KR100202528B1 - Enzyme for dissolving cell wall of streptomyces mutans - Google Patents

Abstract

The present invention relates to a cell wall lytic enzyme that selectively acts on the cell wall of the bacteria Streptcoccus mutans and causes microorganisms to dissolve bacteria.

The present inventors searched for lytic enzymes that dissolve the cell wall of Streptococcus mutans for the purpose of use as additives such as food and toothpaste or medicinal products. Microorganisms producing cell wall lytic enzymes were isolated from soil and identified as Bacillus subtilis, and the characteristics of the strains and enzymes were examined and found to have different characteristics from the existing production strains. The present invention has been completed on the basis of this. The enzyme is a protein with a single subunit of molecular weight 38,000 Daltons (Da). The optimum pH of the reaction is in the range of pH 6 and 8, and the optimum temperature is in the range of 30 ° C and 40 ° C. It is stable and has a stable property in the temperature range of 0 ℃ -50 ℃. Production medium includes carbon sources such as sucrose, fructose, glucose and starch, nitrogen sources such as NH ₄ ⁺ salts, urea, protein and soy flour, amino acid and corn immersion. Liquids, peptones, natural media such as gravy, Mg ⁺ salts, phosphates and the like could be used as the medium. This enzyme is an endopeptidase that breaks down the amino acid bonds of peptidoglycan in the cell wall components of Streptococcus mutans, and is produced by the existing Bacillus subtilis ATCC6051 Enacetyl muti (30,000 Daltons) produced by Streptococcus mutans cell wall lytic enzyme (L-alanine amidase, Bacillus subtilis 168, molecular weight 50,000 daltons) N-acetyl muramoyl-L-alanine amidase and other new enzymes.

Description

Lysis enzymes and their producing microorganisms that dissolve the cell walls of Streptococcus mutans

This strain of FIG. 1 is inoculated on a plate in which the Streptococcus mutans, which is a substrate strain, is inoculated to show lysis.

FIG. 2 is a photograph showing microorganisms producing Streptococcus mutans cell wall lyase in agar medium.

3 is a diagram showing the enzyme production according to the culture time of the strain.

4 is a photograph of SDS-polyacrylamide gel electrophoresis of the purified enzyme protein.

5 is a diagram showing the effect of pH on the activity of purified cell wall lyase.

6 shows the effect of pH on the stability of purified cell wall lyase.

7 shows the influence of temperature on the activity of purified cell wall lyase.

8 shows the influence of temperature on the stability of purified cell wall lyase.

9 is a diagram showing the degradation products of the Streptococcus mutans cell wall according to the reaction time.

The present invention relates to a cell wall lytic enzyme that selectively acts on the cell wall of Streptococcus mutans, a bacterium that causes dental caries, to dissolve bacteria and microorganisms producing the same.

Among the diseases occurring in the oral and maxillofacial region, dental caries is still recognized as a disease with high morbidity. In the meantime, as part of efforts to prevent this, various types of preventive and therapeutic methods have been developed, which have reduced the incidence rate in the United States and Western Europe, but in Korea, most of the dental diseases in children and adults still exist. Occupies. Moreover, the development of dental caries is different from person to person, so this disease is considered to be an important part of the dental field.

Dental caries caused by hard tissue caries is an infectious disease accompanied by destruction. It is a multifactorial disease caused by the interaction of bacteria, foods and saliva in the plaque. Streptococcus mutans cause dental caries through attachment, proliferation, and acid production to the tooth surface. That is, the Streptococcus mutans attach to the acquisition film of the tooth surface and synthesize a glucose-insoluble mutan from sucrose. Synthesized mutans increase the bonds between bacteria that grow on the tooth surface. In this way, the Streptococcus mutans attached to the tooth surface are localized on the tooth surface to form plaques. Aggregation in specific areas is thought to provide the primary cause of dental caries. Plaque, which is a caries-causing substance, contains a very large amount (2 × 10 ⁸ ) of bacteria, and as a result, sucrose is broken down into glucose and fructose and the pH drops below 5.5 to demineralize hard tissue teeth. Conditions that can be established. In other words, if the acid production process is repeated by reducing the pH, microdegradation of enamel is continued and demineralization causes dental caries lesions. As such, dental caries is an infectious disease caused by bacteria, and the type of food ingested and time-dependent variables determine the progress of the disease, but the resistance to demineralization and the large and small salivary glands of the teeth themselves are determined. Saliva flowing from the carcinogenesis is evaluated as an important factor in preventing dental caries progression. As such, dental caries is thought to be suppressed at the beginning due to resistance to tooth demineralization or saliva washing. Nevertheless, dental caries is on the rise, so many other factors work in combination. Think of it as a magnetic disease. Therefore, various methods have been used to suppress such dental caries. Among them, the methods proposed to date include 1) a method using antibiotics such as spiramycin hydrochloride vancomycin HCL and chlorohexidine, and 2) caries bacteria by organic or inorganic fluorine. Inhibition, 3) removal of plaque using enzymes such as glucan degrading enzymes, dextran degrading enzymes, and the like.

