KR20220111845A - Novel streptococcus strain and hyaluronidase derived from the same - Google Patents

Abstract

The present invention relates to a novel Streptococcus equi sub. zooepidemicuskdk BST909 strain (accession number: KCTC 14238BP) which produces a hyaluronic acid degrading enzyme and to a method for producing low-molecular-weight hyaluronic acid using the hyaluronic acid degrading enzyme obtained therefrom. The Streptococcus equi sub. zooepidemicuskdk BST909 strain of the present invention has no hemolytic toxicity and produces novel hyaluronidase having a different molecular weight from hyaluronidase produced by strains of other species belonging to the genus Streptococcus. The enzyme degrades high-molecular-weight hyaluronic acid into low-molecular-weight hyaluronic acid through high substrate specificity for hyaluronic acid. Accordingly, hyaluronidase produced from the strain can be very useful for producing low-molecular-weight hyaluronic acid applicable to various industries such as food, cosmetics, and pharmaceuticals.

Description

New microorganism Streptococcus strain and hyaluronidase derived therefrom {NOVEL STREPTOCOCCUS STRAIN AND HYALURONIDASE DERIVED FROM THE SAME}

The present invention relates to a novel Streptococcus juepidemicus strain, hyaluronidase produced by the strain, and a method for producing low molecular weight hyaluronic acid using the same.

Hyaluronic acid is a polysaccharide of various lengths formed by repeating disaccharide subunits of D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid is abundant in animal skin and has high viscosity and hydrophilicity. In addition, hyaluronic acid is an important component in skin tissue, articular cartilage tissue, etc., and helps to connect and support cells. In addition, due to the moisturizing and lubricating effects of hyaluronic acid, it is applied in a wide range of fields such as pharmaceuticals (joint lubricant), food (hyaluronic acid health functional food), cosmetics (moisturizer, plastic surgery filler), etc. (KR10-1654810 B1).

The molecular weight of hyaluronic acid varies from one hundred Da to several million Da. In addition, the aqueous solution of hyaluronic acid has viscosity, elasticity and moisture retention, which varies depending on the molecular weight of hyaluronic acid and its concentration. Polymeric hyaluronic acid has a disadvantage in that it lacks functionality when used in product production due to its high viscosity and low skin penetration (Eunji Shin et al., the effect on collagen synthesis, anti-inflammatory and skin absorption according to the molecular weight of sodium hyaluronate., Journal of the Korean Cosmetic Society., 2016, 105, 235-245). For this reason, various studies have been made on a method for decomposing high molecular weight hyaluronic acid into low molecular weight hyaluronic acid.

Methods for decomposing hyaluronic acid can be largely divided into high temperature heating, physical decomposition such as ultrasonic waves, chemical decomposition such as acid hydrolysis, and biological decomposition such as decomposing enzymes. However, during high-temperature thermal decomposition, there is a possibility of denaturation due to high temperature, and during acid hydrolysis using a strong acid or decomposition using ultrasonic waves, a lot of monosaccharides are generated and the size of the molecules is not uniform. On the other hand, when a degrading enzyme is used, hyaluronic acid of various molecular weights can be produced by controlling reaction conditions such as temperature and reaction time, and it has the advantage of less risk and environmental pollution during decomposition because strong acids and strong bases are not used ( KR10-1736790 B1).

US 10-1654810 B1 US 10-1736790 B1

Eunji Shin et al., Effect of Sodium Hyaluronate on Collagen Synthesis, Anti-Inflammation and Skin Absorption according to Molecular Weight Size., Journal of the Korean Cosmetic Society., 2016, 105, 235-245.

Accordingly, the researchers studied to develop a hyaluronic acid degrading enzyme (hyaluronidase) that can degrade hyaluronic acid. Microorganisms capable of producing ronidase and the corresponding enzyme were identified, and the present invention was completed by confirming that low molecular weight hyaluronic acid could be produced using the hyaluronidase obtained therefrom.

In order to achieve the above object, one aspect of the present invention provides a Streptococcus equi subsp. zooepidemicus BST909 strain (Accession No.: KCTC 14238BP).

Another aspect of the present invention provides a hyaluronic acid degrading enzyme comprising the amino acid represented by SEQ ID NO: 2.

Another aspect of the present invention, Streptococcus equi subsp. zooepidemicus comprising the step of culturing the BST909 strain (Accession No.: KCTC 14238BP) and purifying the hyaluronic acid degrading enzyme from the culture medium, A method for producing a hyaluronic acid degrading enzyme is provided.

Another aspect of the present invention provides a method for producing low molecular weight hyaluronic acid comprising the step of treating the hyaluronic acid degrading enzyme to high molecular weight hyaluronic acid.

The present invention is a novel Streptacoccus equi subsp. zooepidemicus BST909 strain (Accession No.: KCTC 14238BP) that produces a hyaluronic acid degrading enzyme, and the production of low molecular weight hyaluronic acid using the hyaluronic acid degrading enzyme obtained therefrom it's about how The Streptococcus juepidemicus BST909 strain of the present invention has no hemolytic toxicity and produces a novel hyaluronidase having a molecular weight different from that produced by strains of other species belonging to the genus Streptococcus. The enzyme decomposes high molecular weight hyaluronic acid into low molecular weight hyaluronic acid through high substrate specificity for hyaluronic acid. Therefore, the hyaluronidase produced from the strain can be very usefully used to produce low molecular weight hyaluronic acid applicable to various industrial fields such as food, cosmetics, and pharmaceuticals.

