JPH07135973A - Enzyme originated from periodontic bacteria, its determination and antibody against the enzyme - Google Patents

Abstract

PURPOSE:To provide an enzyme from periodontic bacteria enabling sure determination of the progress and activity of periodontosis without using the experience of skilled person and expectable as a preventing and treating agent for periodontosis in addition to the use as an analytic reagent. CONSTITUTION:This invention relates to an enzyme originated from Porphyromonas gingivalis and having the following enzymological properties, a determination method for the enzyme and an antibody against the enzyme. (1) Action, decomposing periodontal tissue and inhibiting inflammatory cells; (2) substrate specificity, exhibiting high decomposition activity against various proteins such as collagen and immunoglobulin and strong specific decomposition activity against synthetic fluorescent substrate; (3) optimum pH of 7-8 and stable pH of 4-9; (4) suitable working temperature range, from room temperature to 37 deg.C; (5) molecular weight, about 50kDa by gelfiltration and about 44kDa by SDS get electrophoresis; (6) isoelectric point, pH 5 to 5.5.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to an important disease in dentistry,
In particular, it relates to an enzyme derived from periodontopathic bacteria, a method for measuring the same, and an antibody against the enzyme, which are useful in the diagnosis and treatment of periodontal disease.

[0002]

2. Description of the Related Art Periodontal disease is the most important disease in the dental field in today's aging society. The desire to eat things with your own teeth for the rest of your life becomes more and more intense, while the periodontal disease that causes tooth loss is steadily increasing with age. Periodontal examination and diagnosis to understand the pathological condition are extremely important for the progress of periodontal treatment.

Currently, clinical parameters used for periodontal examination and diagnosis include (1) the depth of the periodontal pocket (prob
ing depth) and attachment loss, (2)
Tooth mobility, (3) Gingival inflammation index (gingival)
index), (4) plaque ind showing the cumulative state of plaque
ex), (5) Gingival bleeding inde
Items such as x), (6) alveolar bone resorption from radiographs, and (7) gingival crevicular fluid (GCF volume) are known.

However, these clinical parameters have been problematic in the following points and have not been fully satisfactory. First, the measurement of the depth of the periodontal pocket and the amount of attachment loss, and the determination of the degree of alveolar bone resorption from X-ray photographs
It is widely used as a useful clinical parameter to know the degree of periodontal tissue destruction, which is the most important pathological condition of periodontal disease, but these are only for showing the results of periodontal tissue destruction due to past inflammation. , It cannot be a clue to know the current activity of periodontal tissue destruction and its influence on pulp.

In addition, the gingival index, plaque index, gingival bleeding
index), degree of tooth sway, and amount of gingival crevicular fluid (GCF)
volume) is a parameter that reflects the current state of illness, but the criteria for judgment are extremely rough, and a special measuring device is required (GCF volume). As clinical parameters for diagnosing activity and determining the necessity of treatment, accuracy, reproducibility, and objectivity are lacking. As described above, at present, there is no objective examination method or accurate diagnosis method for the diagnosis of periodontal disease, which is a global problem in the field.

Recently, Porphyromonas gingivali, which is one of the periodontopathic bacteria, is used.
Although a method for diagnosing periodontal disease has been found by measuring a plurality of enzymes derived from (s) using an appropriate substrate and / or activator (International Application Publication WO09: W09).
2/07086), the method of the present invention has the drawback that the enzyme is not specified, the specificity and sensitivity of the substrate used for the measurement are low, and the enzyme activity measured by these methods is Various inhibitors (endogeneou
s inhibitors such as serpins and cystatins) is unknown, and if these enzymes are affected by the inhibitor, the amount of enzyme measured by the method of the invention cannot be said to be accurate, and Was still insufficient for use in the diagnosis of periodontal disease.

[0007]

Regarding diagnosis of periodontal disease and the like, it is very important to objectively know the progress of the condition, but it is very important to judge how to perform the subsequent treatment. As described above, a method that is not sufficiently satisfactory has not yet been provided, and development of a method that can easily determine the progress of periodontal disease has been earnestly desired.

[0008]

[Means for Solving the Problems] The present inventors have found that P. gingivalis, which is one of the periodontopathic bacteria.
As a result of studying the action of Escherichia coli on periodontal disease, it was found that the bacterium produces a novel enzyme which is a protease, and this enzyme is involved in periodontal disease.

