JP2000175694A - Production of mutan by enzymatic synthesis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing mutan by an enzymatic synthesis capable of controlling its molecular weight. SOLUTION: This method for producing mutan by an enzymatic synthesis using sucrose as a raw material comprises controlling the molecular weight of the mutan by changing the concentrations of the raw material and/or the initial reaction product of a reaction system, changing the solubility of reaction products to a reaction liquid or both of them.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing mutan by enzymatic synthesis capable of controlling the molecular weight.

[0002]

2. Description of the Related Art Mutane is a water-insoluble high-molecular-weight polysaccharide in which glucose is linked by an α-1,3-linkage.
It exists in the form of glucan (a mixture of mutan and dextran) produced by sobrinus (Streptococcus sobrinus) and the like.

Conventionally, in order to obtain this mutan, a method has been used in which oral streptococci are cultured in the presence of sucrose to obtain glucan, and α-1,6-glucan is decomposed and removed with dextranase. (Caries and Periodontal Disease Vol. 21)
982, Dental Review Company).

[0004] In order to more efficiently carry out the conventional method, the present inventors have taken out the glucosyltransferase-I (hereinafter referred to as GTF-I) gene from oral streptococci, and have obtained a more efficient method using a recombinant enzyme. (Japanese Patent Application No. 10-27431)
No. 0).

[0005] Recently, mutan has been used as an oral composition having an effect of inhibiting plaque formation (JP-A-10-18).
No. 2382) and use as a humectant (Japanese Patent Application No.
No. 157888), and is a polymer polysaccharide that has been attracting attention as a very unique material that is expected to be applied to carriers and the like due to its water-insoluble property.

When mutan is used for these purposes, its molecular weight has a great influence on the physical properties. Therefore, it is preferable to control the molecular weight and the molecular weight distribution to produce the desired mutan. Did not exist, and there was no method other than fractionation using a column or the like to obtain a mutan having a desired molecular weight.

[0007]

SUMMARY OF THE INVENTION The present invention provides a method for producing mutan, which has been impossible so far, and which enables control of the molecular weight in the synthesis of mutan.

[0008]

According to the present invention, in a method for producing mutan by enzymatic synthesis using sucrose as a raw material, the concentration of the raw material and / or initial reaction product in the reaction system and / or the reaction solution A method for producing mutan, characterized in that the molecular weight of mutan is controlled by changing the solubility of a reaction product with respect to methane.

[0009] α-1,3-glucan (mutane) can be synthesized by a transglycosylation reaction using GTF-I with sucrose as a substrate. At this time, if a primer is present in the reaction system, the reaction rate will increase. It is known to rise sharply. It is also known that the elongation of the mutan chain is closely related to the solubility of the reaction product in the reaction solution. To achieve the above object, the present inventors have studied whether or not the molecular weight of generated mutan can be controlled by utilizing these properties of this enzyme. The inventors have found that it is possible to change the sucrose concentration and the reaction solvent, and to change the initial reaction product concentration in the reaction system by adding a mutan primer to the reaction solution, and complete the present invention. Reached.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for producing mutan of the present invention, known GTF-I can be used. For example, Streptococcus mutans (St
reptococcus mutans), Streptococcus rattus (Streptococcus r)
attus), Streptococcus crycetas (St
reptococcus cricetus, Streptococcus sobrinas (Streptococcus)
s sobrinus, Streptococcus ferus, Streptococcus macacae (Streptococcus)
s macacae), Streptococcus downei, Streptococcus salivarius (Streptococ)
cus salivarius) and the like.
I.

[0011] In the present invention, in addition to GTF-I isolated and purified from the above strains by a conventional method, GT derived from a recombinant is used.
FI can also be used. GTF-I derived from the recombinant is
Extraction of GTF-I from recombination is preferable because it has advantages such as not only a large amount of the obtained enzyme but also simple operation without aggregation. In such a recombinant-derived GTF-I, a gene can be easily obtained from the above strain by a PCR method or the like. For example, the gene information can be obtained from the following literature information and the like. S. mutans GS5: Journal of B
actiology, Sept. 1987 p4
263-270. K. KURAMITSU et al. sobrinus 6715: JP-A-5-2318
No. 8 Publication S. downei Mfe28: Journal of
Bacteriology, Sept. 1987
p4271-4278 ROY R.P. B. RUS
SEL et al. mutans MT8148: FEMS
Microbiol. Lett. 161, 199
8 p331-336 S.P. HAMADA and others

Next, as a host incorporating the gene,
Escherichia col
i), Streptococcus mirelli (Streptoc)
Bacillus subtilis and other bacteria, such as Saccharomyces cerevisiae (Sacchar).
omyces cerevisiae), Kluyveromyces lacs (Kluyveromyces lac)
tis), Candida maltosa (Candida)
yeasts, such as Aspergillus nidulans (Aspergillus nidulans);
And Streptomyces lividans (S
Streptomyces 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.