1 is a view showing the results of culturing by transplanting the collected strain to a serum solid medium in order to isolate a novel strain without hemolytic toxicity.
2 is a view showing the result of analyzing the 16s rRNA gene base sequence to identify the Streptococcus juepidemicus BST909 strain.
3 is a view showing the results of confirming the fermentation of sugar and enzyme activity in order to identify the Streptococcus juepidemicus BST909 strain.
4 is a view showing the result of comparing the hemolytic properties of Streptococcus juepidemicus ATCC43079 strain (left) and Streptococcus juepidemicus BST909 strain (right).
5 is a suspension and supernatant of Streptococcus juepidemicus ATCC43079 or Streptococcus juepidemicus BST909 cells and culture medium, physiological saline, NCN buffer, 1 % Surfactant (SDS) was reacted with each red blood cell to compare the hemolytic properties.
6 is a graph showing the enzymatic activity of hyaluronidase produced by Streptococcus juepidemicus BST909 strain according to temperature.
7 is a graph showing the enzyme activity for each pH of hyaluronidase produced by Streptococcus juepidemicus BST909 strain.
8 is a graph showing the degree of inhibition of enzyme activity by the metal salt of hyaluronidase produced by the Streptococcus juepidemicus BST909 strain.
9 is a graph showing the results of analyzing the enzyme reaction rate constant of hyaluronidase produced by Streptococcus juepidemicus BST909 strain.
Figure 10 is a hyaluronidase produced by Streptococcus juepidemicus BST909 strain. It is a diagram showing the result of performing SDS-PAGE to confirm the molecular weight.
11 is a view showing the N-terminal amino acid sequencing result of hyaluronidase produced by Streptococcus juepidemicus BST909 strain.
12 is a view showing the decomposition process of the polymer hyaluronic acid according to the reaction time of the hyaluronidase produced by the Streptococcus juepidemicus BST909 strain.
13 is a view showing the results of MALDI-TOF analysis of the final degradation product of high molecular weight hyaluronic acid by hyaluronidase produced by Streptococcus juepidemicus BST909 strain.
14 is a view showing the results of analysis by LC-MS of the final degradation product of high molecular weight hyaluronic acid by hyaluronidase produced by Streptococcus juepidemicus BST909 strain.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a Streptococcus equi subsp. zooepidemicus BST909 strain (Accession No.: KCTC 14238BP). The strain contains 16s rRNA represented by SEQ ID NO: 1, and is characterized in that it is non-hemolytic.

Streptococcus juepidemicus is a hyaluronic acid-producing Gram-positive cocci, a subspecies of Streptococcus equi , causing beta hemolysis (β-hemolysis). The strains belonging to the Streptococcus juepidemicus species include Streptococcus juepidemicus ATCC43079, CCUG 23256, LMG16030, and the like. As used herein, the term "hemolysis" refers to the destruction of red blood cells and dissolution of the contents (cytoplasm) into the surrounding liquid (eg, plasma). Strains are sometimes classified as hemolytic, including alpha, beta, and gamma hemolysis. Alpha hemolysis oxidizes hemoglobin in red blood cells, creating a green stain on the blood agar medium. Species that cause beta hemolysis rupture the blood agar medium by destroying surrounding blood cells, creating transparent stains.

The present inventors selected a strain that does not have hemolysis and produces a hyaluronic acid degrading enzyme, and confirmed that the strain is a novel strain belonging to Streptococcus juepidemicus ( FIGS. 1 to 3 ). The newly identified strain does not show haemolytic toxicity, unlike the Streptococcus juepidemicus ATCC43079 strain ( FIGS. 4 and 5 ). The present inventors named the strain "Streptococcus juepidemicus BST909", and deposited the same strain with the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center as of July 14, 2020, with accession number KCTC 14238BP.

Another aspect of the present invention provides a hyaluronic acid degrading enzyme comprising the amino acid represented by SEQ ID NO: 2. Specifically, in an embodiment of the present invention, the hyaluronic acid degrading enzyme may be one obtained from Streptococcus equi subsp. zooepidemicus BST909 strain (Accession No.: KCTC 14238BP), but is not limited thereto.

As used herein, the term "hyaluronic acid (hyaluronic acid, or hyaluronan, HA)" is one of complex polysaccharides consisting of amino acids and uronic acid having a molecular weight ranging from 50,000 Da to 13,000,000 Da, It is a high molecular compound composed of repeating units, glucuronic acid and N-acetylglucosamine, alternately bonded to (1-3) and (1-4).

Hyaluronic acid has various effects and excellent physical properties, such as moisturizing effect, lubricating effect against physical friction, and protective effect against bacterial invasion. It is widely used in pharmaceuticals, quasi-drugs, food, etc. In addition, it controls the movement of physiologically active substances and acts as a mediator to induce cell differentiation and growth by specific action with cells, and supports tissues such as collagen, elastin, and chondroitin sulphate in the extracellular matrix. It is known to serve as a support for proteins and glycoproteins. However, when hyaluronic acid is used in plants and cosmetics, hyaluronic acid having a large molecular weight has a disadvantage in that it is not easily absorbed into the body and into the skin. Such hyaluronic acid is contained in a large amount in the eyes of cattle, chicken bones, buffer tissues of animals, placenta, cancer cells and skin.

As used herein, the term "hyaluronic acid degrading enzyme" is an enzyme that hydrolyzes hyaluronic acid. The enzyme is found in various living things and is divided into three types according to the mechanism of action. Hyaluronate 4-glycanohydrolase (EC 3.2.1.35) is an endo-β-N-acetylglucosaminidase (hyaluronoglucosaminidase), a hydrolase acting on the β-1,4-glucosidic bond. to be. It mainly produces tetrasaccharides and is distributed in testes, lysosomes and bee venom. Hyaluronate 3-glycanohydrolase (EC 3.2.1.36) is an endo-β-glucuronidase, a hydrolase acting on the β-1,3-glucosidic bond. Produces mainly tetrasaccharides and is found in leeches or hookworms. Hyaluronate lyase (EC 4.2.2.1) is a hyaluronidase present in bacteria that acts on the β-1,4-glucosidic bond and produces 4,5-unsaturated disaccharides through the β-elimination mechanism. make it

According to an embodiment of the present invention, the It was confirmed that the molecular weight of hyaluronidase obtained from the strain was about 127 kDa (FIG. 10). In addition, the hyaluronidase may include the N-terminal sequence of SEQ ID NO: 2 (FIG. 11). In the present invention, hyaluronidase purified from Streptococcus juepidemicus BST909 strain (Accession No.: KCTC 14238BP) may be described in combination as "BST909 hyaluronidase". In addition, BST909 hyaluronidase may include a hyaluronidase precursor, a mature hyaluronidase, and a truncated form thereof having activity.

