KR102780203B1 - Paenibacillus strain and new enzyme derived from the same - Google Patents

Abstract

The present invention relates to a Paenibacillus residui BSSK58 strain (Accession Number: KCTC15643BP) producing a novel enzyme, and a method for producing low-molecular-weight hyaluronic acid and low-molecular-weight chondroitin sulfate using the enzyme obtained therefrom. The Paenibacillus residui BSSK58 strain of the present invention is a Penibacillus genus that does not have hemolytic toxicity and does not contain pathogenic genes, and produces a novel hyaluronidase having a molecular weight different from that of hyaluronidases produced by other strains. 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 and low-molecular-weight chondroitin sulfate, which can be applied to various industrial fields such as foods, cosmetics, and pharmaceuticals.

Description

{PAENIBACILLUS STRAIN AND NEW ENZYME DERIVED FROM THE SAME}

The present invention relates to a Paenibacillus residui BSSK58 (KCTC15643BP) strain, a novel enzyme produced from the strain, and a method for producing low-molecular-weight hyaluronic acid and low-molecular-weight chondroitin using the same.

Hyaluronic acid is a polysaccharide of various lengths composed of repeating disaccharide subunits of D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid is abundant in animal skin and has the characteristics of high viscosity and hydrophilicity. In addition, hyaluronic acid is an important component of skin tissue, joint cartilage tissue, etc., and helps connect and support cells. In addition, due to the moisturizing and lubricating effects of hyaluronic acid, it is used in a wide range of fields such as pharmaceuticals (joint lubricants), foods (hyaluronic acid health functional foods), and cosmetics (moisturizers, plastic surgery fillers) (Korean Patent Publication No. 1654810).

The molecular weight of hyaluronic acid varies from hundreds of Da to millions of Da. In addition, hyaluronic acid aqueous solutions have viscosity, elasticity, and moisturizing properties, which vary depending on the molecular weight of hyaluronic acid and its concentration. High molecular weight hyaluronic acid has high viscosity and low skin penetration, which makes it insufficient in functionality when used in product production. For this reason, various studies are being conducted on methods for decomposing high molecular weight hyaluronic acid into low molecular weight hyaluronic acid. Chemical methods for decomposing high molecular weight hyaluronic acid include methods using acids, alkalis, oxidation catalysts, and organic solvents. In addition, physical methods for decomposing high molecular weight hyaluronic acid include methods using heat treatment and ultrasound. However, when decomposing hyaluronic acid into low molecular weight products using these methods, it is difficult to produce various low molecular weight products, and problems such as impurity generation and environmental pollution occur (Korean Patent Publication No. 0665916).

Existing hyaluronidase is extracted and purified from the testes of mammals such as sheep and used as a medicine. It is used for the purpose of increasing the diffusion of local anesthetics and steroids during surgery. However, it is sold at a very high price because it is difficult to purify and mass-produce it to a high purity for use as a medicine.

Meanwhile, chondroitin sulfate is a polysaccharide belonging to glycosaminoglycan (GAG) that exists in both vertebrates and invertebrates as a mucopolysaccharide. Its structural characteristics are that it is a polysaccharide composed of disaccharide sequences in which glucosamine residues linked by β-1,3 bonds and N-acetyl-D-galactosamine (GalNAc) residues alternate and are sulfated at various positions. The chondroitin sulfate exists in the connective tissue of the human body, and is a major component of cartilage tissue along with collagen, and is a biological material closely related to the nervous system and immunity. In other words, it maintains moisture in the skin and is a structural component of the ECM (Extra-cellular Matrix) as a matrix component between cells. In addition, as a core component of the dermal tissue, it can be said to be an essential component for maintaining skin homeostasis and maintaining lubrication in joints. Such chondroitin sulfate is isolated and used from terrestrial animals and marine fish species. When administered orally, chondroitin sulfate is absorbed by the intestinal mucosa in the small intestine and distal tracts, but the mechanism has not been clearly elucidated. However, in order to increase bioavailability, it is essential to reduce the molecular weight of chondroitin sulfate.