In the present invention, GTF-I derived from a recombinant used for the production of mutan can be obtained by culturing in a usual manner and extracting from the cells. In addition, as this method, the method described in the above-mentioned literature information or the like can be exemplified according to the strain.

In the method for producing mutan of the present invention, GT
Regarding the addition amount of FI, basically, any amount of GTF-I of 1 U / ml or more may be added, but preferably 1 to 5000 U / ml, more preferably 5 U / ml. It is better to add ~ 1000 U / ml GTF-I. If the amount is less than 1 U / ml, a sufficient effect is not exhibited, and if it exceeds 5000 U / ml, no further effect is observed. The GTF-I activity here is replaced by sucrose-decomposing activity, and the amount of an enzyme that decomposes 1 μg of sucrose per minute when a reaction is performed using sucrose as a substrate is defined as one unit.

As the enzyme reaction solution, any composition can be used as long as it can react with the above GTF-I.
A buffer such as a phosphate buffer and an acetate buffer, and an organic solvent such as DMSO can be used. The pH may be in any range as long as the GTF-I can react, but preferably p
H4 to pH 7.5, more preferably pH 5 to 7 is suitable. The salt concentration may be any concentration as long as it has a buffering capacity such that the pH of the reaction solution does not deviate from the suitable range during the reaction, but is preferably 5 mM to 1 M, more preferably 25 mM to 250 mM. Is preferred. The temperature may be any temperature at which the above-mentioned GTF-I can react, but is preferably 25 to 45 ° C, more preferably 30 to 40 ° C. Furthermore, it is possible that the amount of sucrose to be added is 0.001 to 60% 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.

Next, a specific method for controlling the molecular weight in the method for producing mutan of the present invention will be described. The method for controlling the molecular weight of the present invention includes the following methods, which can be used alone or in combination of two or more. 1. 1. A method for changing the sucrose concentration of the charged raw material solution 2. A method of changing the concentration of the initial reaction product in the reaction system by adding a primer. A method of changing the solubility of a reaction product in a reaction solution by selecting a reaction solvent

Hereinafter, the above methods 1 to 3 will be specifically described. 1. Method for changing the sucrose concentration of the raw material solution By increasing the sucrose concentration of the raw material solution, low-molecular-weight mutan can be produced. On the other hand, by decreasing the sucrose concentration, high-molecular-weight mutan is produced. can do. When the sucrose concentration is extremely low, the amount of sucrose determines the rate of the reaction, and a very low molecular weight mutan oligomer can be produced.

2. Method of changing the concentration of the initial reaction product in the reaction system by adding a primer When the concentration of the initial reaction product in the reaction system is increased, low molecular weight mutan can be produced. In order to increase the concentration of the initial reaction product in the reaction system, a primer is added. As the primer, dextran or mutan oligomer can be used.
A range of weight% is preferred.

3. A method of changing the solubility of the reaction product in the reaction solution by selecting a reaction solvent. The solvent used in the reaction is preferably an aqueous buffer such as a phosphate buffer or an acetate buffer from the viewpoint of reaction efficiency. By using a solvent that dissolves water-insoluble mutan, which is a substance, it becomes possible to synthesize higher molecular mutan. The solvent used can be arbitrarily selected depending on the solubility of the mutan, and for example, DMSO or the like is preferable.

In the present invention, after completion of the reaction, the mutan is
Since it 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.

[0021]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples.

<Preparation of GTF-I by recombinant bacteria>
A 2 L BHI medium (manufactured by BBL) is charged into a jar fermenter and sterilized in an autoclave. Escherichia coli having the GTF-I gene incorporated therein was inoculated into this medium, and the temperature was adjusted to 3 ° C.
The cells are cultured for 16 hours under the conditions of 2 ° C., a rotation speed of 300 rpm, and an aeration rate of 1 vvm. After the culture, the cells are centrifuged (8000 r).
pm × 20 minutes), and then 100
ml of a 6 M urea-EDTA solution (6 M urea, 10 m
MEDTA, 0.1 M Tris-HCl) is added and left at room temperature for 1 hour. After standing, centrifugation (18000 rpm × 2
(0 min) and collect the supernatant as an enzyme solution. GTF
-I titers are measured as follows. That is, a 0.1 M phosphate buffer containing 10% (W / W) sucrose (pH
6) Take 0.1 ml, add 0.1 ml of the enzyme solution thereto, and incubate at 35 ° C. for 10 minutes to react. After the reaction, 0.4 ml of a dinitrosalicylic acid solution is added to stop the reaction, and the amount of reducing sugar released by the dinitrosalicylic 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 the amount of the enzyme that decomposes 1 μg of sucrose per minute under the above conditions.