According to an embodiment of the present invention, BST909 hyaluronidase may exhibit decomposition activity at 15°C to 45°C. Preferably it may be 40 ℃. As the reaction temperature of hyaluronidase increased to 40 °C, the decomposition activity of the enzyme increased, and the activity decreased rapidly at 45 °C. Through this, it was confirmed that the optimal reaction temperature of BST909 hyaluronidase was 40 °C (FIG. 6).

In one embodiment of the present invention, BST909 hyaluronidase may exhibit degradation activity at pH 4.0 to pH 9.0. Preferably, the pH may be 7.0 (FIG. 7).

According to an embodiment of the present invention, the decomposition activity of BST909 hyaluronidase may be inhibited by any one selected from Ag ²⁺ , Ni ²⁺ , Fe ²⁺ , Zn ²⁺ and Cu ²⁺ . Specifically, the enzyme is Ag ²⁺ , Ni ²⁺ , Fe ²⁺ , Ba ²⁺ , Ca ²⁺ , Zn ²⁺ , Cu ²⁺ , Mn ²⁺ , Mg ²⁺ , K ⁺ , Na ⁺ or EDTA As a result of the reaction with the polymer hyaluronic acid using the containing buffer solution, the decomposition activity of the enzyme reacted with Ag ²⁺ , Ni ²⁺ , Fe ²⁺ , Zn ²⁺ or Cu ²⁺ was greatly reduced. Through this, it was confirmed that Ag ²⁺ , Ni ²⁺ , Fe ²⁺ , Zn ²⁺ and Cu ²⁺ inhibit BST909 hyaluronidase activity ( FIG. 8 ).

In another embodiment of the present invention, BST909 hyaluronidase can specifically degrade hyaluronic acid. Specifically, the enzyme is reacted with a polysaccharide substrate solution such as hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulgate, xanthan or agarose to decompose polysaccharides. The degree was compared. As a result, it was confirmed that only hyaluronic acid was specifically decomposed. Through this, it can be seen that BST909 hyaluronidase exhibits substrate specificity for hyaluronic acid (Table 1).

In another embodiment of the present invention, BST909 hyaluronidase has a Michaelis reaction rate constant (Km) of about 0.0015 mM and a maximum reaction rate (Vmax) of about 50 mM/min. Specifically, the enzyme was reacted with a polymer hyaluronic acid substrate solution for each concentration under the conditions of 35° C., 150 rpm, and 1 hour, and the absorbance was measured at 232 nm. N-acetyl-D-glucosamine, a unit of polysaccharide hyaluronic acid, was The decomposition rate per hour was calculated by comparing the absorbance with the measured value at 232 nm for each concentration. As a result, it was confirmed that the Km value was about 0.0015 mM, and the affinity for the substrate was high, and the Vmax value was about 50 mM/min, confirming that the reaction rate was high (FIG. 9).

In another aspect of the present invention, Streptococcus equi subsp. zooepidemicus hyaluronic acid comprising the step of culturing the BST909 strain (Accession No.: KCTC 14238BP) and purifying the hyaluronic acid degrading enzyme from the culture medium A method for preparing a ronic acid lyase is provided.

Another aspect of the present invention provides a method for producing low molecular weight hyaluronic acid comprising the step of treating the hyaluronic acid degrading enzyme to high molecular weight hyaluronic acid.

The polymeric hyaluronic acid may be prepared by extraction from biological tissues or fermentation from microorganisms according to a conventional method, and may be commercially available polymeric hyaluronic acid. The polymer hyaluronic acid may have a size of 12,00 kDa to 2,600 kDa, preferably 1,200 kDa to 2,200 kDa. However, it is not limited thereto.

In addition, hyaluronic acid in the present specification may include hyaluronate. The "hyaluronate" is in the form of a salt of hyaluronic acid, and is produced by removing lipids, proteins and nucleic acids from hyaluronic acid, so that the molecular size is smaller than that of hyaluronic acid. Examples include, but are not limited to, sodium salts, potassium salts, magnesium salts, calcium salts and zinc salts of hyaluronic acid.

Specifically, in the method for producing low molecular weight hyaluronic acid, the treatment of BST909 hyaluronic acid is performed by adding 3.5 unit/mL of BST909 hyaluronidase to high molecular weight hyaluronic acid at 30° C., 200 rpm, pH 7.0 in 10 mM phosphate buffer solution for 0.5 hour to It can be processed for 3 hours, but is not limited thereto.

In addition, the low molecular weight hyaluronic acid may be 300 Da to 100 kDa. Alternatively, the low molecular weight hyaluronic acid may be 380 Da to 50,000 Da. Alternatively, the low molecular weight hyaluronic acid may be 380 Da to 10,000 Da. Alternatively, the low molecular weight hyaluronic acid may be 380 Da to 5,000 Da. Preferably, the produced low molecular weight hyaluronic acid may be a tetrasaccharide of about 760 Da. In addition, the produced low molecular weight hyaluronic acid may be a disaccharide of about 380 Da to 400 Da.

Specifically, as a result of treating BST909 hyaluronidase under the above conditions in one embodiment of the present specification, it was confirmed that the polymer hyaluronic acid was decomposed into the disaccharide hyaluronic acid during a reaction for 3 hours ( FIGS. 12 to 14 ).

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1. Search and identification of non-hemolytic strains

In order to isolate non-hemolytic strains, natural substances such as sand and grass were inoculated in THB (todd hewitt broth) liquid medium and cultured at 35° C. and 150 rpm. Then, the culture medium was smeared on THB solid medium to obtain colonies. Each colony was transplanted into a serum solid medium (blood agar plate, BAP, Asan Pharmaceutical) and cultured at 35° C., and then non-hemolytic bacteria that did not form a clear ring around the transplanted colony were isolated (FIG. 1). For THB, BD Difco's product was used, and agar (BD Difco) was mixed at a concentration of 15 g/L to prepare a solid medium, sterilized, and then dispensed into Petri dishes by 20 mL each and hardened.

