JP5054279B2 - Mutanase - Google Patents

Description

  The present invention relates to a mutanase derived from a bacterium belonging to the genus Bacillus, and a gene encoding the same.

Periodontal disease and dental caries, which are two major diseases in the dental field, are known to be closely related to lifestyle habits. Therefore, it is extremely important to develop products related to oral hygiene that can effectively prevent these diseases.
Caries are caused by bacteria such as Streptococcus mutans producing acids such as lactic acid and acetic acid from saccharides in dental plaque. Streptococcus mutans in the oral cavity is said to synthesize high-molecular glucan using sucrose in the diet as a substrate, which is involved in the initiation of plaque formation. The synthesized glucan includes dextran mainly composed of water-soluble α-1,6 bonds and mutan mainly composed of insoluble α-1,3 bonds. Is considered to have a large involvement (Non-Patent Documents 1 and 2). That is, it is considered that decomposition of mutan can prevent plaque formation and suppress induction of caries.

Therefore, development of a mutanase that degrades mutane is very important, and mutanase or a mutanase-producing bacterium has been reported (Patent Documents 3-6). Moreover, it has been reported that not only mutanase but also dextranase that degrades dextran is more effective for the degradation of dental plaque (Non-patent Document 7).
Shigeyuki Hamada How can teeth be made? Iwanami Shinsho 1982 Yamashita et al., Infect. Immun. 61, 3811-3817, 1993 Shimada et al., Journal of Oral Hygiene, 33, 89-98, 1983 JP-A-47-9743 JP-A-63-301788 JP 9-37773 A Japanese Patent Laid-Open No. 10-201483

  An object of the present invention is to find a mutanase useful as an enzyme for oral hygiene products, to obtain a gene encoding them, and to establish a method for producing a single and a large amount of mutanase using the gene.

  The present inventor screened a production strain of a glucanase involved in plaque degradation, particularly an enzyme (mutanase) that degrades mutan which is an α-1,3 glucan, and found a mutanase having specific properties. Furthermore, the gene encoding the mutanase can be cloned and used to produce the mutanase by genetic engineering, and the protein comprising a part of the amino acid sequence of the mutanase amino acid sequence has mutanase activity. I found.

That is, the present invention provides a mutanase having the following enzymological properties.
1. Action:
Hydrolysis of α-1,3-glucan.
2. Substrate specificity:
Decomposes mutan and nigeran, especially mutan.
3. Optimum reaction pH:
When reacted at 40 ° C. using mutan as a substrate, it exhibits high activity in the pH range of 6 to 6.5.
4). pH stability:
When treated with mutan as a substrate at 30 ° C. for 30 minutes, the pH is stable in the range of 5 to 10.
5. Optimal reaction temperature:
When reacting at pH 6.5 using mutan as a substrate, it exhibits high activity at 45-50 ° C.
6). Heat-resistant:
When heat-treated for 15 minutes at pH 6.5 using mutan as a substrate, it is stable up to 50 ° C.
7). Molecular weight:
The molecular weight by SDS-polyacrylamide gel electrophoresis is about 120 kDa.
8). Isoelectric point:
The isoelectric point measured by isoelectric focusing is 6.0 to 7.0.

The present invention also provides a mutanase having the following enzymological properties.
1. Action:
Hydrolysis of α-1,3-glucan.
2. Substrate specificity:
Decomposes mutan and nigeran, especially mutan.
3. Optimum reaction pH:
When reacted at 40 ° C. using mutan as a substrate, it exhibits high activity in the pH range of 6 to 6.5.
4). Molecular weight:
The molecular weight by SDS-polyacrylamide gel electrophoresis is about 40 kDa.
5. Amino-terminal amino acid sequence:
The sequence of 19 amino acid residues on the amino terminal side is
Ala-Arg-Gly-Ala-Val-Met-Pro-Tyr-Ser-Arg-Tyr-Asp-Thr-Glu-Asp-Ala-Thr-Leu-Gly.

The present invention also provides any of the following proteins (a) to (c) and a gene encoding the same.
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (b) an amino acid sequence shown in SEQ ID NO: 1 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and a mutanase activity (C) a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having a mutanase activity

The present invention also provides a gene comprising any of the following polynucleotides (a) to (c).
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2 (b) encoding a protein that hybridizes with the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and has a mutanase activity (C) a polynucleotide comprising a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 2 and encoding a protein having mutanase activity

The present invention also provides any of the following proteins (a) to (c) and a gene encoding the same.
(A) a protein consisting of amino acids 650 to 1217 of the amino acid sequence shown in SEQ ID NO: 1 (b) a sequence consisting of amino acids 650 to 1217 of the amino acid sequence shown in SEQ ID NO: 1; A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having mutanase activity (c) 80% of amino acids 650 to 1217 in the amino acid sequence represented by SEQ ID NO: 1 A protein having an amino acid sequence having the above identity and having a mutanase activity

The present invention also provides a gene comprising any of the following polynucleotides (a) to (c).
(A) a polynucleotide comprising the base sequence of 1948 to 3651 of the base sequence shown in SEQ ID NO: 2 (b) consisting of the base sequence of 1948 to 3651 of the base sequence of SEQ ID NO: 2 A polynucleotide that hybridizes with a polynucleotide under stringent conditions and encodes a protein having mutanase activity (c) a polynucleotide comprising a nucleotide sequence from the 1948th base to the 3651st base sequence of the base sequence represented by SEQ ID NO: 2 A polynucleotide encoding a protein comprising a nucleotide sequence having 80% or more identity with a nucleotide and having a mutanase activity

  The present invention also provides a recombinant vector containing the above gene and a transformant having the recombinant vector.