However, the first method is an effective method for preventing caries, but it can cause side effects such as the occurrence of resistant strains due to the use of antibiotics, diarrhea and vomiting, and inhibits the growth of all bacteria in the oral cavity. Therefore, the balance of the normal oral bacterial layer is unbalanced and has the possibility of evaporative infection or other diseases caused by pathogenic bacteria from the outside. The second method of using fluoride is also the most widely used method. Fluoride is known as a substance that suppresses demineralization and promotes remineralization, and it is also evaluated as having an antibacterial effect. There is a difficulty in the purpose of preventing dental caries. Finally, methods using enzymes such as glucan degradase and dextran degrading enzyme have been evaluated as the most probable and potential methods, but this is the second method to remove tartar. It can be called the way.

Therefore, in order to prevent dental caries, a new substance that is strongly selective for dental caries and specifically works is required. From this point of view, the use of natural products exhibiting non-specific antimicrobial action is necessary to reconsider, and the search for dental caries cell wall lytic enzymes that selectively and specifically act on dental caries is urgently needed.

The most prominent lytic phenomena present in microorganisms are induced by bacteriophage, and the growth of phage and release of newly produced phage always involve dissolution of the host cell, which is produced by the host cell. It is due to virolysin, which is a component of cell wall lytic enzymes or phage itself. Spore forming by sporelysin is also part of this phenomenon in spore-forming bacteria, and the cell wall can be degraded by extracellular enzymes that are believed to be produced for a series of defenses.

Bacterial cell wall lyase can be used to analyze the reaction products to reveal the structure of the bacterial cell wall due to the high specificity of the cell wall peptidoglycan, plant preservatives, bacteria that protect food from microbial contamination It can be used as a growth inhibitor and can also be used to isolate and study useful substances in cells. However, studies on the fundamental elimination of tartar-forming bacteria using lytic enzymes are still weak. Despite the relative stability of enzymes in humans, studies on tartar-forming bacteria are insignificant. Therefore, it is possible to develop safer and more effective oral hygiene agents by searching for and developing lytic enzymes for them.

The present inventors searched for lytic enzymes soluble in the cell wall of Streptococus mutans for use as additives such as food and toothpaste or pharmaceuticals, and new cell walls different from the known cell wall lytic enzymes. Microorganisms producing lytic enzymes were isolated from soil and identified as Bacillus subtilis, and the characteristics of the strain and the characteristics of the enzyme were examined and found to have different characteristics from existing production strains. The present invention has been completed. Production media of this enzyme include carbon sources such as sucrose, fructose, glucose and starch, nitrogen sources such as NH ₄ ⁺ salts, urea, proteins and soy flour, amino acids, corn steep liquor, peptone, natural media such as gravy, Mg ⁺ salts, Phosphates and the like could be used as the medium. Accordingly, it is an object of the present invention to provide a bacterial cell wall lyase and a microorganism producing the enzyme selectively acting and lysing the cell wall of Streptococcus mutans.

Isolation of Strains with Lytic Activity

In order to search for strains with bacterial cell wall lysis activity, microorganisms isolated from soil samples collected at home and abroad were inoculated into agar plate medium containing Streptococcus mutans KCTC3283 cell, which is a substrate strain. A strain having a clear zone in the grown conoly region was selected as a strain having cell wall lytic activity (see FIG. 1).

Identification of Strains

The strain of the present invention is a gram-positive bacterium in form, and compared with the characteristics of the microorganisms and the morphological characteristics of the strain as a bacillus forming spores, the strain of the present invention is different from the existing cell wall lytic enzyme producing strain As microorganisms with other morphological characteristics, we have identified this strain as a novel strain and named Bacillus subtilis YU-1004 (see Table 1, Figure 2).

This strain was deposited with the KFCC10893 accession number on April 3, 1996, to the Korean spawn association. Composition of the culture medium for culturing the strain Bacillus subtilis KFCC10893 is 1% glucose, 0.5% polypeptone, 0.5% yeast extract mole, the pH of the medium was 6.0, the culture temperature was 37 ℃ It was suitable. In addition, for the production of the enzyme was obtained the maximum enzyme production when incubated for 24 hours under aerobic conditions of 37 ℃ in the medium.

The cell wall lyase produced by Bacillus subtilis KFCC10893 of the present invention has a single subunit having a molecular weight of about 38,000 Daltons as shown in Examples 3 and 4, and the optimum reaction pH and temperature are pH 8.0, As shown in Example 6 as an enzyme at 37 ° C., it is an endopeptidase that degrades amino acid bonds in the peptidoglycan component of the Streptococcus mutans cell wall.

The strain of the present invention and the cell wall lyase produced by the strain will be described in more detail with reference to the following examples.

Table 1

Example 1

In order to measure the enzyme activity, the substrate strain was first cultured. Streptococcus mutans KCTC3283, a substrate strain, was cultured in BHI (Brain Heart Infusion, Difco) medium at 37 ° C for 3 days, collected by centrifugation, washed twice with physiological saline, -20 It was stored at ℃ and used as a substrate cell. The substrate cells were suspended in a buffer of pH 8.0 so that the absorbance was 1.0 at 660 nm, and 0.2 ml of enzyme solution was added to 2 ml of the cell suspension, followed by reaction at 37 ° C. for 30 minutes, and then the absorbance decrease at 660 nm was measured. At this time, the enzyme activity was set to 1 unit (Unit) the amount of enzyme that decreases the absorbance 0.001 at 660nm for 1 minute given conditions.