To date, various strains producing chondroitin sulfate decomposition enzymes are known. For example, chondroitinase ABC (EC 4.2.2.4) produced by microorganisms such as Proteus vugaris , Flavobacterium heparinum , and Bacteria sulfolifolia is known. Another decomposition enzyme is hyaluronic acid decomposition enzyme (Hyaluronidase sheep testes Type II, EC 3.2.1.35). This enzyme, extracted from the testes of male sheep, is known to randomly decompose the N-acetylhexosamine-(1→4)-glycosidic bond of hyaluronic acid, chondroitin, and chondroitin sulfate.

As methods for reducing chondroitin sulfate to lower molecular weights, acid hydrolysis, alkaline hydrolysis, and enzymatic methods are known. Chemical decomposition methods using acids and alkalis produce a lot of monosaccharides during hydrolysis, resulting in low conversion efficiency, and many sulfate groups, which are functional groups, are removed. In addition, a neutralization process is essential for using it as a raw material, which may cause environmental pollution. To replace the shortcomings of chemical decomposition methods, methods using enzymes are being used as an alternative, and enzymatic hydrolysis methods have mild reaction conditions, so pollution is reduced and can be easily controlled, and enzymatic hydrolysis does not destroy sulfate groups, so it has advantages in industrial production processes.

KR 10-1654810 B1 KR 10-0665916 B1

Accordingly, the present researchers conducted research to develop a hyaluronic acid-decomposing enzyme (hyalunidase) capable of decomposing hyaluronic acid, and as a result, identified a microorganism capable of producing a novel enzyme capable of decomposing hyaluronic acid and chondroitin sulfate, and confirmed that low-molecular-weight hyaluronic acid and low-molecular-weight chondroitin sulfate could be produced using the enzyme obtained therefrom, thereby completing the present invention.

To achieve the above purpose, one aspect of the present invention provides Paenibacillus residui BSSK58 strain (Accession Number: KCTC15643BP).

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

Another aspect of the present invention provides a method for producing low molecular weight hyaluronic acid and its salts, comprising the step of treating high molecular weight hyaluronic acid with the enzyme.

Another aspect of the present invention provides a method for producing low molecular weight chondroitin and its salts, comprising the step of treating chondroitin sulfate with the enzyme.

The present invention relates to a soil microorganism, Paenibacillus residui BSSK58 strain (Accession Number: KCTC15643BP), and a method for producing low-molecular-weight hyaluronic acid and low-molecular-weight chondroitin using a novel hyaluronic acid-decomposing enzyme obtained therefrom. The Paenibacillus residui BSSK58 strain of the present invention has no hemolytic toxicity and exhibits activity for decomposing β-1,4 linkages of hyaluronic acid, like those derived from mammals or microorganisms. In particular, the Paenibacillus residui BSSK58 strain, which is a hyaluronidase-producing strain, can be safely used as a non-pathogenic microorganism.

Figure 1 is a phylogenetic diagram of Paenibacillus residui BSSK58 strain.
FIG. 2 illustrates a process for screening the strain of the present invention on a solid plate medium (left side of FIG. 2), and is a photograph showing the culturing of the Penicillium residuum BSSK58 strain selected through this process (right side of FIG. 2).
Figure 3 shows whether the Penicillium recidui BSSK58 strain produces hemolytic toxin. As shown in the figure, the Penicillium recidui BSSK58 strain does not produce hemolytic toxin (left side of Figure 3), but Staphylococcus warnen produces hemolytic toxin (right side of Figure 3).
Figure 4 shows the results showing that seven virulence genes were not detected in the Penicillium recidui BSSK58 strain.
Figures 5a and 5b are graphs showing the optimal culture temperature and optimal pH of the Penicillium residuum BSSK58 strain.
Figure 6 shows the Native-PAGE electrophoresis results of the enzyme produced from the Penicillin-Bacillus Recidui BSSK58 strain.
Figures 7a and 7b are graphs showing the optimal reaction pH and pH stability of the enzyme produced by Penicillium residui BSSK58 strain.
Figures 8a and 8b are graphs confirming the activity temperature range and thermal stability of the enzyme produced by Penicillium residui BSSK58 strain.
Figure 9 is a graph showing the enzyme reaction rate constant for hyaluronic acid.
Figures 10a and 10b are graphs showing enzyme reaction rate constants for chondroitin sulfate (derived from bovine trachea or shark cartilage).
Figure 11 is a graph showing the change in hydrolysis of high molecular weight hyaluronic acid according to the reaction time of the enzyme produced by Penicillium rhizobacillus residue BSSK58 strain.
Figure 12 is a graph showing the change in hydrolysis of chondroitin sulfate (derived from bovine trachea) according to the reaction time of the enzyme produced by Penicillium residui BSSK58 strain.
Figure 13 is a graph showing the change in hydrolysis of chondroitin sulfate (derived from shark cartilage) according to the reaction time of the enzyme produced by Penicillium residui strain BSSK58.
Figure 14 shows the results of amplifying the gene of an enzyme produced by the Penicillium recidui BSSK58 strain using PCR.
Figure 15 is a recombinant vector produced by combining enzyme genes produced by the Penicillium recidui BSSK58 strain obtained through a PCR reaction.
Figure 16 shows the results of SDS-PAGE confirmation of hyaluronidase expressed and purified in cells transformed with a recombinant vector constructed by combining the genes of enzymes produced by Penicillium recidui BSSK58 strain.
Figure 17 is a graph showing the enzyme activity of hyaluronidase obtained from cells transformed with a recombinant vector constructed by combining the gene of an enzyme produced by Penicillium recidui BSSK58 strain.