  The present invention also provides a method for producing a mutanase, which comprises culturing the transformant and collecting the mutanase.

  If the gene of the present invention is used, mutanase and miniaturized mutanase useful as an enzyme for blending into oral hygiene products for preventing dental plaque formation and preventing dental caries can be produced in a single and large amount. Further, this mutanase and miniaturized mutanase can be blended into foods such as gums and candies.

The mutanase of the present invention is a novel enzyme and has the following enzymological properties.
(1) Action Acts on mutan and hydrolyzes it. That is, the α-1,3-glucoside bond of mutan is decomposed.
(2) Substrate specificity Decomposes mutan and nigeran, and particularly degrades mutan. That is, the mutanase solution (0.004 unit / mL) of the present invention was added to a reaction solution comprising standard reaction conditions (50 mM MOPS buffer (pH 6.5), 0.5 mM cobalt chloride, 0.2% mutan). When the solution is added and reacted at 40 ° C. for 60 minutes), various polysaccharides (0.2% mutan, nigeran, dextran, pullulan, starch, β-glucan, chitosan) are added and the reaction is carried out. Is decomposed well and mutan is decomposed particularly well.
(3) Optimum pH
Acetic acid (pH 4-6), citric acid (pH 4.3-6.4), MOPS (pH 6-7.8), phosphoric acid (pH 6.4-8), sodium glycine hydroxide (pH 7.3-10), The mutanase of the present invention was added to a 0.2% mutan solution adjusted to each pH with each buffer solution of carbonic acid (pH 9.6-11.2) so as to be 0.004 unit / mL, and reacted at 40 ° C. for 60 minutes. The activity is measured, and the relative activity at each pH when the activity at the optimum pH is set to 100% is determined, the action is optimum in the vicinity of pH 6, particularly in the range of pH 6 to 6.5. High activity is shown (FIG. 1).
(4) Working pH
Acetic acid (pH 4-6), citric acid (pH 4.3-6.4), MOPS (pH 6-7.8), phosphoric acid (pH 6.4-8), sodium glycine hydroxide (pH 7.3-10), The mutanase of the present invention was added to a 0.2% mutan solution adjusted to each pH with each buffer solution of carbonic acid (pH 9.6-11.2) so as to be 0.004 unit / mL, and reacted at 40 ° C. for 60 minutes. The activity was measured, and when the relative activity at each pH when the activity at the optimum pH was 100% was determined, the activity was 90% or more at pH 5.5 to 7.0, and pH 5.0 It shows 80% or more activity at ˜8.0 (FIG. 1).
(5) pH stability The mutanase of the present invention is converted to 25 mM acetic acid (pH 4-6), citric acid (pH 4.3-6.4), MOPS (pH 6-7.8), phosphoric acid (pH 6.4-8). , Glycine sodium hydroxide (pH 7.3-10), carbonic acid (pH 9.6-11.2), phosphoric acid (pH 11-12.5) added to 0.01 units / mL in each buffer solution When the residual activity after measuring at 30 ° C. for 30 minutes was measured, the residual activity was 80% or more in the range of pH 5 to 10 and stable (FIG. 4).
(6) Optimal reaction temperature The mutanase of the present invention was added to 50 mM MOPS buffer of pH 6.5 containing 0.2% of mutan so as to be 0.003 unit / mL, and reacted at each temperature for 60 minutes. When the activity at 100 ° C. is taken as 100% and the relative activity at each temperature is determined, the reaction rate is highest at 45 ° C. to 50 ° C. (FIG. 2).
(7) Heat resistance 0.004 unit / mL of the mutanase of the present invention is added to a 25 mM MOPS buffer (pH 6.5) and left at 30 ° C. to 70 ° C. for 15 minutes. Thereafter, it was rapidly cooled and the residual activity was measured using standard reaction conditions. As a result, it showed a residual activity of 90% or more at 50 ° C. and is stable (FIG. 3).
(8) Molecular weight The molecular weight of the enzyme purified by 10% SDS polyacrylamide electrophoresis is about 120 kDa (however, in SDS polyacrylamide electrophoresis, it may be increased or decreased by about 10 kDa depending on the measurement conditions).
(9) Isoelectric point The isoelectric point measured by the isoelectric focusing method is 6.0 to 7.0. The isoelectric point measured using a PAG plate (Pharmacia) is around 6.5.

The mutanase of the present invention also includes a mutanase having the following enzymological properties.
(1) Action It hydrolyzes α-1,3-glucan.
(2) Substrate specificity Decomposes mutan and nigeran, and particularly degrades mutan.
(3) Optimum reaction pH
When reacted at 40 ° C. using mutan as a substrate, it exhibits high activity in the pH range of 6 to 6.5.
(4) Molecular weight The molecular weight by SDS-polyacrylamide gel electrophoresis is about 40 kDa (however, it may go up and down by about 5 kDa).
(5) Amino-terminal amino acid sequence The amino-terminal 19 amino acid residue sequence is Ala-Arg-Gly-Ala-Val-Met-Pro-Tyr-Ser-Arg-Tyr-Asp-Thr-Glu-Asp- Ala-Thr-Leu-Gly. This amino acid sequence corresponds to the amino acid sequence from the 650th amino acid to the 668th amino acid of the amino acid sequence shown in SEQ ID NO: 1.

