, 5ephacel, manufactured by Pharmacia), and the like.

In addition, as hydroxyapatite, Blo-Ge1
IITP [manufactured by Bio-Rad] etc. can be used.

Purification using an anion exchanger involves contacting the extract (crude protein) contained in a crude extraction sample with an anion exchanger, and then extracting the target enzyme from both components adsorbed to the anion exchanger. This can be done by selectively eluating and fractionating both components.

A fraction containing the target enzyme can be obtained, for example, by adjusting the salt concentration of the eluate.

This salt concentration varies depending on the type of anion exchanger used, elution conditions, etc., but may be determined by selecting a concentration range that allows selective fractionation of both components having GTF(-) activity.

For example, when DEAE-Sephacel is used, the Na1L concentration in the phosphate buffer is 0.45 to 1. A fraction having GTF(-1) activity within the OM range can be obtained.

The sample to be brought into contact with the anion exchanger was precipitated crude protein obtained from a crude extraction sample by a precipitation method using a salt solution such as ammonium sulfate, ethanol precipitation, or an organic solvent, and recovered by centrifugation. Afterwards, the precipitate can be prepared by dissolving it in a suitable solvent and treating it, if necessary, with dialysis or the like to remove low molecular weight impurities from the precipitate.

When the treatment with the anion exchanger is completed, GTF (
-1) Fractions having activity are collected, desalted by means such as dialysis, and then brought into contact with hydroxyapatite.

Next, CA-
By selectively separating and eluting GTF-1, GA-GTF-
Obtain the I fraction, treat the fraction with dialysis etc. as necessary,
A purified sample of CA-GTF-1 can be obtained.

The purified CA-GTF-1 sample thus obtained was
5DS-PAGE gives a single band at a position corresponding to a dalton with a molecular weight of 150k N165.

Further, according to the above operation, purification to a specific activity of 6.96 U/mg protein is possible, for example, as shown in the Examples below.

Furthermore, CA-GTF-1 of the present invention can be produced by performing the above-mentioned bacterial cell culture and extraction operations. The obtained extract may be further purified until a desired degree of purification is obtained.

[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 (Study of localization of GTF in various serotypes of S. mutans)
(Obtained from Osaka University School of Dentistry) individually at 50aIL BHI
3 in medium and TH (Todd-Hewitt) medium.
The cells were cultured at 7°C for 18 hours.

After the culture was completed, the bacterial cells and the culture supernatant were separated by centrifugation, and the resulting bacterial cells were washed with physiological saline.

Next, the GTF activity of each of the bacterial cells and culture supernatant was measured according to the following method. The results are shown in Table 1.

Method for measuring GTF activity: 1) In the case of sample bacteria used for measurement: After washing the collected cultured bacteria with physiological saline, the total amount of bacteria was reduced to 5I.
The sample was a cell suspension obtained by suspending the cells in IIL's 10 mM phosphate buffer (pu6.o) and dispersing them by sonication.

In the case of Joname The culture supernatant was used as a sample.

2) Measurement of GTF activity 10μL of each sample was added to 20mM [+40-
Glucose] Sucrose (["C-glucosel5
ucrose, 0.2 containing 0.05 Ci/mol)
Mix with 10 μM of M phosphate buffer (p116.0);
After reacting at 37°C for 1 hour, the entire reaction solution was filtered with filter paper (1,Ox2
．． After washing it with methanol,
The amount of radioactivity incorporated into the methanol-insoluble glucan remaining on the filter paper was measured, and the GTF activity was calculated.

As is clear from the results shown in Table 1, d mural and g
In type seedlings, when cultured in BHI medium, that is, in the absence of sucrose, most of the GTF is present in the culture supernatant, but when cultured in TH medium, that is, in the presence of sucrose, most of the GTF is in the bacterial cell-bound form. It becomes.

On the other hand, in the C-type, e-type, and f-type seedlings, no major changes were observed as in the d-wall paintings and the g-type seedlings.

Example 2 (Extraction of CA-GTF-1 from bacterial cells) Mutans
178148 strains (1 & L clear type C) were transferred to TTY of IIL.
The cells were cultured in a medium at 37° C. for 18 hours, the cells were collected by centrifugation, and the collected cells were washed twice with physiological saline.

Next, a portion of the bacterial cells were suspended in various extraction solutions, and the resulting suspension was extracted while stirring.

After the extraction procedure is completed, bacterial cells are separated from each extract by centrifugation, and the resulting supernatant is diluted with 10 mM phosphate buffer (po
Dialyzed against e,o).

After the dialysis was completed, insoluble matter generated in each supernatant was removed by centrifugation, and then the GTF activity and protein content of each supernatant were measured.

In addition, GTF activity was determined using the method described in Example 1.
Further, the protein content was measured by the method of Lowry et al. [bovine serum albumin (BSA) was used as a standard], and the specific activity was determined.

Table 2 shows typical examples of the extraction solution and extraction conditions used in the extraction operation, and the GTF specific activity of the extract.