The bacteria isolated through the above method were named "BST909", and Bionics Co., Ltd. was requested to identify the bacteria through 16s rRNA gene sequencing analysis. As a result, by confirming that the first priority identification result was Streptococcus equi subsp. zooepidemicus ATCC43079, BST909 was found to be a strain belonging to the Streptococcus juepidemicus ATCC43079 strain (FIG. 2).

In addition, the characteristics of the strain were confirmed using the API STREP20 kit (Biomerieux) and the API ZYM kit (Biomerieux). As a result of analysis using the API STREP20 kit, it was identified as Streptococcus equi subsp. zooepidemicus with 99.6% ID (99.6% ID, T Index 0.31) (FIG. 3). In this case, % ID indicates relative proximity, and the higher this value, the higher the reliability of the identification result. In addition, the T Index indicates the proximity to biochemical properties, and the closer this number is to 1, the more typical it means.

Example 2. Confirmation of non-hemolytic properties of Streptococcus juepidemicus BST909 strain

The Streptococcus juepidemicus BST909 strain (Accession No.: KCTC 14238BP) identified through Example 1 is characterized as having non-hemolytic properties. However, the strain to which the strain belongs, Streptococcus juepidemicus ATCC43079 strain, is a strain having beta-hemolytic properties. Therefore, the hemolytic difference between the Streptococcus juepidemicus ATCC43079 and the Streptococcus juepidemicus BST909 strain was confirmed again through the experiment.

First, each colony of Streptococcus jeuepidemicus ATCC43079 strain or Streptococcus jeuepidemicus BST909 strain was transplanted into a serum medium (Asan Pharmaceutical) and cultured at 35° C., and then the generation of clear rings around the transplanted colonies was confirmed. . As a result, it was confirmed that a clear ring was generated in the colony of the Streptococcus strain ATCC43079, whereas the clear ring was not generated in the colony of the Streptococcus strain BST909 (FIG. 4).

In addition, the cell suspension (cell), the supernatant (sup.) and the culture medium ( THB), physiological saline (saline, 0.89% NaCl aqueous solution), and NCN buffer (3 mM sodium citrate, 0.9% NaCl, pH 6.8) were reacted with 1% red blood cell solution at 35° C. for 18 hours. At this time, a 1% red blood cell solution was prepared by diluting a 10% sheep red blood cell solution purchased from Innovative Research in NCN buffer solution.

After completion of the reaction, the supernatant was separated by centrifugation, and the color of the reaction solution (top) and absorbance at 540 nm (bottom) were measured to compare the degree of hemolysis of red blood cells. As a negative control, the 1% red blood cell solution was a red blood cell solution that did not react with any solution, and a solution in which the red blood cell solution was completely dissolved using 0.1% SDS (surfactant) was used as a positive control, and compared with the experimental group solution. For specific test methods, refer to "Purification and characterization of hemolysin from Porphyromonas gingivalis A7436" (FEMS Microbiol Lett., 176, 387-394).

As a result, the reaction with 0.1% SDS solution as a positive control was the most red, and the reaction with the Streptococcus strain ATCC43079 culture medium showed a deep red color. In contrast, it was confirmed that the color of the Streptococcus juepidemicus BST909 strain culture solution or general test solution (normal physiological saline, NCN buffer solution, culture medium) was not relatively red (Fig. 5, top). When comparing the absorbance of the reactants, the reactant of Streptococcus strain ATCC43079 caused about 50% hemolysis, whereas the reactant of the Streptococcus strain BST909 had a similar degree of hemolysis to that of normal saline, NCN buffer, and culture medium. This was observed. In addition, it was confirmed that the reactant of the Streptococcus juepidemicus BST909 strain had a lower absorbance at 540 nm than the 1% red blood cell solution itself used for the reaction (FIG. 5, bottom).

Therefore, it can be seen that the novel Streptococcus juepidemicus BST909 strain is a strain having a new characteristic without hemolysis unlike the general Streptococcus juepidemicus ATCC43079 strain.

Example 3. Preparation of novel hyaluronidase

Example 3.1. Streptococcus juepidemicus BST909 strain culture

Streptococcus strain BST909 in 20 mL of sterile THB broth (BD Difco) The strain was inoculated and cultured under conditions of 35° C., 150 rpm, and 6 hours. About 20 mL of the culture medium cultured under the above conditions was inoculated into 200 mL of sterile THB liquid medium and cultured at 35° C., 150 rpm, and 12 hours. About 150 mL of the culture solution cultured under the above conditions was inoculated into 3.5 L of sterile hyaluronidase production medium. The composition per 1 L of hyaluronidase production medium is tryptone 13.5 g, yeast extract 7.0 g, soytone 6.5 g, K ₂ SO ₄ 1.4 g, Na ₂ HPO ₄ 6.5 g, antiform 1.0 g, MgSO ₄ 2.5 g, Glucose is 72.0 g.

After inoculation of the hyaluronidase production medium, culture conditions were adjusted to 35° C., pH 7.0 (adjusted by adding 5 M NaOH), 300 rpm, and air flow rate of 1.5 L/min (lpm) using a fermenter, and cultured for 12-14 hours and then ended.

Example 3.2. Hyaluronidase obtained from Streptococcus juepidemicus BST909 strain

The culture solution obtained in Example 3.1 was centrifuged at 4° C., 8000 rpm, and 30 minutes to remove the settled cells and only the supernatant was recovered. Protein was precipitated from the recovered supernatant using ammonium sulfate, and the protein was purified through an ion exchange column to finally obtain hyaluronidase (hereinafter, referred to as BST909 hyaluronidase).

Specifically, the protein was precipitated by adding ammonium sulfate to the supernatant at a final concentration of 60% (g/L) saturated by centrifugation. The precipitated protein was recovered by centrifuging a 60% saturated solution of ammonium sulfate at 4°C, 8000 rpm, and 30 minutes. The recovered precipitated protein was suspended in 10 mM Tris-HCl (pH 7.5) buffer, and then dialyzed using a 12-14 kDa membrane for about 24 hours.

Using a Hi Prep DEAE FF 16/10 column (GE), AKTA pure FPLC (Cytiva) instrument, the protein solution obtained by dialysis was added to a 10 mM Tris-HCl (pH 7.5) buffer solution with a linear gradient from 0 M to 1 M NaCl. was eluted in this way.