Further, the present inventors have found that the above-mentioned enzyme activity is increased in the gingival crevicular fluid of clinical patients with periodontal disease in proportion to the progress of the periodontal disease, and the disease state of periodontal disease and the activity of the enzyme We found a new finding that the degree of increase is correlated.

In particular, the enzyme has a collagenase activity that strongly decomposes type I collagen, which is a major component of periodontal tissue, and causes direct destruction of periodontal tissue and inflammatory cells such as neutrophils. It becomes clear that it has properties and functions closely related to the onset and progression of periodontal disease, such as causing functional impairment and destroying the biological defense system,
It has been found that measuring the enzyme derived from P. gingivalis, which is conventionally known, is more significant in measuring the progress of periodontal disease than in measuring the enzyme.

The present invention has been completed based on the above findings, and provides the above-mentioned enzyme produced by P. gingivalis and a method for detecting this enzyme.

The enzyme of the present invention can be obtained, for example, as follows using a microorganism belonging to P. gingivalis. First, a microorganism belonging to P. gingivalis is cultivated, and ammonium sulphate is added to the culture supernatant to make it 70% saturated, and the resulting precipitate is collected by means of centrifugation or the like. Dialyzed against phosphate buffer containing. Then, the centrifugal dialysis supernatant is applied to a column such as DEAE Sephacel equilibrated with a phosphate buffer, the resulting non-adsorbed fraction is concentrated, and further applied to a column such as CM-Toyopearl to elute the active fraction. Finally, the elution fraction was concentrated and dialyzed, and then subjected to isoelectric point separation in the range of pH 3.5 to 10, the active fractions of pH 5.0 to 5.5 were collected, and after further concentration and dialysis, TSK gel G2000SW
It can be obtained as a purified enzyme by subjecting it to gel filtration.

The novel enzyme (protease) of the present invention thus obtained has the following specific enzymological properties.

(1) Action: It has a direct degrading effect on periodontal tissue mainly composed of collagen, and also has a damaging activity on inflammatory cells such as neutrophils. The decomposition mode of this enzyme is relatively non-specific and acts as an endoproteinase. (2) Substrate specificity: A synthetic fluorescent substrate t-butyloxycarbonyl-L-phenylalanyl-L-seryl-L-arginine-4-methylcoumaryl, which has high resolution for various proteins such as collagen and immunoglobulin. -7-amide (Boc-Phe-Ser-Arg-MCA) and carbobenzoyl-L-phenylalanyl-L-arginine-
Arginyl endopeptidase (arginyle) that specifically decomposes 4-methylcoumarylamide (Z-Phe-Arg-MCA)
ndopeptidase) activity.

(3) Optimum pH and stable pH: As shown in FIG. 1, the optimum pH is 7 to 8 for both protein substrates and synthetic substrates, and is stable in the range of pH 4 to 9. (4) Optimum temperature range of action; room temperature to 37 ° C (Fig. 2
reference).

(5) Activation: As shown in Table 1, it is remarkably activated by SH group reducing agents such as cysteine, 2-mercaptoethanol and dithiothreitol.

(6) Inhibitors: As shown in Table 2, chymostatin, leupeptin, E-64, antipain,
It is strongly inhibited by EDTA, TPCK, TLCK, etc. However, it is completely unaffected by the cystatin group (eg egg white cystatin and human cystatin S).

(7) Molecular weight: The apparent molecular weight determined by gel filtration is about 50 kDa, and the molecular weight determined by SDS gel electrophoresis is about 44 kDa. (8) Others; isoelectric point pH 5-5.5

The above enzyme is presumed to have a structure represented by the formula below or a peptide sequence similar thereto. In the formula, the base sequence encoding the peptide sequence is also shown.

[0020]

[Chemical 1]

[Chemical 2]

The antibody that specifically recognizes the enzyme of the present invention, which can be used for the analysis of the enzyme of the present invention, is prepared by a known method using the enzyme of the present invention, for example, using Freund's complete adjuvant. be able to. The outline of the method for obtaining the antibody is as follows.

That is, the purified enzyme solution (about 1 mg) prepared as described above was mixed with an equal amount of Freund's complete adjuvant, and the suspension was subcutaneously injected into a rabbit or the like at several sites. , This is repeated twice every two weeks. Then, the booster is performed once to collect the antiserum. An antibody can be obtained as an IgG fraction by subjecting the collected antiserum to ammonium sulfate treatment and protein A-Sepharose (Pharmacia, Sweden) column chromatography.