  The mutanase of the present invention includes (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, or (b) an amino acid in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of (a) A protein consisting of a sequence is included.

  Here, in the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which one or several amino acids are deleted, substituted, or added means an amino acid sequence equivalent to the amino acid sequence of SEQ ID NO: 1, respectively. An amino acid sequence in which several, preferably 1 to 10 amino acids have been deleted, substituted or added, and still retains mutanase activity. For addition, 1 to several amino acids at both ends Is included.

  As a result of comparing the identity between the amino acid sequence of the mutanase shown in SEQ ID NO: 1 of the present invention and the amino acid sequence of the known mutanase, 75.6% of the mutanase produced by the Bacillus sp. Showed the identity. The identity with other known mutanases was extremely low, suggesting that the mutanase of the present invention is a novel enzyme.

  Therefore, when the sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 is appropriately aligned, a protein having an identity of 80% or more, preferably 85% or more, more preferably 90% or more and having a mutanase activity is It is equivalent to the above mutanase and is included in the present invention.

  Here, having mutanase activity means the activity of acting on mutan and decomposing the α-1,3-glucoside bond, and the degree of the activity is represented by SEQ ID NO: 1 as long as it exhibits its function. It may be higher or lower than that of a protein having the same amino acid sequence.

  Moreover, the protein equivalent to the above mutanase may have additional properties as long as it has mutanase activity. Such properties include, for example, the property of being superior in stability compared to the protein consisting of the amino acid sequence represented by SEQ ID NO: 1, the property of having different functions only at low temperature and / or high temperature, and the property of different optimum pH Etc.

  A protein consisting of amino acids 650 to 1217 in the amino acid sequence shown in SEQ ID NO: 1 has the same enzymatic properties as the above mutanase. (1) hydrolyzing α-1,3-glucan, (2) decomposing mutan and nigeran, particularly degrading mutan well, (3) when reacting at 40 ° C. with mutan as a substrate, pH 6 (4) Stable at pH 5 to 10 when treated with 30% at 30 ° C. using mutan as a substrate, (5) Reaction at pH 6.5 using mutan as a substrate (6) When it is heat treated at pH 6.5 for 15 minutes using mutan as a substrate, it is stable up to 50 ° C. (7) Measured by isoelectric focusing The isoelectric point is 6.0 to 7.0. Hereinafter, this low molecular weight mutanase-like protein is referred to as “miniaturized mutanase”.

That is, the following (a) to (c) are included in the miniaturized mutanase.
(A) a protein consisting of amino acids 650 to 1217 of the amino acid sequence shown in SEQ ID NO: 1 (b) a sequence consisting of amino acids 650 to 1217 of the amino acid sequence shown in SEQ ID NO: 1; A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having mutanase activity (c) 80% of amino acids 650 to 1217 in the amino acid sequence represented by SEQ ID NO: 1 A protein comprising an amino acid sequence having the above identity, preferably 85% or more, more preferably 90% or more, and having mutanase activity

  The gene of the present invention encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or a protein consisting of an amino acid sequence equivalent to the amino acid sequence, (a) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 (B) a polynucleotide that hybridizes with the gene under stringent conditions and encodes a protein having mutanase activity; (c) has a identity of 80% or more to the nucleotide sequence represented by SEQ ID NO: 2 A gene comprising a nucleotide sequence and a polynucleotide encoding a protein having mutanase activity is preferred. That is, the gene of the present invention is a DNA whose nucleotide sequence is partially changed by treatment with a mutagen, random mutation, specific site mutation, deletion or insertion in the DNA represented by the nucleotide sequence of SEQ ID NO: 2. Furthermore, these DNA variants include a gene consisting of a polynucleotide that hybridizes with a DNA represented by the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having a mutanase activity.

Here, "stringent conditions" are, for example, ^{Molecular cloning -a laboratory manual, 2 nd} edition (Sambrook et al., 1989), and the conditions described. That is, 6XSSC (1XSSC composition: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5X Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C. Examples include conditions of constant temperature for 8 to 16 hours and hybridization.

  In addition, the gene represented by (b) or (c) above has, for example, a higher expression level of mRNA, higher stability of the mRNA, and stability of the translated protein than the gene represented by (a). It may have additional properties such as excellent properties.

  In addition, the present invention provides a gene relating to a miniaturized mutanase. Examples of such a gene include a protein (miniaturized mutanase) consisting of amino acids 650 to 1217 in the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence. As long as it encodes a protein consisting of an amino acid sequence equivalent to the above, preferably (a) a polynucleotide consisting of the base sequence from 1948 to 3651 of the base sequence shown in SEQ ID NO: 2, and (b) the sequence A polynucleotide that hybridizes with a polynucleotide comprising the base sequence from 1948 to 3651 of the base sequence represented by No. 2 under a stringent condition and encodes a protein having a mutanase activity, (c) a sequence 1948th salt of the base sequence shown by No. 2 To a polynucleotide consisting of a nucleotide sequence having the identity of 80% or more, preferably 85% or more, more preferably 90% or more, and a polynucleotide encoding a protein having mutanase activity. Gene.