The hyaluronic acid (HA) decomposition activity and protein content of the elution solution by concentration were compared while eluting at a flow rate of 2 mL/min. To remove the NaCl component by collecting the elution solution of the section with high hyaluronic acid decomposition activity, dialysis was performed using a 10 mM Tris-HCl (pH 7.5) buffer solution and a 12-14 kDa membrane.

The solution exhibiting hyaluronic acid decomposition activity obtained by the purification and dialysis process was mixed with 10 mM Tris-HCl (pH 7.5) buffer solution using the FPLC device and Hi Trap Butyl FF column (GE) with ammonium sulfate 40% (g/L) was eluted in a linear concentration gradient manner from 0% (g/L) to 0% (g/L). While eluting at a flow rate of 1.5 mL/min, the hyaluronic acid decomposition activity and protein content of the elution solution by concentration were compared. To remove the ammonium sulfate component by collecting the elution solution of the section with high hyaluronic acid decomposition activity, dialysis was performed using a 12-14 kDa membrane in 10 mM Tris-HCl (pH 7.5) buffer. During the purification process, the progress was confirmed using SDS-PAGE.

Example 4. Confirmation of optimal activity conditions for hyaluronidase produced by Streptococcus juepidemicus BST909 strain

Optimal reaction time of hyaluronidase (hereinafter referred to as BST909 hyaluronidase) obtained from Streptococcus strain BST909 by the method of Example 3, optimal reaction temperature, activity difference depending on the pH of the substrate, Activity inhibition, polysaccharide decomposition ability, reaction rate constant, and molecular weight were confirmed. The hyaluronic acid solution used in the experiment was dissolved in 10 mM SPB (sodium phosphate buffer, pH 7.0, containing 3% NaCl) at a concentration of 1% of high molecular weight hyaluronic acid (sodium hyaluronate, Kewpie, HA-LQH) was used as a substrate solution. .

Example 4.1. Confirmation of optimal reaction temperature of BST909 hyaluronidase

In order to confirm the optimal reaction temperature of BST909 hyaluronidase, BST909 hyaluronidase solution was reacted with 1% high molecular weight hyaluronic acid (Kewpie, HA-LQH) substrate solution in a constant temperature water bath for 1 hour at each temperature environment. Then, the degree of decomposition of hyaluronic acid was compared by measuring the absorbance at 232 nm.

As a result, it was confirmed that the decomposition activity of the enzyme increased as the reaction temperature increased to 40 °C, but the activity was rapidly decreased at 45 °C (FIG. 6). Therefore, it was found that the optimal reaction temperature of this enzyme was 40 °C.

Example 4.2. Confirmation of optimal reaction pH of BST909 hyaluronidase

In order to confirm the optimal reaction pH of BST909 hyaluronidase, 1% high molecular weight hyaluronic acid (Kewpie, HA-LQH) solution to be used as a substrate was prepared by pH using buffers prepared for each pH, and 35 ° C with the enzyme solution. , 150 rpm, and reacted under the same conditions for 1 hour. The substrate solution according to each pH is 10 mM sodium acetate, pH 4.0, pH 5.0, pH 6.0 buffer solution, 10 mM sodium phosphate buffer (SPB), pH 6.0, pH 7.0, pH 8.0 or 10 mM Tris-HCl, pH 8.0, It was prepared using a pH 9.0 buffer solution. After the reaction was completed, the degree of decomposition of hyaluronic acid was compared by measuring the absorbance at 232 nm.

As a result, it was confirmed that BST909 hyaluronidase had the highest reaction activity with a 1% polymer hyaluronic acid solution prepared using 10 mM SPB (pH 7.0) (FIG. 7). Therefore, it was found that the optimal pH of the enzyme was 7.0.

Example 4.3. Comparison of inhibition of activity of BST909 hyaluronidase by metal salt

In order to compare the degree of inhibition of the activity of BST909 hyaluronidase by the metal salt, the enzyme solution was mixed with a 5 mM solution of each metal salt in a ratio of 1:1, and reacted at 30° C., 150 rpm, and 1 hour. Thereafter, after further reaction with a 1% polymer hyaluronic acid (Kewpie, HA-LQH) solution at 30° C., 150 rpm, and 1 hour conditions, the reaction was stopped by heating in a constant temperature water bath at 80° C. for 20 minutes. Thereafter, the degree of decomposition of hyaluronic acid was compared by measuring the absorbance at 232 nm.

As a result, when reacted with metal salts such as barium (Ba ²⁺ ), calcium (Ca ²⁺ ), manganese (Mn ²⁺ ), magnesium (Mg ²⁺ ), potassium (K ⁺ ), sodium (Na ⁺ ) and EDTA Although the ^{degree of hyaluronic} acid ^{decomposition activity} was similar to It was confirmed that the decomposition activity of the reacted enzyme was greatly reduced (FIG. 8).

Example 5. Confirmation of resolution for polysaccharides of BST909 hyaluronidase

In order to confirm the decomposition ability of BST909 hyaluronidase on other polysaccharides, first, polysaccharides such as high molecular weight hyaluronic acid (Kewpie, HA-LQH), chondroitin sulfate A/C, dermatan sulfate, xanthan, and agarose were 259 at a concentration of 1%. A polysaccharide substrate solution was prepared by dissolving it in mM SPB (pH 7.0). 100 μl of the prepared polysaccharide substrate solution, 600 μl of distilled water, 200 μl of 259 mM SPB (pH 7.0), and 100 μl of an enzyme solution were mixed and reacted at 37° C., 150 rpm, 16 hours, and then in a water bath at 100° C. for 10 The enzymatic reaction was stopped by reacting for 10 minutes in cold water. The reaction solution was centrifuged at 4° C., 15000 g, for 15 minutes, and the supernatant was recovered and absorbance was measured at 232 nm to compare the degree of decomposition of polysaccharides.