Furthermore, the previously obtained bacterial cells were added to 1mM phosphate buffer (p) 16
．． 0) and the suspension was added to 20 Kl (z, 200
Table 2 also shows the results of measuring the GTF activity of the supernatant after preparing a bacterial cell disruption mixture by subjecting it to ultrasonic disruption treatment for 1.3 minutes and removing the insoluble fraction by centrifugation. Ta.

Table 2 *Indicates the recovery rate of insolubilized substances generated during dialysis treatment.

As is clear from the results shown in Table 2, proteins with GTF activity can be extracted from bacterial cells by each extraction operation, and in order to obtain an extract with high yield and high specific activity,
It was found that an extraction operation using 8M urea solution at 25° C. for 1 hour is optimal.

Example 3 (Preparation of TF-1 purified wire product) In Example 2, it was found that the extraction operation using 8M urea at 25°C for 1 hour was optimal, so the same procedure as in Example 2 was carried out. MT8148 was cultured in TTY medium (8ffi) under the following conditions, and the resulting bacterial cells were collected and washed,
mff1 was suspended in an 8M urea solution. The dry weight of the cultured bacterial cells obtained was 9.7 g.

Next, the suspension was stirred at 25° C. for 1 hour to perform extraction.

After the extraction process, the bacterial cells were removed from the extract by centrifugation.
0).

After completion of the dialysis, insoluble substances generated in the supernatant were removed by centrifugation to obtain a C^-GTF-I crude extraction sample.

Next, a precipitate with ammonium sulfate saturated at 60% was prepared from the crude extracted sample, and this precipitate was further dissolved in 20 tsl of 505M phosphate buffer (p17.5), and the resulting solution was diluted with 50mM Dialysis was performed against phosphate buffer (pH 7.5).

After the dialysis was completed, insoluble materials generated during the dialysis treatment were removed from the solution by centrifugation, and the supernatant was applied to a DEAE-5ephacel (DEAE-5ephacel, manufactured by Pharmacia) column of 2.5 x 13 cm. .

Both fractions adsorbed on the column were added to phosphate buffer (pH 7.5).
selectively eluted with a concentration gradient of NaCl in

The GTF activity of each eluted fraction was measured by the method described in Example 1, and the protein content was measured by an ultraviolet absorption method at 280 nm. As shown in FIG. 1, the GTF activity was 0.45 to 1.
Significant GTF activity was observed in the 0M NaClttl fraction.

Collect the elution fractions with 0.45-1.0 M NaCl, dialyze it against l0mM phosphate buffer (pH 6,0), and then dialyze it against l0mM phosphate buffer (pH6,0).
Hydroxyapatite [8io Ge
t HTP, Biorad mouth 1o-1? ad) company]
column (1, Ox 13 cm).

The two adsorbed portions of the column were 0.01M, 0.2M,
0.26M and 0.5M phosphate buffer (pH 6,0
), respectively.

The GTF activity and protein content of each portion were measured in the same manner as described above, and as shown in FIG.
GTF activity was observed in both minutes with 5M phosphate buffer.

Next, both eluates with 0.5M phosphate buffer, which shows high activity, were collected and added to 10mM phosphate buffer (pH 6, 0).
) and used as a purified enzyme preparation.

In addition, GTF obtained in each step of the above extraction and purification
GTF activity, protein content, GTF in both active fractions
Table 3 shows the TF specific activity and yield.

Table 3 Furthermore, this purified enzyme preparation was subjected to 5DS-PAG under the following conditions.
E When the protein was stained with Kumasi Brilliant Blue (CBB), a single band was detected at a molecular weight of 156 Daltons, indicating that the purified enzyme preparation was an enzyme protein with a molecular weight of 156 Daltons. confirmed.

5DS-PAGE operating conditions: 5O5-PAGE was performed according to the method of Laemmli et al.

That is, crude extracted preparations and purified preparations (0.2 to 35μ
g) were individually treated at 100° C. for 3 minutes in 62.5 mM Tris-HCl buffer (pH 6,8) containing 2% SOS, 5% 2-mercaptoethanol and 20% glycerol.

Electrophoresis was performed in a 7.5% separation gel containing 0.1% SDS and a 4% concentration gel at room temperature, 10 mA, and 2 hours.

In addition, as a molecular weight marker, ferritin (ferr
itin, 220 Dalton), phosphorylase (p
1ase, 94 Dalton), bovine serum albumin (bovine serum albumin)
in, Dalton at 67), catalase (catalas
e, Dalton on 60), of77L/Bumin (oval
bumin, 43 to Dalton) and lactate hydrogen enzyme (
1actate dehydrogenase, 36 Dalton) (both manufactured by Pharmacia) were used.

Next, an amount of the purified enzyme preparation corresponding to the amount of 5G+sU enzyme activity was added to a 0.1M phosphate buffer (pH 6,0
), the volume was adjusted to 3 ml with distilled water, the mixture was mixed, and the enzymatic reaction was carried out at 37°C for 18 hours. Note that the enzyme reaction was stopped by cooling the reaction solution with ice to 4°C.

