JP2000083665A - Production of mutan - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently and industrially advantageously obtain mutan, having an effect of inhibiting bacterial plaque formation, and useful for compositions for oral cavities, humectant and the like, by adding a recombinant-derived mutan synthetase to saccharose to synthesize mutan. SOLUTION: This mutan is efficiently and industrially advantageously obtained by a series of steps: (A) obtaining GTF-I gene by the PCR method after preparing a primer with 30 bases each on upstream and downstream portions, based on the gene arrangement of mutan synthetase (glucosyltransferase I: GTF-1) of Streptococcus downei, (B) obtaining recombination Escherichia coli holding the GTF-I gene by inserting the above gene into the vector to phenotypically transform it into Escherichia coli (JM 109 stock), (C) culturing the above recombinant in a medium, (D) centrifugally treating the above culture system to recover the mycobiont therefrom, and extracting the mycobiont with a 6M urea-EDTA solution, (E) recovering the supernatant liquid as the enzymatic solution, and (F) adding the above solution to saccharose, allowing them to react with each other at 35 deg.C for 16 h.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing mutan by an enzymatic method which can efficiently produce mutan and which is industrially advantageous.

[0002]

2. Description of the Related Art Mutane is a water-insoluble high molecular weight polysaccharide in which glucose is linked by α-1,3-linkage, and Streptococcus mutans (naturally known as a causative bacteria of caries). Strepto
coccus mutans) and Streptococcus.
Sobrinus (Streptococcus sobri)
nu) and the like in the form of glucan (a mixture of mutan and dextran).

Recently, mutan has been used as an oral composition having an effect of inhibiting plaque formation (JP-A-10-182382).
JP-A-10-157) and use as a humectant (Japanese Patent Application No. 10-157).
No. 884), and is a polymer polysaccharide that has attracted attention as an extremely unique material that is expected to be applied to carriers and the like due to its water-insoluble nature.

Conventionally, in order to obtain this mutan, oral streptococci are cultured in the presence of sucrose to obtain glucan.
A method of decomposing and removing α-1,6-glucan with dextranase has been used (Caries and Periodontal Disease Vol. 2,
1982, Dental Review).

However, in the conventional fermentation method generally used, since living cells are used, the concentration of sucrose, which has the greatest effect on the production, is a limiting condition for the growth of the bacteria. Since sugar cannot be added, the yield per medium is low, and it takes days to cut and remove the α-1,6-chain by dextranase, so naturally it is necessary to increase productivity. There was a limit. In addition, in order to remove the cells from the generated glucan, it is necessary to solubilize the glucan by performing an alkali treatment once, but at this time, the cell components, especially iodine-soluble intracellular glucan are extracted and mixed by the alkali. In addition, it is not easy to completely remove metabolites of bacteria entrapped in mutan.

Further, such a manufacturing method has a drawback that since the steps are long and complicated, the quality of each lot varies widely.

[0007] In view of the above, the present inventors added dextranase to a culture solution of oral streptococci as described above to produce glucan and dextran in order to perform the conventional method more efficiently. A method for efficiently producing mutan by simultaneously performing a stranase treatment was developed (Japanese Patent Application No. 9-9).
No. 1667), but a more efficient method is desired.

By the way, apart from the fermentation method, a mutan synthase (glucosyltransferase-I; hereinafter abbreviated as GTF-I) is obtained from the cells. In some documents, GTFP3 is described. The expressed genes may be described as gtfI and gtfB depending on the bacterial species.
However, in the present specification, all enzymes having a function of synthesizing mutan are defined as GTF-I. It is theoretically possible to separate and purify only) and use it for the production of mutan, but in practice it is very difficult due to the aggregation, adsorption and proteolysis of enzymes.

The present invention has been made in view of the above circumstances,
It is an object of the present invention to provide a method for producing pure mutan with high availability as an industrial product by improving the conventional problems of a small amount of α-1,6-glucan and low yield.