As a result, it was confirmed that BST909 hyaluronidase had a decomposition activity only in sodium hyaluronic acid, and had no decomposition activity in other polysaccharides (Table 1).

temperament RA (%) One sodium hyaluronate 100.00 2 chondroitin sulfate A 0.812 3 chondroitin sulfate C 1.753 4 dermatan sulfate 1.596 5 xanthan -1.070 6 agarose -3.859

Example 6. Analysis of rate constant of BST909 hyaluronidase

To confirm the reaction rate constant of BST909 hyaluronidase, high molecular weight hyaluronic acid (Kewpie, HA-LQH) at a concentration of 0% to 1% was dissolved in 10 mM SPB (pH 7.0, 3% NaCl) and used.

The polymeric hyaluronic acid substrate solution for each concentration was reacted with BST909 hyaluronidase under the conditions of 35°C, 150 rpm, and 1 hour. The decomposition rate per hour was calculated by comparing the absorbance measured at 232 nm with the value measured at 232 nm for each concentration of N-acetyl-D-glucosamine, a unit of polysaccharide hyaluronic acid.

The Reinweaver-Burke equation was prepared using the concentration of the substrate (polymer hyaluronic acid) obtained through the above reaction and the decomposition rate value thereof, and the Michaelis reaction rate constant (Km) and the maximum reaction rate (Vmax) were calculated. . As a result, it was confirmed that the Km value was 0.0015 mM, indicating a high affinity for the substrate, and the Vmax value was 50 mM/min, indicating a high reaction rate (FIG. 9).

Example 7. Confirmation of molecular weight of BST909 hyaluronidase

In order to confirm the molecular weight of BST909 hyaluronidase, the molecular weight of BST909 hyaluronidase obtained in Example 3.2 was roughly confirmed through SDS-PAGE. As a result, it was confirmed that the molecular weight of BST909 hyaluronidase was about 127 kDa (FIG. 10).

The protein isolated through the SDS-PAGE was requested to the Gyeonggi Bio Center, and N-terminal sequence analysis was performed. As a result, it was confirmed that the hyaluronate lyase precursor HylB ( Streptococcus equi subsp. zooepidemicus SzAM60) has a similarity of 86.5% (Fig. 11). In addition, when the N-terminal sequence was searched using NCBI BLAST, the N-terminal LWLSPMATGTEKK sequence (SEQ ID NO: 3) was hyaluronate of various Streptococcus juepidemicus strains including the hyaluronate lyase precursor HylB. It was confirmed that it was present in the amino acid sequence of the enzyme. However, the molecular weight of hyaluronidase of several species belonging to the genus Streptococcus is 116 kDa ( Streptococcus Agalactiae , Streptococcus Dysglactiae ), 54 kDa ( Streptococcus Uberis ), 55 kDa ( Streptococcus equi subsp. zooepidemicus ), etc. It is different from Ronnie Days.

Through this, it was confirmed that the BST909 hyaluronidase isolated from the Streptococcus juepidemicus BST909 strain is a novel enzyme different from the hyaluronidase of the general Streptococcus juepidemicus strain.

Example 8. Method for producing low molecular weight hyaluronic acid using BST909 hyaluronidase

The BST909 hyaluronic acid obtained in Example 3.2 was prepared and used at 35 unit/mL, and for the enzymatic reaction, an enzyme solution of 10% (v/v) of the substrate amount was added (the final titer of the enzyme was 3.5 unit/mL ) was carried out while stirring at 30 °C at 200 rpm. HPLC analysis was performed at UV absorbance 210 nm using a YMC-Pack Polyamine II (PB12S05-2546WT, YMC) column, and 1 M NaH ₂ PO ₄ was used as the mobile phase, in a linear gradient method from 5 mM to 1 M. eluted.

In addition, the flow rate was measured at 1.0 mL/min. The decomposition process of polymer hyaluronic acid according to the reaction time of BST909 hyaluronidase is shown in FIG. 12, and the results of analyzing the final degradation products by MALDI-TOF and LC-MS at the Gyeonggi Bio Center are shown in FIGS. 13 and 14. It was.

As a result, as shown in FIGS. 12 to 14 , when reacted with BST909 hyaluronidase for 3 hours, high molecular weight hyaluronic acid of 1,200 kDa to 2,200 kDa was decomposed into disaccharides of about 380 Da to 400 Da and tetrasaccharides of about 760 Da. Confirmed.

Therefore, it was found that BST909 hyaluronic acid can produce low molecular weight hyaluronic acid by decomposing high molecular weight hyaluronic acid.