  In addition, the identity of an amino acid sequence and a base sequence can be performed by comparing amino acid sequences using known algorithms such as Lipman-Pearson method (Science, 227, 1435, 1985). Specifically, the identity can be obtained using a GENTYTYX-WIN (software development) search homology or maximum matching program.

  The mutanase of the present invention is produced and obtained by, for example, culturing a microorganism belonging to the genus Bacillus, a Bacillus sp. KSM-M86 strain, or a fungus having the same bacteriological and physiological properties as the present bacterium, by a known method. be able to. The bacteriological and physiological properties of Bacillus sp. KSM-M86 strain are shown below.

(A) Shape and size of cells: Neisseria gonorrhoeae (0.53 to 0.73 × 2 to 2.7 μm)
(B) Cell polymorphism: None (c) Motility: Existence (d) Spore: Existence, Center, Swelling (0.67-1.0 × 1.3-2.0 μm)
(E) Gram staining: positive Physiological properties (a) nitrate reduction: negative (b) catalase: positive (c) VP test: positive (d) indole production: negative (e) starch hydrolysis: positive (f) dihydroxyacetone production: positive (G) Oxidase: Positive (h) Acid production from glucose: Positive (i) Acid production from arabinose: Negative (j) Acid production from xylose: Positive (k) Acid production from mannitol: Negative (l) Gas production from glucose: negative (m) lecithinase: negative (n) use of citrate: positive (o) use of propionic acid: negative (p) degradation of tyrosine: negative (q) deamination of phenylalanine: negative ( r) Casein degradation: positive (s) Gelatin degradation: positive (t) Anaerobic growth: grow (u) Salt tolerance: 2% or less (v) Growth temperature: 15 ° C-35 ° C
(W) YM agar medium: weak growth (x) Nutrient agar medium: grows

  As a result of examining the bacteriological and physiological properties of the KSM-M86 strain described above according to the description in Bergey's Mannual of Systematic Bacteriology (Williams & Wilkins, 1984), it was clear that the strain belongs to the genus Bacillus. The species was not completely consistent with known Bacillus bacteria. Therefore, it was deposited as a new microorganism as Bacillus SP KSM-M86 (FERM P-19721) at the National Institute of Advanced Industrial Science and Technology.

  In order to produce the mutanase of the present invention from Bacillus sp. KSM-M86, it is inoculated into a medium supplemented with 0.005% to 10% (w / v) mutan under an aerobic condition at an appropriate temperature. What is necessary is just to culture. Purification of mutanase from the culture can be performed according to a conventional method. That is, after centrifuging the culture solution and removing the cells, the supernatant can be concentrated by adding ammonium sulfate to precipitate the enzyme, or using an ultrafiltration membrane or the like. Furthermore, it can be prepared in various forms by spray drying, freeze drying, crystallization and the like.

  The mutanase of the present invention obtained from Bacillus sp. KSM-M86 has a molecular weight measured by SDS-polyacrylamide electrophoresis of about 40 kDa (however, it may rise and fall by about 5 kDa), and mutan as a substrate at 40 ° C. When reacted, it exhibits high activity in the pH range of 6 to 6.5. Further, the sequence of 19 amino acid residues on the amino terminal side is Ala-Arg-Gly-Ala-Val-Met-Pro-Tyr-Ser-Arg-Tyr-Asp-Thr-Glu-Asp-Ala-Thr-Leu- Gly.

Although the mutanase of the present invention can be obtained from nature as described above, the gene can be cloned from the chromosomal DNA of the microorganism, and the mutanase can be produced and obtained in large quantities.
As a method for cloning the mutanase gene, for example, the DNA encoding the mutanase of the present invention can be stabilized by ligating it to a DNA vector that can stably amplify the gene, or introducing it onto a chromosomal DNA that can stably maintain the gene. And a method for introducing mutanase by introducing the gene into a host where the gene can be expressed stably and efficiently.

  Further, in order to produce a recombinant vector containing the mutanase gene of the present invention, it is possible to maintain replication in the host cell, to stably express the enzyme, and to a vector that can stably hold the gene. A gene should be incorporated. Examples of such expression vectors include pUC18, pBR322, pHY300PYL (Takara Bio) and the like when Escherichia coli is used as a host, and pUB110, pC194, pHY300PYL and the like when Bacillus subtilis is used as a host.

  When obtaining the miniaturized mutanase of the present invention, the miniaturized mutanase is obtained by performing PCR using appropriate primers designed based on the chromosomal chromosome of the microorganism and the mutanase gene sequence cloned from the chromosomal DNA of the microorganism. The DNA encoding the miniaturized mutanase of the present invention can be stably obtained by obtaining a gene and, for example, ligating it to a DNA vector capable of stably amplifying the gene or introducing the gene onto a chromosomal DNA capable of stably maintaining the gene. A method for amplifying and introducing a gene into a host capable of stably and efficiently expressing the gene and producing a miniaturized mutanase can be employed.