[0010]

Means for Solving the Problems and Embodiments of the Invention The present inventors have conducted intensive studies in order to achieve the above object. As a result, in the process of producing mutan, a fermentation method using oral streptococci was used instead of a fermentation method. By using a transformant,
It has been found that conventional problems can be solved by producing mutan by an enzymatic method using only GTF-I. In particular, the extraction of GTF-I from the recombinant is very different from the separation and purification from the original strain, in that not only the amount of the obtained enzyme is large but also it can be easily performed without aggregation. The present invention was found to be an excellent method, and led to the present invention.

Hereinafter, the present invention will be described in more detail.

The method for producing mutan of the present invention is based on an enzymatic method in which a recombinant mutan synthase (GFT-I) is added to sucrose.

In the present invention, known GTF-I can be used, for example, Streptococcus mutans (Streptococcus mutans).
tans), Streptococcus rattus (Stre)
ptococcus rattus, Streptococcus crycetas (Streptococcus cr)
icetus, Streptococcus sobrinas (S
Streptococcus sobrinus, Streptococcus ferras (Streptococcus)
s ferus), Streptococcus macacae (S
streptococcus macacae, Streptococcus downey (Streptococcus)
downei), Streptococcus salivarius (Streptococcus salivariu)
s) and the like.

The genes obtained from these strains are obtained by PCR.
For example, the gene information can be obtained by the following literature information. S. mutans GS5: Journal of Bacteriology,
Sept. 1987p4263-4270H.
K. KURAMITSU et al. sobrinus 6715: JP-A-5-23188. downei Mfe28: Journal of Bacteriology,
Sept. 1987p4271-4278 ROY
R. B. RUSEL et al. mutans MT8148: FEMS Microbiol. Lett. 16
1, 1998 p331-336 S.M. HAMAD
A

[0015] Hosts incorporating the gene include Escherichia coli,
Streptococcus mirelli
usmilleri), Bacillus subtilis (Bac)
bacteria such as illus subtilis, Saccharomyc
es cerevisiae), Kluyveromyces lactis
s), Candida maltosa
yeast such as Aspergillus nidulans, and molds such as Aspergillus nidulans, and Streptomyces lividans (Str.
Actinomycetes such as C. eptomyces lividans can be used. As hosts, Streptococcus mirelli, a bacterium of the same genus, and Escherichia coli, which has ample basic information, are easy to handle.

GTF-I used for the production of mutan can be obtained by culturing this recombinant by a conventional method and extracting it from the cells. In addition, as this method, the following method can be illustrated according to a bacterial strain. S. mutans GS5: Journal of Bacteriology,
Sept. 1987p4263-4270H.
K. KURAMITSU et al. sobrinus 6715: JP-A-5-23188. downei Mfe28: Journal of Bacteriology,
Sept. 1987p4271-4278 ROY
R. B. RUSEL et al. mutans MT8148: FEMS Microbiol. Lett. 16
1, 1998 p331-336 S.M. HAMAD
A

Next, in the production of mutan, GTF-I
As for the amount of addition of GT, basically, GT of 1 U / ml or more
Any amount of FI may be added, but preferably 1 to 1.
It is preferable to add ＴＦ5000 U / ml, and more preferably, to add GTF-I at 5５〜1000 U / ml. When the amount is less than 1 U / ml, a sufficient effect is not exhibited.
Further, even if added in excess of 5000 U / ml, no further effect is seen.

The GTF-I activity mentioned here is replaced by sucrose-decomposing activity, and when the reaction is carried out using sucrose as a substrate, the amount of an enzyme capable of decomposing 1 μg of sucrose per minute is reduced by one.
Unit.

As the enzyme reaction solution, any composition can be used as long as it can react with the above-mentioned GTF-I.
Buffers such as phosphate buffer and acetate buffer are preferred.

The pH may be in any range as long as the above-mentioned GTF-I can react, but it is preferably pH4 to pH7.
5, more preferably pH 5-7.