The synthetic substrate that specifically binds to the enzyme of the present invention can be found from commercially available synthetic fluorescent substrates, for example, as follows. That is, 10 μM
Reaction solution containing 20 mM substrate, 5 mM cysteine and the enzyme (20 mM phosphate buffer, pH 7.5) at 40 ° C.
After heating for 0 minutes, 10 mM iodoacetic acid solution (pH 5)
The reaction is stopped by the addition of 4-methylcoumaryl-7-amide (AM) at an excitation wavelength of 460 nm and a fluorescence wavelength of 380 nm.
The release of C) is measured, and the substrate that releases AMC may be a synthetic substrate that specifically binds to the enzyme of the present invention.

Table 3 shows the decomposition activity under the same conditions when various synthetic fluorescent substrates were used, that is, the substrate specificity of the enzyme of the present invention. From this result, as the substrates specifically recognized by the enzyme of the present invention, Z-Phe-Arg-MCA, B
oc-Phe-Ser-Arg-MCA, Boc-Gl
n-Ala-Arg-MCA or the like is selected. These substrates can be purchased from Peptide Institute (Osaka).

[0025]

For the enzyme activity produced by the above-mentioned P. gingivalis present in the periodontal region, for example, a synthetic substrate that specifically recognizes the enzyme and / or an antibody that specifically recognizes the enzyme is used by the following method. Can be measured.

The method for measuring the enzyme of the present invention existing in the periodontal region can be specifically performed as follows.

(1) Collection of test liquid: As a subject for collecting the test liquid, a clinical patient having mild to severe periodontitis without systemic disease is suitable, and the test site is Select periodontal pockets that show a clear vertical resorption of alveolar bone on radiographs.

The gingival crevicular fluid (GCF) was collected by first simply removing moisture and then carefully removing the supragingival plaque, and then removing periopaper strips (Harco Electronics, Canada).
To collect GCF. The sampling is continuously performed for 30 seconds using three paper strips.
For the GCF amount, the amount of liquid that permeates per unit time is measured by applying a second paper strip to PERIOTRON 6000 (Harco Electronics, Canada).

(2) Measurement of enzyme activity; 300 μl of phosphate buffered saline (pH 7.
Soak 3 paper strips in 4),
After standing for 6 hours, ultrasonic treatment is carried out for 5 minutes, and the centrifugal supernatant is used as a sample solution. First, a part of the test liquid is used, and a certain amount of a substrate (for example, Z-Phe-Arg) is used.
-MCA, Boc-Phe-Ser-Arg-MCA
Etc.) and cysteine, and after heating at about 40 ° C., an iodoacetic acid solution or the like is added to stop the reaction. Then, the release of 4-methylcoumaryl-7-amide (AMC) was measured at an excitation wavelength of 460 nm and a fluorescence wavelength of 380 nm,
The enzymatic activity can be determined from the decomposition activity for the above substrate.

At this time, the amount of activity that is inhibited by the presence of EDTA (10 mM) and leupeptin (50 μM) is the amount of the enzyme as a standard (see FIG. 3). However, the amount of enzyme at this stage is such that the substrate used is not recognized only by the enzyme, and may be degraded by other proteases. Therefore, further
Add a specific antibody against the enzyme to a part of the test solution,
After reacting for 10 minutes at 0 ° C, the reaction solution is centrifuged to separate into a solid phase (immune complex) and a liquid phase, and the amount of activity transferred to the solid phase is measured by measuring the decomposition activity against the above-mentioned substrate in the liquid phase. By calculating, the more accurate amount of the enzyme is determined.

Therefore, the following steps may be taken in order to obtain a more accurate enzyme activity. (1) A part of the sample is reacted with a substrate of the enzyme of the present invention in the presence of EDTA and leupepsin to measure the enzyme activity. (2) After a part of the sample is reacted with an antibody against the enzyme of the present invention, The reaction solution is separated into a solid phase and a liquid phase, and then the enzyme activity is measured according to the above (1), (3)
The enzyme activity of step 2 is subtracted from the enzyme activity of step 1 to calculate the amount of enzyme activity of the present invention.