  In order to transform a host bacterium using the obtained recombinant vector, a calcium chloride method, a protoplast method, a competent cell method, an electroporation method, or the like can be used. Examples of the host bacterium include Bacillus subtilis, Escherichia coli, mold, yeast, actinomycetes, etc., preferably Bacillus subtilis, and more preferably Bacillus subtilis lacking or deleting the protease gene.

  The transformant may be cultured under appropriate conditions using a medium containing a carbon source, a nitrogen source, an inorganic metal salt, a vitamin and the like that can be assimilated by the organism. From the culture solution, the enzyme is collected and purified by a general method, and a necessary enzyme form can be obtained by lyophilization, spray drying, and crystallization.

Furthermore, the mutanase and miniaturized mutanase of the present invention can be prepared by using known techniques such as site-directed mutagenesis to improve productivity and specific activity, and to prepare mutant enzymes. For example, any of the commonly used methods of random mutation and partial mutation specific to a gene encoding mutanase or miniaturized mutanase can be employed. More specifically, for example, it can be performed using Site-Directed Mutagenesis System Mutant-Super Express Km kit manufactured by Takara Shuzo. In addition, a recombinant PCR (polymerase chain reaction) method (PCR protocols, Academic press, New York, 1990) can also be used.
The mutanase and miniaturized mutanase of the present invention prepared as described above are useful as enzymes for blending into various oral hygiene products, particularly products such as dentifrices or gums and candies.

Example 1 Screening for Mutanase-Producing Bacteria 0.5 g of soil samples from various parts of Japan were suspended in physiological saline (5 mL) and treated at 80 ° C. for 20 minutes. Thereafter, 0.1 mL of the heat-treated supernatant was added to a liquid medium (4 mL) having the composition shown in Table 1, and shaking culture was performed at 30 ° C. for 7 days. The culture solution in which the fungus grew was inoculated with 0.1 mL of a new medium having the same composition, and further cultured with shaking for 7 days. This operation was further repeated once, and then the bacterial solution was smeared on a trypticase soy agar medium, followed by stationary culture at 30 ° C. for 3 days. The grown bacteria were selected and purified, then inoculated into a liquid medium, cultured at 30 ° C. for 3 days with shaking, and the mutanase activity in the supernatant was measured. Bacillus sp. KSM-M86 strain was selected from strains with high mutanase production.

Example 2 Production and Purification of Mutanase Bacillus sp. KSM-M86 strain was converted to 1.5% (w / v) Polypeptone S (Nippon Pharmaceutical), 0.5% yeast extract (Difco), 1.0% fish meat extract (Wako) Inoculate a 70 mL liquid medium consisting of 0.1% dipotassium phosphate, 0.01% magnesium sulfate heptahydrate, 0.001% manganese sulfate, and 0.1% mutan. went. The culture solution (0.084 U / mL) is centrifuged (8,000 × g, 10 minutes, 4 ° C.), and ammonium sulfate is gradually added to 2 L of the obtained supernatant solution so as to be 80% saturation, followed by centrifugation ( The precipitate was collected and dialyzed against 10 mM Tris-HCl buffer (pH 7.5) for one day and night. The dialyzed internal solution was applied to a SuperQ Toyopearl column that had been equilibrated in advance with 50 mM Tris-HCl buffer (pH 7.5). The mutanase activity was obtained in the non-adsorbed fraction, then applied to a Butyl Toyopearl column previously equilibrated with 10 mM Tris-HCl buffer (pH 7.5) containing 0.75 M ammonium sulfate, and then 0.75 M to 0 M ammonium sulfate. The adsorbed protein was eluted by the linear concentration gradient method. The fraction of mutanase activity was collected, concentrated and desalted by ultrafiltration, and applied to a DEAE Toyopearl column that had been equilibrated in advance with 10 mM MOPS buffer (pH 6.0). The adsorbed protein was eluted by a linear concentration gradient method using potassium chloride up to 0.1 M, and the active fractions were collected and adsorbed onto a hydroxyapatite column that had been equilibrated in advance with 10 mM phosphate buffer. Activity was observed in the fractions eluted with the same buffer, which were collected and concentrated. When the concentrated solution was subjected to SDS electrophoresis, a uniform protein band was confirmed around a molecular weight of 40 kDa. As a result of analyzing the amino terminal amino acid sequence of this band, Ala-Arg-Gly-Ala-Val-Met-Pro-Tyr-Ser-Arg-Tyr-Asp-Thr-Glu-Asp-Ala-Thr-Leu-Gly The 19 amino acid sequence was determined.