As long as the salt concentration has a buffering capacity such that the pH of the reaction solution does not deviate from the suitable range during the reaction, any concentration can be used, but preferably 5 mM to 1 M, more preferably 25 mM to 250 mM. A range is preferred.

The temperature may be any temperature at which the above-mentioned GTF-I can react, but preferably 25 to 45 ° C, more preferably 30 to 40 ° C.

The amount of sucrose to be added can be 1 to 50% by weight, but preferably 10 to 30% by weight. In addition, there is no hindrance to the reaction if used at such a high concentration that sucrose remains undissolved, but the yield to sugar is not increased and it is not efficient.

Since the mutan produced under the above conditions is insoluble in water, it can be easily recovered by centrifugation. Depending on the purpose, even lower molecular weight mutan can be recovered by adding an organic solvent such as ethanol, methanol or acetone to the reaction solution. The obtained precipitate is washed with water to remove impurities such as fructose released as a result of mutan synthesis, and can be stored as a dry powder by a suitable method such as freeze-drying or vacuum drying.

[0025]

According to the present invention, mutan, which has been difficult to obtain, can be efficiently produced, which is industrially advantageous.

[0026]

EXAMPLES The present invention will be specifically described below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[Example 1] (1) Acquisition of recombinant bacterium GT of Streptococcus downei reported previously
FI gene sequence (Journal of Bacte
riology, Sept. 1987 p4271
-4278 ROY R.R. B. Based on RUSEL et al., Primers of 30 bases each for upstream and downstream were prepared, and the target gene was obtained by PCR. Next, the resulting gene was inserted into a vector (pUC118) and transformed into Escherichia coli (JM109 strain) to obtain GTF-
A recombinant E. coli carrying the I gene was obtained.

(2) Preparation of GTF-I by Recombinant Bacterium A 2 L BHI medium (BB
L company) and sterilized in an autoclave.

The Escherichia coli recombinant strain obtained by the above method was inoculated into this medium and cultured for 16 hours at a temperature of 32 ° C., a rotation speed of 300 rpm, and an aeration of 1 vvm.

After the culture, the cells were centrifuged (8000 rpm ×
20 minutes), 100 ml of a 6 M urea-EDTA solution (6 M urea, 10 mM ED
TA, 0.1 M Tris-hydrochloric acid) was added and left at room temperature for 1 hour. After standing, the supernatant was recovered by centrifugation (18000 rpm × 20 minutes) to obtain an enzyme solution.

(3) Production of Mutan Next, a phosphate buffer containing 20% sucrose (pH 6.
5) Add 750000 U of enzyme solution to 5 L,
The reaction was performed at 35 ° C. for 16 hours. Further, the mutan produced as a result of the enzymatic reaction is collected by centrifugation (8000 rpm × 20 minutes), and the conductivity when suspended in distilled water 10 times the wet weight becomes 50 μs / cm (in terms of salt). Washing with distilled water was repeated until then, followed by freeze-drying to obtain a powder of mutan. With this method, 25 g of mutan was obtained per liter of a 20% sucrose solution.

When the mutan produced under the above conditions was analyzed by NMR under the following conditions, no α-1,6-bond was detected as shown in FIG. Analysis conditions Sample concentration: 15% Measurement solvent: 1N-sodium hydroxide / 20% heavy water Measurement magnetic field: 67.9 MHz Measurement temperature: 22 ° C.

The GTF-I titer used here is measured as follows. That is, 0.1 ml of a 0.1 M phosphate buffer (pH 6) containing 10% (w / w) of sucrose is added, and 0.1 ml of the enzyme solution is added thereto, followed by incubation at 35 ° C. for 10 minutes to react. After the reaction, 0.4 ml of a dinitrophthalic acid solution is added to stop the reaction, and the amount of reducing sugar released by the dinitrophthalic acid method is measured.

As a control, an enzyme previously inactivated by heating at 100 ° C. for 10 minutes is treated in the same manner as described above.

One unit of the enzyme is defined as the amount of the enzyme that decomposes 1 μg of sucrose per minute under the above conditions.