In order to more easily measure the enzyme of the present invention, (a) an antibody that specifically binds to the enzyme of claim 1, and (b) specifically recognizes the enzyme of claim 1. Substrates, (c) leupeptin and (d) EDT
A kit for measuring the enzyme derived from periodontopathic bacteria containing A may be used.

The present invention is based on the fact that the enzyme of the present invention is unique to P. gingivalis, as shown in Table 4 below. The relationship between the increased activity of the enzyme of the present invention in the periodontal pockets of periodontal disease patients and the symptoms of periodontal disease is as follows. That is, it can be seen that the amount of the enzyme in the exudate collected from the gingival sulcus of a patient with periodontitis increases in proportion to the increase in the exudate amount per unit time (increase in periotron value) (see FIG. 4). . That is, the amount of the enzyme activity at a stage where the amount of exudate is slight is extremely low, but the amount of the activity increases as the amount of exudate increases, and in moderate symptoms, the amount increases significantly, and when it becomes severe, it further increases. You can see that it will increase.

It is generally reported that there is a correlation between the increase in the amount of exudate and the condition of periodontitis (Cimasoni
G .: Crevicular Fluid Updated. In: Myers HM, ed. M
onographs in Oral Sciences. Basel, Karger, pp.1-15
2, 1983) and the relationship between exudate volume and other clinical parameters (gingival index and plaque index), the above results are produced by the periodontopathic bacterium P. gingivalis. It shows that there is a correlation between the activity amount of the enzyme and the condition of periodontal disease. Therefore, based on the measured value of the enzyme activity derived from periodontopathic bacteria obtained using the method of the present invention, it becomes possible to make a more objective judgment on the condition of each periodontal disease patient.

The enzyme is a known 50 kDa cysteine protease derived from P. gingivalis (gingipain; Chen Z, Potempa J, Polanows.
ki A, Wikstrom M, Travis J: Purification and charac
terization of a 50-kDa cysteine proteinase (gingip
ain) from Porphyromonas gingivalis. J. Biol. Chem.
267: 18896-18901, 1992) in enzymatic properties such as optimum pH and sensitivity to inhibitors, but different in properties such as substrate specificity and thermostability.
In addition, the enzyme has a very important property closely related to periodontal disease, which has not been clarified by the above-mentioned known enzymes, and is clearly a novel enzyme.

That is, proteins such as type I collagen and immunoglobulin are well decomposed (see FIG. 5), they are not easily inhibited by important protease inhibitors of biological origin such as serpin and cystatin, and the function of polymorphonuclear leukocytes. Concentration-dependently and time-dependently (see FIG. 6 and FIG. 7), and common to multiple strains of P. gingivalis with different serotypes, but other teeth The enzyme is not present in the culture supernatant of bacteria called enteropathogenic bacteria or enterobacteria (see Table 4), etc., and the enzyme is a periodontopathic factor peculiar to P. gingivalis, and direct destruction of periodontal tissues and protection of the living body. It has been clarified that it has an important role in the destruction of the system. Therefore, measuring the enzyme of the present invention in the gingival crevicular fluid of patients with periodontal disease is useful in the diagnosis of periodontal disease.

[0037]

Table 4 shows the amount of the enzyme activity contained in the culture supernatants of a plurality of strains of P. gingivalis having different serotypes and in the culture supernatants of other periodontopathic bacteria and enterobacteria. It shows the results of measurement using two kinds of synthetic substrates, and it is shown that the enzyme of the present invention is peculiar to P. gingivalis.

Therefore, since the enzyme according to the present invention has the above-mentioned enzymatic properties, it should be purified from the culture supernatant of P. gingivalis normally present in the human mouth by using the properties as an index. Thus, the desired enzyme can be obtained.

[0040]

The present invention will be described in detail below with reference to examples.

Example 1 Purification and Enzymatic Properties of P. gingivalis Derived Enzyme:
The enzyme of the present invention was purified as follows. Ammonium sulfate was added to the culture supernatant of the P. gingivalis strain 381 to make it 70% saturated. The precipitate was collected by centrifugation and dialyzed against 10 mM phosphate buffer (A) containing 0.05% bridge 35 of nonionic detergent. The centrifugal dialysis supernatant was applied to a DEAE Sephacel column equilibrated with solution A, the resulting non-adsorbed fraction was concentrated, and then the solution was equilibrated with solution A C.
It was applied to an M-Toyopearl column (Fig. 8).