Example 3 Cloning of Mutanase Gene Primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4) were designed based on the 19 amino acid sequence obtained in Example 2.
Bacillus sp. KSM-M86 strain was inoculated into 10 mL of liquid medium consisting of 1.0% (w / v) bactotripton (Difco), 0.5% yeast extract (Difco), 1.0% NaCl, and 30 After shaking culture at 1 ° C. for 1 day, chromosomes were extracted from the cells using Gen Toru-kun (for yeast) (Takara Bio). According to the LA PCR in vitro Cloning kit (Takara Bio), 5 μg of the obtained chromosome was treated with HindIII restriction enzyme, then ligated with a cassette (attached to the kit), and PCR was performed using primer 1 and primer C1 (attached to the kit). . Using the obtained amplified fragment as a template, PCR was further performed using Primer 2 and Primer C2 (attached to the kit) to obtain a PCR amplified fragment of about 0.8 kb. The amplified DNA fragment was ligated with pT7Blue T-Vector (Takara Bio), and competent cells of E. coli JM109 strain (Takara Bio) were transformed. Plasmid DNA was extracted from the white transformant, sequenced using primer 2 and primer C2, and the partial sequence of the mutanase gene was analyzed. Primers were constructed based on the obtained base sequence, and a 4863 bp base sequence containing a gene encoding mutanase was determined by the LA PCR in vitro Cloning kit.
The promoter region of the spoVG gene of Bacillus subtilis was used for expression of the mutanase of the present invention. Primer 3 (SEQ ID NO: 5), primer 4 (SEQ ID NO: 6), and primer 5 (SEQ ID NO: 7) were designed based on the known gene sequence of Bacillus subtilis and the determined mutanase gene sequence. PCR was performed using Primer 3 and Primer 4 using the chromosomal DNA of Bacillus subtilis 168 strain (BGSC 1A1) as a template to obtain a promoter region of the spoVG gene, an amplified fragment of about 0.2 kb. PCR was performed using LA Taq (Takara Bio), denaturing the template DNA at 94 ° C for 1 minute, followed by 30 cycles of 94 ° C for 1 minute, 56 ° C for 30 seconds, 72 ° C for 1 minute, Incubated at 72 ° C. for 2 minutes. Next, PCR was performed using the chromosomal DNA of Bacillus sp. KSM-M86 strain as a template and the obtained DNA fragment of about 0.2 kb and primer 5. PCR was performed by denaturing the template DNA at 94 ° C. for 1 minute, followed by 30 cycles of 94 ° C. for 1 minute, 56 ° C. for 30 seconds, 72 ° C. for 3 minutes, and further incubated at 72 ° C. for 2 minutes. The amplified about 3.9 kb DNA fragment was treated with restriction enzymes SpeI and BglII, similarly ligated with plasmid pHY300PLK (Takara Bio) treated with SpeI and BglII, and Escherichia coli JM109 strain (Takara Bio) was used using the reaction solution. Transformed. A plasmid into which a mutanase gene was introduced was selected from a tetracycline resistant strain. A plasmid containing a mutanase gene was extracted from the transformant, and Bacillus subtilis ISW1214 strain (Yakult Head Office) lacking alkaline protease was transformed.

Example 4 Production of Miniaturized Mutanase (Δ) PCR was performed using primer 6 (SEQ ID NO: 8) and primer 5 (SEQ ID NO: 7) designed based on the mutanase gene sequence of Bacillus sp. KSM-M86 strain. PCR was performed using LA Taq, denaturing the template DNA at 94 ° C for 1 minute, followed by 30 cycles of 94 ° C for 1 minute, 55 ° C for 30 seconds, 72 ° C for 3 minutes, and 2 cycles at 72 ° C. Keep warm for a minute. The amplified DNA fragment of about 3.4 kb was purified, then treated with restriction enzymes SalI and BglII, and ligated with plasmid pHY300PLK (Takara Bio) similarly treated with SalI and BglII. Using the reaction solution, E. coli HB101 strain competent cell (Takara Bio) was transformed, and a plasmid in which a miniaturized mutanase gene region was introduced from tetracycline-resistant bacteria was selected.
As in Example 3, the promoter region of the spoVG gene of Bacillus subtilis was used for the expression of the miniaturized mutase. That is, Primer 7 (SEQ ID NO: 9) and Primer 8 (SEQ ID NO: 10) were designed based on the publicly available chromosomal DNA of Bacillus subtilis 168 strain. Furthermore, as the SD sequence and the signal sequence, the sequence of the alkaline cellulase gene (GenBank M84963) of Bacillus sp. KSM-64 strain was used, and primer 9 (SEQ ID NO: 11) and primer 10 (SEQ ID NO: 12) were designed. In addition, primer 11 (SEQ ID NO: 13) and primer 12 (SEQ ID NO: 14) were designed in consideration of binding to the signal sequence based on the miniaturized mutanase gene sequence.
Using the chromosomal DNA of Bacillus subtilis 168 as a template, PCR of the promoter region using primer 7 and primer 8, and the SD sequence and signal region using the chromosomal DNA of Bacillus sp. KSM-64 strain as a template and primer 9 and primer 10 PCR of the miniaturized mutanase region was performed using primers 11 and 12 using the chromosomal DNA of Bacillus sp. KSM-M86 as a template. PCR was performed by denaturing the template DNA at 94 ° C. for 1 minute, followed by 30 cycles of 94 ° C. for 1 minute, 55 ° C. for 30 seconds, 72 ° C. for 1 minute, and further incubated at 72 ° C. for 2 minutes. SOE-PCR using the amplified DNA fragment was performed, followed by PCR with addition of primer 7 and primer 12 to obtain an amplified fragment of about 0.5 kb in which the three fragments were bound. The obtained DNA fragment was treated with restriction enzymes BamHI and HaeII, and similarly, a binding reaction was performed with a plasmid into which the downstream region of the mutanase gene treated with BamHI and HaeII was introduced. The reaction solution was used to transform E. coli HB101 competent cells, and a plasmid into which a miniaturized mutanase gene was introduced from a tetracycline resistant strain was selected. A plasmid containing a miniaturized mutanase gene was extracted from the transformant and transformed into Bacillus subtilis ISW1214 strain (Yakult Head Office), which lacked the alkaline protease.