Korea Institute of Bioscience and Biotechnology KCTC14238BP 20200714

<110> Biostream Technologies Co., Ltd. <120> NOVEL STREPTOCOCCUS STRAIN AND HYALURONIDASE DERIVED FROM THE SAME <130> SPD20-149BST <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1503 <212> RNA <213> Streptococcus equi <400> 1 ctcaggacga acgctggcgg cgtgcctaat acatgcaagt ggaacgcaca gatgatacgt 60 agcttgctac aattatctgt gagtcgcgaa cgggtgagta acgcgtaggt aacctagctt 120 atagcggggg ataactattg gaaacgatag ctaataccgc ataaaagtgg ttgacccatg 180 ttaactattt aaaaggagca acagctccac tatgagatgg acctgcgttg tattagctag 240 ttggtagggt aaaggcctac caaggcgacg atacatagcc gacctgagag ggtgaacggc 300 cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttcgg 360 caatgggggg aaccctgacc gagcaacgcc gcgtgagtga agaaggtttt cggatcgtaa 420 agctctgttg ttagagaaga acagtgatgg gagtggaaag tccatcatgt gacggtaact 480 aaccagaaag ggacggctaa ctacgtgcca gcagccgcgg taatacgtag gtcccgagcg 540 ttgtccggat ttattgggcg taaagcgagc gcaggcggtt tgataagtct gaagttaaag 600 gcagtggctt aaccattgta tgctttggaa actgttaaac ttgagtgcag aaggggagag 660 tggaattcca tgtgtagcgg tgaaatgcgt agatatatgg aggaacaccg gtggcgaaag 720 cggctctctg gtctgtaact gacgctgagg ctcgaaagcg tggggagcaa acaggattag 780 ataccctggt agtccacgcc gtaaacgctg agtgctaggt gttaggccct ttccggggct 840 tagtgccgga gctaacgcat taagcactcc gcctggggag tacgaccgca aggttgaaac 900 tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 960 gcgaagaacc ttaccaggtc ttgacatccc gatgctattc ttagagataa gaagttactt 1020 cggtacattg gagacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1080 taagtcccgc aacgagcgca acccttattg ttagttgcca tcattaagtt gggcactcta 1140 gcgagactgc cggtaataaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1200 tatgacctgg gctacacacg tgctacaatg gttggtacaa cgagtcgcaa gccggtgacg 1260 gcaagctaat ctctgaaagc caatctcagt tcggattgta ggctgcaact cgcctacat 1320 aagtcggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt ccggggcctt 1380 gtacacaccg cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg aggtaaccgt 1440 taaggagcca gccgcctaag gtgggataga tgattggggt gaagtcgtaa cagggtaacc 1500 gta 1503 <210> 2 <211> 1059 <212> PRT <213> Artificial Sequence <220> <223> BST909 hyaluronidase N-terminal sequence <400> 2 Met Lys Gln Arg Lys Lys Leu Ser Ser Ser Val Cys Ser Ser Ile Ile 1 5 10 15 Leu Ala Cys Leu Phe Arg Ala Pro Thr Val Leu Ala Glu Glu Lys Thr 20 25 30 Ala Ala Lys Ala Ile Ser Lys Glu Leu Gln Arg Val Lys Lys Ala Pro 35 40 45 Leu Lys Thr Glu Glu Ala Ser Gln Glu Ser Ser Pro Asp Thr Gln Asp 50 55 60 Ala Ser Ser Val Ala Thr Glu Ser Ser Leu Gly Glu Gln Leu Ile Leu 65 70 75 80 Thr Gly Asp Asn Leu Leu Lys Asn Pro Gln Phe Asp Gln Thr Ser Pro 85 90 95 Ala Gln Glu His Gln Ser Thr Ser Leu Trp Ser Lys Glu Ser Ala Asn 100 105 110 Asp Trp Lys Asp Tyr Lys Asp Ala Ser Lys Ser Gln Gly Cys Pro Lys 115 120 125 Ile Ala Val Ser Asp Asn Lys Leu Thr Met Thr Ser Asp Gly Asn Gln 130 135 140 Arg Phe Arg Gly Cys Val His Gln Thr Val Ala Ile Asn Pro Asp Lys 145 150 155 160 Gln Tyr Leu Leu Thr Glu Asp Ile Glu Thr Lys Asp Lys Thr Gly Gln 165 170 175 Ala Phe Ala Arg Ile Ile Glu Glu Ile Lys Gln Gly Ser Gly Ala Ala 180 185 190 Lys Glu Gln Arg Leu Trp Leu Ser Pro Met Ala Thr Gly Thr Glu Lys 195 200 205 Lys His Gln Glu Lys Leu Tyr Ile Pro Lys Leu Lys Val Asn Gln Ile 210 215 220 Lys Leu Glu Leu Phe Tyr Glu Ala Gly His Gly Gln Val Val Phe Asp 225 230 235 240 Asn Leu Ser Leu Arg Glu Ala Gly Asp Lys Pro Ser Asp Asp Ile Lys 245 250 255 Ile Ala Ser His Tyr Leu Glu Glu Gln Ile Val Leu Pro Leu Asn Lys 260 265 270 His Tyr Leu Met Glu Met Ala Asp Tyr His Tyr Gln Ile Ala Ala Glu 275 280 285 Ser Ser Asn Ile Val Arg Val Glu Asn Gly Leu Leu Ile Pro Leu Ala 290 295 300 Gln Gly Lys Thr Leu Leu Glu Val Leu Asp Gln Glu Gly Gln Arg Val 305 310 315 320 Val Thr Val Pro Val Glu Ile Leu Ala Ala Glu Asp Pro Gln Thr Thr 325 330 335 Ser Leu Ile Thr Lys Trp Cys Glu Val Ile Leu Gly Ala Glu Asn Phe 340 345 350 Asp Arg Ser Ser Pro Ala Met Val Ala Leu Asn Gln Lys Leu Asp Asp 355 360 365 Ser Val Thr Lys Asn Leu Ala Val Leu Thr Lys Asp Gln Lys Ser Thr 370 375 380 Tyr Leu Trp Ser Asp Leu Ala Asp Leu His Gln Ser Ser His Met Thr 385 390 395 400 Ala Thr Cys Arg Arg Leu Glu Glu Met Ala Lys Gln Val Ser Ser Leu 405 410 415 Ala Ser Arg Tyr Tyr Gln Asp Lys Glu Leu Ile Arg Leu Ile Lys Asp 420 425 430 Lys Leu Ala Trp Leu Thr Leu Asn Tyr Tyr His Pro Gln Lys Asp Ile 435 440 445 Glu Gly Lys Ala Asn Trp Trp Asp Phe Glu Ile Gly Thr Pro Arg Ala 450 455 460 Ile Val Asn Thr Leu Ala Phe Ile Tyr Pro Tyr Val Thr Gln Glu Glu 465 470 475 480 Ile Lys Arg Tyr Thr Lys Gly Ile Ser His Phe Val Pro Asn Pro Arg 485 490 495 Gln Phe Arg Ser Thr Leu Val Asn Pro Phe Lys Ala Ile Gly Gly Asn 500 505 510 Leu Val Asp Met Gly Arg Ile Lys Ile Ile Glu Ala Leu Leu Lys His 515 520 525 Asp Lys Lys Ala Leu Gln Asp Ser Ile Ala Ala Leu Asp Thr Leu Phe 530 535 540 Ala Phe Gln Pro Arg Gly Ser Lys Gly Glu Gly Phe Tyr Glu Asp Gly 545 550 555 560 Ser Tyr Ile Asp His Thr Asn Val Ala Tyr Thr Gly Ala Tyr Gly Asn 565 570 575 Val Leu Ile Asp Gly Leu Ser Gln Leu Val Pro Leu Ile Gln Gln Ser 580 585 590 Ala Ala Ser Leu Asp Gln Lys Lys Leu Glu Ala Met Thr His Trp Ile 595 600 605 Glu Gln Ala Phe Leu Pro Leu Met Val His Gly Glu Leu Met Asp Met 610 615 620 Asn Arg Gly Arg Ser Ile Ser Arg Glu Asn Ala Ser Ser Arg Gln Ala 625 630 635 640 Ala Leu Glu Ala Leu Arg Gly Met Leu Arg Leu Ala Asp Ala Leu Pro 645 650 655 Glu Gln Ala Lys Ile Arg Ile Lys Ala Val Leu Ala Phe His Asn Gln 660 665 670 Glu Ala Ile Leu Glu Ser Leu Ser Ser Tyr Tyr Asp Met Lys Leu Phe 675 680 685 Lys Glu Leu Leu Glu Asp Thr Ala Ile Gln Ala Ser Pro Val Lys Ser 690 695 700 Tyr Leu Ser Leu Phe Asn Gln Met Asp Lys Leu Ala Tyr Tyr Asn Ala 705 710 715 720 Glu Lys Asp Phe Ala Phe Ala Leu Ser Leu His Ser Asn Lys Thr Leu 725 730 735 Asn Phe Glu Ala Met Asn Asn Glu Asn Thr Arg Gly Trp Tyr Thr Gly 740 745 750 Asp Gly Met Phe Tyr Leu Tyr Asn Ser Asp Leu Gly His Tyr Ser Asp 755 760 765 Arg Phe Trp Pro Thr Val Asn Pro Leu Lys Met Ala Gly Thr Thr Glu 770 775 780 Ala Glu Val His Arg Glu Asp Val Thr Val Ala Tyr Leu Lys Lys Leu 785 790 795 800 Thr Asn Asp Tyr Lys Glu Lys Ala Lys Glu Lys Ala Gly Met Ser Thr 805 810 815 Gln Gln Ser Ser Phe Val Gly Ala Ile Lys Ala Gly Asp Lys Thr Ala 820 825 830 Leu Ala Val Met Asp Phe Gln Asn Trp Asp Arg Thr Val Thr Ala Lys 835 840 845 Lys Ser Trp Thr Ile Leu Asp Asp Gln Ile Val Phe Leu Gly Thr Ala 850 855 860 Ile Thr Ser Gln Thr His Gln Ala Val Ser Thr Thr Ile Asp Gln Arg 865 870 875 880 Lys Glu Asn Pro Asp Asn Ser Tyr Thr Leu Phe Ile Asn Gly Gln Glu 885 890 895 Thr Ala Leu Thr Glu Glu Val Leu His Arg Asp Asp Val Thr Ser Leu 900 905 910 Leu Leu Leu Ser Lys Asp Gly Gln Ala Asn Ile Gly Tyr Leu Phe Ala 915 920 925 Lys Pro Thr Ser Leu Ala Leu Ser Arg Lys Val Gln Ser Gly Arg Trp 930 935 940 Ser Glu Ile Asn Thr Asn Ser Lys Asn Glu Asp Leu Ile Ser Gln Ser 945 950 955 960 Phe Ile Thr Ile Ser Gln Ala His Ser Gln Ala Ser Asp Ser Tyr Ala 965 970 975 Tyr Thr Leu Leu Pro Asn Ile Ser Lys Ala Asp Phe Asp Lys Val Cys 980 985 990 Ser Glu Ala Arg Ile Glu Val Leu Gln Asn Asp Ser Lys Leu Gln Leu 995 1000 1005 Ile His Asp Lys Lys Gln Gly Leu Leu Ala Val Val Lys Tyr Asn Gln 1010 1015 1020 Ala Lys Glu Val Val Asn Gly Gln Leu Ser Leu Glu Lys Ser Gly Leu 1025 1030 1035 1040 Tyr Leu Tyr Gln Lys Val Gly Asn Asp Phe Lys Gln Leu Ser Phe Lys 1045 1050 1055 Ala Leu Ser <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> hyaluronidase fragment sequence <400> 3 Leu Trp Leu Ser Pro Met Ala Thr Gly Thr Glu Lys Lys 1 5 10