After thoroughly washing the column with the same buffer, the enzyme active fraction was eluted with the same buffer containing 70 mM sodium chloride. The eluted fraction was concentrated and dialyzed, and then subjected to isoelectric point separation in the range of pH 3.5 to 10 (see FIG. 9). Active fraction (p
H5.0-5.5) was collected, concentrated and dialyzed, then 0.1 M Na was added.
TS equilibrated with 10 mM phosphate buffer containing ₂ SO ₄
Purification was performed by gel filtration of K gel G2000SW (see FIG. 10).

A protocol for a method for purifying an enzyme derived from the periodontopathic bacterium P. gingivalis, and Z-Phe-A as a substrate.
Amount of activity when using rg-MCA (total activity and specific activity, etc.)
Table 5 shows the changes in

[0044]

Example 2 Production of antibody and characteristics: The antibody used in the method for measuring an enzyme of the present invention was produced as follows. The enzyme solution (about 1 mg) purified from the culture supernatant of P. gingivalis was mixed with an equal amount of Freund's complete adjuvant, and the suspension was subcutaneously injected into rabbits at several sites, This was repeated twice at 2-week intervals. After that, the booster was performed once to collect the antiserum.

The antiserum was treated with ammonium sulphate and protein A-Sepharose (Pharmacia, Sweden) column chromatography I
The measurement was performed as a gG fraction. The specificity of the antibody was determined by the fact that the IgG fraction obtained from rabbits administered with only Freund's complete adjuvant without the enzyme used as a control did not neutralize the enzyme activity at all and the non-reactivity in Western blotting. It is confirmed. Further, the antibody against the enzyme does not inhibit any protease activity of the culture supernatant of other periodontopathic bacteria, and is therefore extremely highly specific and useful.

Example 3 Method of Measuring Enzyme: The method of measuring the enzyme derived from P. gingivalis of the present invention was carried out as follows. First,
Z-Phe-Arg-MCA or Boc-P as a substrate
Using he-Ser-Arg-MCA, this substrate solution (10 μM substrate / 5 mM cysteine / 20 mM) was added to the test solution.
Add phosphate buffer, pH 7.5) and incubate at 40 ° C. for 10 minutes. The reaction was stopped by adding the same amount of 10 mM iodoacetic acid solution (pH 5), and the amount of released AMC was measured by fluorescence measurement using a fluorescence spectrophotometer (excitation wavelength: 380 n
m, fluorescence wavelength 460 nm). At this time, EDTA (10 mM) and leupeptin (50 μm
The same measurement is performed in the presence of M), and the activity amount inhibited by any substance is the enzyme amount.

Further, a specific antibody against the enzyme is added to a part of the test solution, reacted at 37 ° C. for 10 minutes, and then the reaction solution is centrifuged to separate it into a solid phase (immune complex) and a liquid phase. ,
The amount of the enzyme can be measured more accurately by measuring the decomposition activity for the substrate in the liquid phase and calculating the amount of activity transferred to the solid phase.

[0049]

Industrial Applicability The present invention provides an objective and simple method for measuring an enzyme derived from periodontopathic bacteria. By using the enzyme measurement method of the present invention, conventionally, it is possible to accurately grasp the progress and activity of periodontal disease, which was difficult to objectively diagnose, without depending on experience, It makes a great contribution to the treatment of dentistry. In addition, the antibody against the enzyme of the present invention inhibits the reaction suppressing effect of polymorphonuclear leukocytes possessed by the enzyme of the present invention, in addition to the reagent for analysis (see FIG. 11), thus preventing periodontal disease
It can be expected to be used as a therapeutic agent.

[0050]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 991 Sequence type: Amino acid Topology: Sequence type: Peptide sequence

[Chemical 3]

[Chemical 4]

[Brief description of drawings]

FIG. 1 is a drawing showing the influence of pH when the enzyme of the present invention decomposes two kinds of synthetic substrates.

FIG. 2 is a drawing showing the thermostability of the enzyme of the present invention.

FIG. 3 is a drawing showing the action of various protease inhibitors on the inhibitory action on the chemiluminescence reaction of polymorphonuclear leukocytes possessed by the culture supernatant of periodontopathic bacteria, P. gingivalis.