Example 5 Production and Purification of Recombinant Mutanase and Miniaturized Mutanase (Δ) The transformants obtained in Example 3 and Example 4 were treated with 8% (w / v) polypeptone S, 1% fish meat extract, 0.5% A medium (50 mL) consisting of% yeast extract, 0.2% potassium phosphate and 0.04% magnesium sulfate was inoculated and cultured at 30 ° C. for 3 days with shaking.
The culture solution (0.28 U / mL, Δ is 0.068 U / mL) was centrifuged (8,000 × g, 10 minutes, 4 ° C.), and ammonium sulfate was saturated 80% with respect to 600 mL of the obtained supernatant solution. The mixture was gradually added to the solution and centrifuged (8,000 × g, 1 minute, 4 ° C.), and the precipitate was collected and dialyzed against 10 mM Tris-HCl buffer (pH 7.5) overnight. The dialyzed internal solution was applied to a SuperQ Toyopearl column that had been equilibrated in advance with 50 mM Tris-HCl buffer (pH 7.5). The mutanase activity was obtained in the non-adsorbed fraction, and then adsorbed to a DEAE Toyopearl column that had been equilibrated in advance with 10 mM MOPS buffer (pH 6), and then adsorbed protein was obtained by a linear concentration gradient method of 0 to 0.1 M potassium chloride. Was eluted. The mutanase activity fraction was collected, added with ammonium sulfate to 1M, and then applied to a Phenyl Toyopearl column that had been equilibrated in advance with a Tris-HCl buffer (pH 7.5) containing 1M ammonium sulfate. The adsorbed protein was eluted by a linear concentration gradient method using 1M to 0M ammonium sulfate, and the active fractions were collected and concentrated by ultrafiltration. The concentrated solution was placed on a Bio-Gel-A 0.5 m gel filtration column that had been equilibrated in advance with 10 mM Tris-HCl buffer (pH 7.5) containing 0.1 M potassium chloride. Fractions in which activity was observed in the eluted fractions were collected and concentrated. When the concentrated solution was subjected to SDS electrophoresis, a uniform protein band was confirmed in the vicinity of a molecular weight of about 120 kDa, and various properties were examined using this concentrated solution. In the case of miniaturized mutanase (Δ), it was completely purified up to the Phenyl Toyopearl column, and SDS electrophoresis was performed on the concentrated fraction of the active fraction. As a result, a uniform protein band was confirmed around a molecular weight of about 57 kDa. Various properties were examined using this concentrated solution.

Example 6 Properties of recombinant mutanase Optimal reaction pH
Acetic acid (pH 4-6), citric acid (pH 4.3-6.4), MOPS (pH 6-7.8), phosphoric acid (pH 6.4-8), sodium glycine hydroxide (pH 7.3-10), Reaction was performed using each buffer solution of carbonic acid (pH 9.6-11.2). As a result, the reaction rate of this enzyme was highest in a citrate buffer around pH 6 (FIG. 1).
2. Optimum reaction temperature The reaction temperature was 30 ° C to 80 ° C under standard reaction conditions. As a result, the reaction rate of this enzyme was highest at around 45 ° C. to 50 ° C. (FIG. 2).
3. Thermostable enzyme was added to 25 mM MOPS buffer (pH 6.5) and left at 30 ° C. to 70 ° C. for 15 minutes. Thereafter, it was quenched and the residual activity was measured using standard reaction conditions. As a result, this enzyme was stable up to 50 ° C. (FIG. 3).
4). pH stability Enzymes were mixed with 25 mM acetic acid (pH 4-6), citric acid (pH 4.3-6.4), MOPS (pH 6-7.8), phosphoric acid (pH 6.4-8), glycine sodium hydroxide ( pH 7.3-10), carbonic acid (pH 9.6-11.2), and phosphoric acid (pH 11-12.5) were added to each buffer solution, and the residual activity was measured after incubation at 30 ° C. for 30 minutes. . As a result, this enzyme was stable in the range of pH 5-10 (FIG. 4).
5. Substrate specificity Various polysaccharides (all 0.2%) were added in place of mutan under standard reaction conditions to carry out the reaction. As a result, this enzyme was able to degrade nigeran in addition to mutan, but did not degrade other polysaccharides other than α-1,3 glucan.
6). Molecular weight The molecular weight of this enzyme was about 120 kDa as determined by 10% SDS polyacrylamide electrophoresis (however, in SDS polyacrylamide electrophoresis, the molecular weight may increase or decrease by about 10 kDa depending on the measurement conditions).
7). Isoelectric point As a result of measuring the isoelectric point of this enzyme using a PAG plate (Pharmacia), it was around 6.5.
The molecular weight of the miniaturized mutanase (Δ) from amino acid 650 to 1217 by 10% SDS polyacrylamide electrophoresis is about 57 kDa, and the isoelectric point measured using a PAG plate (Pharmacia) is around pH 6.5. there were. Properties such as optimum reaction temperature (FIG. 5), optimum reaction pH (FIG. 6), and substrate specificity almost coincided with those of wild-type mutanase.