[Examples 2 to 17] Mutane was prepared using GTF-I derived from each strain produced by the method of Example 1 under the following conditions, and the yield was compared. The results are shown in Tables 1 and 2. The produced mutan was washed and dried according to the method of Example 1.

[0037]

[Table 1]

[0038]

[Table 2]

[Comparative Example] As a comparative example, an example in which mutan is actually produced by a fermentation method is shown below. Culture strain used for culture conditions : sobrinus strain 6715 Medium used: TTY medium Culture temperature: 37 ° C. Culture environment: aerobic Suc concentration: 10% Inoculum size: 1% Culture days: 7 days Medium composition TTY medium Trypticase (BBL) 15 g / L Tryptose (Difco) 4 g / L yeast extract (Difco) 4 g / L K ₂ HPO ₄ 2 g / L Na ₂ CO ₃ 2 g / L NaCl 2 g / L KH ₂ PO ₄ 5 g / L Glucose 10 g / L

Using a 5 L culture bottle, the above-mentioned TTY medium component containing 10% sucrose was dissolved in 3 L of deionized water.
Autoclave treatment was performed at 21 ° C. for 20 minutes. Next, a brain heart infusion medium (B
BL) (pre-cultured overnight)
30 ml of Sobrinus 6715 was inoculated and cultured at 37 ° C. by standing.

On the seventh day of the culture, the obtained precipitate (mixture of glucan and bacterial cells) was collected by centrifugation.

Next, the obtained precipitate was dissolved in a 1N sodium hydroxide solution and left at room temperature for 1 hour. After standing, the bacteria were removed by centrifugation, the resulting supernatant was neutralized with 2N hydrochloric acid, and then left overnight in a low-temperature room to precipitate mutan. The next day, the precipitated mutan was collected by centrifugation, washed thoroughly with deionized water, and then washed with 0.1 M phosphate buffer (pH 6.
0), followed by dextranase treatment, and α-
1,6-glucan was cleaved off.

The dextranase treatment was carried out using O.D.M. when the reducing sugar in the supernatant was measured by the Somogyi-Nelson method. D660nm
Was carried out until the color development became 0.1 or less (the buffer was replaced every three days and dextranase was newly added.
Took weeks).

O. When the color development of D became 0.1 or less, an alkali treatment and a neutralization precipitation operation were performed again, and after washing with deionized water, freeze-drying was performed. By this operation, about 3.5 g of white powder (mutane) was obtained per 1 L of the medium.

When the mutan produced under the above conditions was analyzed by NMR under the conditions of Example 1, 7.2% of the α-1,6-bond was detected. Table 3 shows the results.

[0046]

[Table 3]

From the above results, it is difficult to remove the α-1,6-chain below the detection limit of NMR when prepared by a general fermentation method, and the yield of mutan obtained is 3 g / L. Before and after. Therefore, by using the recombinant enzyme method, it is possible to increase the purity,
By setting the reaction pH to 5.0 to 7.0 and the reaction temperature to 25 to 45 ° C., an increase in the yield by about one digit can be expected.

[Brief description of the drawings]

FIG. 1 is an NMR chart of the mutan obtained in Example 1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) C12R 1:46) (C12N 1/21 C12R 1:19) (C12N 9/10 C12R 1:19) (72 ) Inventor Hiromitsu Kikawa 1-7-7 Honjo, Sumida-ku, Tokyo Inside Rion Co., Ltd. (72) Inventor Yasuko Takino 1-3-7 Honjo, Sumida-ku, Tokyo F-term inside Lion Co., Ltd. 4B024 AA03 BA07 CA02 DA06 EA04 GA11 4B050 CC03 DD02 LL05 4B064 AG01 CA02 CA19 CC24 DA16 4B065 AA26X AA49Y AB01 AC14 BA02 CA27 CA60

Claims (1)

[Claims] 1. A method for producing mutan, comprising adding mutan synthase derived from a recombinant to sucrose to obtain mutan.