Claims (11)

Streptococcus juepidemicus ( Streptococcus equi subsp. zooepidemicus ) BST909 strain (Accession No.: KCTC 14238BP). According to claim 1,
16s rRNA of the strain is characterized in that it comprises SEQ ID NO: 1, Streptococcus juepidemicus BST909 strain (Accession No.: KCTC 14238BP). According to claim 1,
Streptococcus juepidemicus BST909 strain (Accession No.: KCTC 14238BP), characterized in that the strain is non-hemolytic. A hyaluronic acid degrading enzyme comprising the amino acid represented by SEQ ID NO: 2. 5. The method of claim 4,
The hyaluronic acid degrading enzyme is Streptococcus equi subsp. zooepidemicus ) BST909 strain (Accession No.: KCTC 14238BP) that is obtained from the, hyaluronic acid degrading enzyme. 5. The method of claim 4,
The hyaluronic acid degrading enzyme, characterized in that it exhibits high molecular weight hyaluronic acid decomposition activity at 15 ° C. to 45 ° C., hyaluronic acid degrading enzyme. 5. The method of claim 4,
The hyaluronic acid degrading enzyme, characterized in that it exhibits decomposition activity at pH 4.0 to pH 9.0, hyaluronic acid degrading enzyme. 5. The method of claim 4,
The hyaluronic acid degrading enzyme is Ag ²⁺ , Ni ²⁺ , Fe ²⁺ , Zn ²⁺ and Cu ²⁺ Hyaluronic acid degrading enzyme, characterized in that the decomposition activity is inhibited by any one selected from. Streptococcus juepidemicus ( Streptococcus equi subsp. zooepidemicus ) Culturing the BST909 strain (Accession No.: KCTC 14238BP);
purifying the hyaluronic acid degrading enzyme from the culture medium
A method for producing a hyaluronic acid degrading enzyme. A method for producing low molecular weight hyaluronic acid comprising the step of treating the high molecular weight hyaluronic acid with the hyaluronic acid degrading enzyme of claim 4. In claim 10,
The low molecular weight hyaluronic acid has a size 380 Da to 100 kDa, the method for producing low molecular weight hyaluronic acid.

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