Reference Example Method for Measuring Mutanase Activity The enzyme activity in the above enzymatic properties was carried out by the following method.
An enzyme solution appropriately diluted was added to a reaction solution consisting of 50 mM MOPS buffer (pH 6.5), 0.5 mM cobalt chloride, and 0.2% mutan, and the mixture was incubated at 40 ° C. for 60 minutes (total volume: 1 mL). 1 mL of DNS reagent [0.5% (w / v) dinitrosalicylic acid, 0.4N sodium hydroxide, 30% sodium / potassium tartrate] was added, and the mixture was incubated at 100 ° C. for 5 minutes. Quantified. One unit of mutanase (unit) was defined as an amount that produces reducing sugar corresponding to 1 μmol of glucose per minute under the above reaction conditions.

It is a graph which shows the optimal reaction pH of the mutanase of this invention. It is a graph which shows the optimal reaction temperature of the mutanase of this invention. It is a graph which shows the heat resistance of the mutanase of this invention. It is a graph which shows the pH stability of the mutanase of this invention. It is a graph which shows the optimal reaction temperature of the miniaturized mutanase of this invention. It is a graph which shows the optimal reaction pH of the miniaturized mutanase of this invention.

Claims (13)

A mutanase having the following enzymatic properties:
1. Action:
Hydrolysis of α-1,3-glucan.
2. Substrate specificity:
Decomposes mutan and nigeran, especially mutan.
3. Optimum reaction pH:
When reacted at 40 ° C. using mutan as a substrate, it exhibits high activity in the pH range of 6 to 6.5.
4). pH stability:
When treated with mutan as a substrate at 30 ° C. for 30 minutes, the pH is stable in the range of 5 to 10.
5. Optimal reaction temperature:
When reacting at pH 6.5 using mutan as a substrate, it exhibits high activity at 45-50 ° C.
6). Heat-resistant:
When heat-treated for 15 minutes at pH 6.5 using mutan as a substrate, it is stable up to 50 ° C.
7). Molecular weight:
The molecular weight by SDS-polyacrylamide gel electrophoresis is about 120 kDa.
8). Isoelectric point:
The isoelectric point measured by isoelectric focusing is 6.0 to 7.0. A mutanase having the following enzymatic properties:
1. Action:
Hydrolysis of α-1,3-glucan.
2. Substrate specificity:
Decomposes mutan and nigeran, especially mutan.
3. Optimum reaction pH:
When reacted at 40 ° C. using mutan as a substrate, it exhibits high activity in the pH range of 6 to 6.5.
4). Molecular weight:
The molecular weight by SDS-polyacrylamide gel electrophoresis is about 40 kDa.
5. Amino-terminal amino acid sequence:
The sequence of 19 amino acid residues on the amino terminal side is
Ala-Arg-Gly-Ala-Val-Met-Pro-Tyr-Ser-Arg-Tyr-Asp-Thr-Glu-Asp-Ala-Thr-Leu-Gly. Any of the following proteins (a) to (c):
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (b) an amino acid sequence shown in SEQ ID NO: 1 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and a mutanase activity (C) a protein comprising an amino acid sequence having 90 % or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having a mutanase activity   A gene encoding the protein according to claim 3. A gene comprising any of the following polynucleotides (a) to (c):
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2 (b) encoding a protein that hybridizes with the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and has a mutanase activity (C) a polynucleotide comprising a nucleotide sequence having 90 % or more identity with the nucleotide sequence represented by SEQ ID NO: 2 and encoding a protein having mutanase activity Any of the following proteins (a) to (c):
(A) a protein consisting of amino acids 650 to 1217 of the amino acid sequence shown in SEQ ID NO: 1 (b) a sequence consisting of amino acids 650 to 1217 of the amino acid sequence shown in SEQ ID NO: 1; A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having mutanase activity (c) 90 % of amino acids 650 to 1217 in the amino acid sequence represented by SEQ ID NO: 1 A protein having an amino acid sequence having the above identity and having a mutanase activity   A gene encoding the protein according to claim 6. A gene comprising any of the following polynucleotides (a) to (c):
(A) a polynucleotide comprising the base sequence of 1948 to 3651 of the base sequence shown in SEQ ID NO: 2 (b) consisting of the base sequence of 1948 to 3651 of the base sequence of SEQ ID NO: 2 A polynucleotide that hybridizes with a polynucleotide under stringent conditions and encodes a protein having mutanase activity (c) a polynucleotide comprising a nucleotide sequence from the 1948th base to the 3651st base sequence of the base sequence represented by SEQ ID NO: 2 A polynucleotide encoding a protein comprising a nucleotide sequence having 90 % or more identity with a nucleotide and having a mutanase activity   A recombinant vector containing the gene according to claim 4, 5, 7, or 8.   A transformant comprising the recombinant vector according to claim 9.   The transformant according to claim 10, wherein the host is a microorganism.   A method for producing a mutanase, comprising culturing the transformant according to claim 11 and collecting a mutanase.   A microorganism (Bacillus sp. KSM-M86) deposited as FERM P-19721 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

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