In order to produce bacterial cell wall lyase, the strain of the present invention is cultured at a culture temperature of 10-60 ° C. and an initial pH of 3-11 in a medium composed of a carbon source such as corn starch, sugar, soybean meal, and an organic nitrogen source such as yeast extract. When cultured under conditions, the maximum enzyme can be obtained from the culture medium when cultured at 37 ° C. for 24 hours under conditions of 1.0% glucose or 1% starch, 0.5% polypeptone, 0.5% yeast extract, and pH 6.0. have.

Example 2

Bacillus subtilis KFCC10893 of the present invention was cultured under the optimum enzyme production conditions, and the results of observing the growth and enzyme production and pH change of the strains are shown in FIG. 3. As can be seen in FIG. 3, the growth of the fungi increased to 36 hours after inoculation and then stopped. The pH of the medium gradually increased from 18 hours of culture at the first 6.0 to pH 8.6 at 36 hours. The production of enzyme began to increase from the beginning of the culture and showed maximum activity (22U / ml) at 24 hours, but then decreased.

Example 3

The enzyme was purified from a culture medium of 1 L of optimum enzyme production medium by affinity chromatography and gel column chromatography using Sephadex G-100 (from Sigma). SDS-polyacrylamide gel electrophoresis confirmed that it is a kind of enzyme having a single subunit of molecular weight of about 38,000 Daltons. The experimental results are shown in FIG.

Example 4

As a result of examining the characteristics of the purified cell wall lytic enzyme, the optimum pH was 8.0 in the enzyme reaction, and showed 50% or more activity from pH 5.0 to pH 9.0, and was stable from pH 5.0 to pH 11.0. The results are shown in FIGS. 5 and 6.

In addition, the optimum reaction temperature was 37 ℃, up to 40 ℃ 100% activity was maintained even after 1 hour and at 50 ℃ showed an afterglow activity of 64% after 1 hour.

This result is shown in FIG. 7 and FIG.

Example 5

To examine the range of action of cell wall lyase and the selectivity of Streptococcus muttans produced by this strain, various strains of oral and intestinal bacteria and other Gram-positive or Gram-negative bacteria were tested. Used. After incubating each strain for 3 days, the solution was suspended in pH 8.0 buffer at 660 nm to have an absorbance of 1.0, and then 15U of enzyme was added to 2 ml of the reaction solution and reacted at 37 ° C for 30 minutes. The results obtained by measuring the decrease in absorbance at 660 nm as solubility (%) are shown in Table 2.

TABLE 2

Lactobacillus helveticus, a lactic acid bacterium, showed little solubility in Lactobacillus helveticus and the solubility of Streptococcus mutans KCTC 3283 in When 100% of the bacteria showed a lysis of over 50% of all strains, including 61.9% lysis of Streptococcus mutans KFCC 3289. Lytic activity was high for Streptococcus mutans, the causative factor.

Example 6

In order to examine the dissolution site of the Streptococcus mutans cell wall of the enzyme, peptidoglycan, a cell wall component of Streptococcus mutans, was isolated and reacted under optimal reaction conditions. The amounts of free amino acids and reducing sugars were quantified and the absorbance was measured.

As shown in FIG. 9, as the reaction proceeded, the free amino acid gradually increased with decreasing absorbance, and the amount of reducing sugar was almost constant. Therefore, the bacterial cell wall lytic enzyme was found to be an endopeptidase that breaks down the amino acid bond of the cell wall component peptidoglycan.

This is a molecular weight of 50,000 Daltons (Da), L-alanine amidase, Bacillus subtilis, a Streptococcus mutans cell wall lytic enzyme produced by Bacilln subtilis ATCC 6051. N-acetyl muramoyl-alanine amidase, a molecular weight of 30,000 Daltons produced by Bacillus subtilis 168, and other enzymes. do.

Claims (3)

Bacillus subtilis YU-1004 (KFCC-10893), which produces cell wall lytic enzymes. A method for preparing cell wall lyase using Bacillus subtilis YU-1004 (KFCC-10893), the molecular weight of the enzyme is about 38,000 Daltons, the optimum pH of the reaction is about 7.5, the optimum temperature of the reaction It is about 35 ℃, stable in the pH range 5-11 and at the same time stable in the temperature range 0 ℃-50 ℃ characterized in that the cell wall lytic enzyme production method. Cell wall lysase produced by Bacillus subtilis YU-1004 (KFCC-10893), the molecular weight of the enzyme is about 38,000 Daltons, the optimum pH of the reaction is about 7.5, the optimum temperature of the reaction is 35 It is about ℃, the cell wall lytic enzyme characterized in that it is stable in the range of pH 5-11 and at the same time stable in the range of 0 ℃ -50 ℃.