After the enzymatic reaction was completed, the reaction product was centrifuged at 1600xg and separated into a precipitated water-insoluble fraction and a supernatant.

The water-insoluble fraction was washed twice with distilled water and suspended in 3 atll of distilled water to obtain a water-insoluble glucan sample.

In addition, 2.5 times the volume of ethanol was added to the supernatant,
After standing at 4°C for 1 hour, the resulting precipitate was collected by centrifugation at 1600xg, dissolved in 3 wit distilled water,
Furthermore, the same precipitation treatment was performed again, and the recovered precipitate was dissolved again in 3IIIL distilled water to obtain a water-soluble glucan sample.

Furthermore, we quantitatively analyzed the amount of glucan contained in these preparations using the Anthrone method to investigate the types of glucans synthesized by the action of the purified enzyme preparations, and found that the purified enzyme preparations synthesized water-insoluble glucans. CA- which has the effect of
It became clear that it was GTF-1.

Furthermore, in the method for measuring GTF activity in Example 1,
By varying the temperature and pH, the optimum temperature and optimum pt+ for the water-insoluble glucan synthetase activity of the purified enzyme preparation were determined.

Further, the I value was also measured according to a conventional method.

Table 4 shows the results obtained in the above measurements and tests.

For comparison, the method of Baba et al. [Carbohydrate
Research, 158.147-155 (198
Similar measurement results for 0F-GTF-5 obtained by [6)] are added to Table 4.

Table 4 *Measurement impossible due to strong aggregation within the gel Furthermore, immunoblotting was performed using antiserum prepared from mice immunized with the GA-GTF-1 of the present invention.
It was confirmed that an antibody that reacts with CA-GTF-1 of the present invention was obtained in the antiserum.

On the other hand, regarding the antigenicity of CA-GTF-1 and F-GTF-5 obtained by the above procedure,
Mouse antiserum against [CA- obtained from strain 178148]
[Prepared from mice immunized by subcutaneous injection of GTF-1 with FCA (complete Freund's adjuvant)] and monoclonal antibody against F-GTF-5 [prepared from the culture supernatant of strain 178148 by the method of 5ato et al.
Sato, S., ni oga.

T. and Inoue, M., Carbohyd
rateResearch, 134, 293-304 (
A monoclonal antibody that reacts with water-soluble glucan synthetase prepared according to 1984) was used, and alkaline phosphatase-labeled goat anti-mouse I was used as a secondary antibody.
The gA+IgG◆IgM antibody was examined by ELISA using 2-sodium p-nitrophenyl phosphate as a substrate, and the results shown in Table 5 were obtained. That is, mouse antiserum against CA-GTF-I and CF-G
It did not react with TF-5, and the monoclonal antibody against CF-GTF-5 did not react with CA-GTF-1, indicating that they have different antigenicities.

Table 5 Note that all percentages of the various solutions prepared by the above operations are weight/volume %.

[Effects of the Invention] The present invention shows that the serotypes having water-insoluble glucan synthase activity are e, e, or f of mutans (:A-G
TF-I was provided.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the protein content (A28°), GTF activity and F Ta5e (D-fructosyltransferase) activity in each elution fraction from DEAE-Sephacel in Example 3, and FIG. Protein content (A2ao), GTF activity and F T in each elution fraction from hydroxyapatite in Example 3
It is a graph showing a5e activity.

Claims (1)

[Scope of Claims] 1) A bacterial cell-bound glucosyltransferase having the following physicochemical properties. (1) Action and substrate specificity: Acts on sucrose and synthesizes water-insoluble glucan. (2) Optimum pH; pH 6.7 to 7.0 (3) Suitable temperature range for action; 15 to 50°C (4) Inactivation conditions: Inactivation is achieved by treatment at 80°C for 5 minutes. (5) Molecular weight; The molecular weight measured by SDS-polyacrylamide gel electrophoresis is 150 k to 165 k Daltons. (6) Immunogenicity: Acts as an immunogen in animals and can generate specific antibodies against the enzyme. 2) Streptococcus mutans whose serotype is c, e, or f produces cell-bound glucosyltransferase that catalyzes the reaction of synthesizing water-insoluble glucan from sucrose.
1. A method for purifying a bacterial cell-bound glucosyltransferase having water-insoluble glucan synthase activity, which comprises the steps of extracting and purifying the glucosyltransferase from cultured bacterial cells of Mutans mutans. 3) The method according to claim 2, wherein the purification is performed by a combination of a purification process using an adsorption method using an anion exchanger and a purification process using an adsorption method using hydroxyapatite. 4) Streptococcus mutans (￥Streptococcus
1. A method for producing a cell-bound glucosyltransferase having water-insoluble glucan synthetase activity, which comprises the steps of cultivating the glucosyltransferase from the resulting cultured cells.