FIG. 4 is a drawing showing the amount of the enzyme of the present invention contained in the gingival crevicular fluid of a patient with periodontal disease. Boc-Ph as a substrate
e-Ser-Arg-MCA was used. In the figure, 1 unit means the amount required to release 1 μmol of AMC per minute at 40 ° C., which is 1 of the periotron value.
The unit corresponds to 0.005 μl of GCF.

FIG. 5 is a drawing showing the degradation patterns of human-derived collagen type I (lane 2, 3) and type IV (lane 4, 5) by the enzyme of the present invention. Each collagen and the enzyme 20
After incubation at 0 ° C (A) or 37C (B) for 10 hours, SDS gel electrophoresis was performed. Lane 1
Was treated with a molecular weight marker, lanes 2 and 4 were treated in the absence of the enzyme, and lanes 3 and 5 were treated in the presence of the enzyme (1 μg).

FIG. 6 is a drawing showing the results of time-course examination of the inhibitory effect on the chemiluminescence reaction of polymorphonuclear leukocytes (2 × 10 ⁶ cells) using the purified enzyme of the present invention (5 μg).

FIG. 7 is a drawing showing the concentration dependency of the chemiluminescence reaction inhibitory effect of polymorphonuclear leukocytes by the enzyme of the present invention.
Black circles indicate the enzyme to which 1 mM cysteine was added, open circles indicate the enzyme alone, and black triangles indicate the enzyme to which 1 mM cysteine and 40 μg / ml leupeptin were added.

FIG. 8 is a drawing showing a gel filtration pattern of the enzyme of the present invention on CM Toyopearl.

FIG. 9 is a drawing showing a distribution pattern of the enzyme of the present invention by column isoelectric focusing.

FIG. 10 is a view showing a gel electrophoresis pattern of the purified enzyme of the present invention. The presence of a single band indicates that the enzyme was purified homogeneously.

FIG. 11 is a view showing that a specific antibody against the enzyme of the present invention inhibits the chemiluminescence reaction inhibitory effect of polymorphonuclear leukocytes possessed by the enzyme. that's all

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Claims (4)

[Claims] 1. Porphyromonas gingivalis having the following enzymatic properties:
Derived enzyme: (1) Action: It has a direct degrading effect on periodontal tissue mainly composed of collagen, and has a damaging activity on inflammatory cells such as neutrophils. (2) Substrate specificity: A synthetic fluorescent substrate t-butyloxycarbonyl-L-phenylalanyl-L-seryl-L-arginine-4-methylcoumaryl, which has high resolution for various proteins such as collagen and immunoglobulin. -7-amide (Boc-Phe-Ser-Arg-MCA) and carbobenzoyl-L-phenylalanyl-L-arginine-
It shows a strong specific decomposition activity against 4-methylcoumarylamide (Z-Phe-Arg-MCA). (3) Optimum pH and stable pH; the optimum pH is 7 to 8 for both protein substrate and synthetic substrate, and pH 4 to 4
It is stable in the range of 9. (4) Optimum temperature range of action: room temperature to 37 ° C. (5) Activation; remarkably activated by SH group reducing agents such as cysteine, 2-mercaptoethanol and dithiothreitol. (6) Inhibitors: chymostatin, leupeptin, E-
64, strongly inhibited by antipain, EDTA, TPCK, TLCK, etc. (7) Molecular weight: The apparent molecular weight determined by gel filtration is about 50 kDa, and the molecular weight determined by SDS gel electrophoresis is about 44 kDa. (8) Others; isoelectric point pH 5-5.5 2. A method for measuring an enzyme according to claim 1, which comprises the following steps: (1) A part of a sample is reacted with a substrate for the enzyme according to claim 1 in the presence of EDTA and leupepsin. And (2) allowing a specific antibody against the enzyme of claim 1 to act on a part of the sample, separating the reaction solution into a solid phase and a liquid phase, and then performing the step (1) above. (3) Subtracting the enzyme activity of step 2 from the enzyme activity of step 1 to calculate the enzyme activity according to claim 1). 3. An antibody which specifically binds to the enzyme of claim 1. 4. The following (a) to (d) (a) an antibody which specifically binds to the enzyme of claim 1, (b) a substrate which is specifically recognized by the enzyme of claim 1, A kit for measuring an enzyme derived from periodontopathic bacteria, which comprises c) leupeptin and (d) EDTA.