Example 1 Control of molecular weight by addition of mutan oligomers Phosphate buffer containing 20% by weight of sucrose (pH 6.5)
To 4.5 L, 500 ml of a mixed solution of a mutan oligomer was added, and an enzyme solution equivalent to 750000 U was further added, followed by reaction at 35 ° C. for 16 hours. As a comparative example, a product obtained by adding 500 ml of the same phosphate buffer instead of the mutan oligomer was used. After completion of the reaction, the mutan generated as a result of the enzyme reaction is collected by centrifugation (8000 rpm × 20 minutes),
Washing with distilled water was repeated until the conductivity when suspended in distilled water 10 times the wet weight became 50 μs / cm (converted to salt). Thereafter, freeze-drying yielded a mutan powder. When the molecular weight of the obtained mutan was measured under the following conditions, the molecular weight of sample a to which the mutan oligomer was added was 5,000, and the molecular weight of sample b without the addition was 10,000. The chart is shown in FIGS. Analysis conditions Column: CarboPac PA1 (manufactured by Dionex) Mobile phase: A 0.2 mM sodium hydroxide B 0.2
mM sodium hydroxide / 0.2 mM sodium nitrate Flow rate: 0.85 mL / min Detector: Electrochemical detector PAD mode Standard product: Pullulan

Example 2 Control of Molecular Weight in Dextran Addition Using the above-prepared GTF-I, mutan was prepared under the following conditions, and its molecular weight was measured. To 5 L of a phosphate buffer (pH 6.5) containing 20% by weight of sucrose, 1% by weight and 3% by weight of dextran T-10 (manufactured by Pharmacia) were added, and an enzyme solution equivalent to 750,000 U was further added thereto 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 is 50 μs / cm (in terms of salt). Until
Washing with distilled water was repeated. Thereafter, freeze-drying yielded a mutan powder. The molecular weight of the obtained mutan was determined according to Example 1.
As a result, the molecular weight of the 1% by weight dextran-added sample was 17,500, and that of the 3% dextran-added sample was 15,000.

Example 3 Control of Molecular Weight by Sucrose Concentration Using the produced GTF-I, mutan was prepared under the following conditions and its molecular weight was measured. To each of 5 L of a phosphate buffer (pH 6.5) containing 10, 20, and 30% by weight of sucrose, an enzyme solution equivalent to 750000 U was added and reacted 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 is 50 μs / cm (in terms of salt). Washing with distilled water was repeated until it was. Thereafter, freeze-drying yielded a mutan powder. When the molecular weight of the obtained mutan was measured under the conditions of Example 1, the molecular weight of the sample having a sucrose concentration of 10% by weight was 30,000, the sample having a sucrose concentration of 20% by weight was 10,000, and the sample having a sucrose concentration of 30% by weight was 5%.
000.

Example 4 Control of Molecular Weight Using Organic Solvent Using the produced GTF-I, mutan was prepared under the following conditions and its molecular weight was measured. To 5 L of a DMSO solution containing 10% by weight of sucrose, an enzyme solution equivalent to 750000 U was added and reacted at 35 ° C. for 16 hours. Further, the mutan generated as a result of the enzymatic reaction is centrifuged (8000 rpm ×
20 minutes), and the washing with distilled water was repeated until the conductivity when suspended in distilled water 10 times the wet weight became 50 μs / cm (converted to salt). Thereafter, freeze-drying yielded a mutan powder. When the molecular weight of the obtained mutan was measured under the conditions of Example 1, the molecular weight was 1
00000.

[0027]

According to the present invention, when producing mutane by enzymatic synthesis using sucrose as a raw material, the sucrose concentration of the raw material in the reaction system is changed, so that the initial reaction is achieved by adding a primer. Controlling the molecular weight of the product mutan by changing the concentration of the product, or by changing the solubility of the reaction product, mutan by selecting a solvent, or by combining them Thus, it is possible to produce a mutan having a desired molecular weight according to the purpose and application.

[Brief description of the drawings]

FIG. 1 is a chart showing the molecular weight distribution of mutan (sample a) obtained by Example 1 of the present invention (addition of a mutan oligomer).

FIG. 2 is a chart showing the molecular weight distribution of mutan (sample b) obtained in a comparative example (without addition of a mutan oligomer).

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Hiromitsu Kigawa, Inventor 1-3-7 Honjo, Sumida-ku, Tokyo Inside Lion Corporation (72) Inventor Isao Shimotsuura 1-3-7, Honjo, Sumida-ku, Tokyo No. Lion Corporation (72) Inventor Yasuko Takino 1-37 Honjo, Sumida-ku, Tokyo F-Term (Lion Corporation) 4B064 AF12 CA02 CA19 CA21 CC03 CC09 CD09 CD19 DA01 4C083 AD211 AD212 FF01 4C090 AA03 AA09 BA23 BB12 BB32 BB35 BB52 BB92 BD04 DA23

Claims (1)

[Claims] 1. A method for producing mutan by enzyme synthesis using sucrose as a raw material, wherein the concentration of the raw material and / or initial reaction product in the reaction system and / or the solubility of the reaction product in the reaction solution are changed. A method for producing a mutan, comprising controlling the molecular weight of the mutan by means of a method.