Hereinafter, the present invention will be described in detail.

New strain

One aspect of the present invention provides a Paenibacillus residui BSSK58 strain (accession number: KCTC15643BP). The strain comprises 16s rRNA represented by sequence number 1 and is characterized in that it is non-hemolytic.

The above Penibacillus residuei BSSK58 strain is a microorganism of the genus Paenibacillus, which can directly promote crop growth by producing the plant hormone indole-3-acetic acid (IAA) and releasing siderophores that enable iron acquisition. In addition, Penibacillus is known to produce various antimicrobial agents. Specifically, Penibacillus-derived antimicrobial agents include polymyxins, which are nonribosomal lipopeptides first isolated from the Paenibacillus polymyxa strain, and fusaricidins, which have been widely applied in the medical field. Other useful molecules derived from Penibacillus include exopolysaccharides (EPS) and enzymes, such as amylase, cellulase, hemicellulase, lipase, pectinase, oxygenase, dehydrogenase, lignin-modifying enzyme, and mutanase. They can be applied in detergents, food and feed, textiles, paper, biofuels and healthcare.

In addition, Phenibacillus is known to produce various enzymes. Specifically, among the Phenibacillus genus that has been approved by the European Food and Drug Administration (EMA) to produce enzymes for food additives , the Paenibacillus lentus DSM280288 strain produces endo-1,4-mannanase, the Paenibacillus macerans strain produces Cyclomaltodextringlucanotransferase, and the Paenibacillus Illinois strain produces Cyclomaltodextrinotransferase and are sold. In particular, the Phenibacillus Recidui of this invention can be safely used as a non-pathogenic microorganism.

As used herein, the term "hemolysis" means the destruction of red blood cells, causing their contents (cytoplasm) to dissolve into the surrounding fluid (e.g., plasma). Hemolytic strains are sometimes classified as alpha, beta, and gamma hemolytic. Alpha hemolytic strains oxidize the hemoglobin in red blood cells, producing a green stain on blood agar plates. Beta hemolytic strains rupture blood agar plates by destroying surrounding blood cells, producing a clear stain.

The present inventors selected a strain that is non-hemolytic and produces an enzyme that decomposes hyaluronic acid or chondroitin sulfate, and confirmed that the strain is a novel strain belonging to the genus Paenibacillus (Figs. 1 to 3). In addition, the strain was named " Paenibacillus residui BSSK58", and the same strain was deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on October 16, 2023 under the deposit number KCTC15643P.

new enzyme

Another aspect of the present invention provides an enzyme or a fragment thereof comprising an amino acid represented by SEQ ID NO: 2. Specifically, in one embodiment of the present invention, the enzyme may be obtained from, but is not limited to, a Penicillium lactis strain BSSK58. In addition, the enzyme may be hyaluronidase. In addition, hereinafter, the enzyme may be used interchangeably with "BSSK58 hyaluronidase".

The term "hyaluronic acid (or hyaluronan, HA)" as used herein is a polymer compound consisting of a complex polysaccharide composed of hyaloid and uronic acid, with a molecular weight of 50,000 Da to 13,000,000 Da, and in which repeating units of glucuronic acid and N-acetylglucosamine are alternately bonded in a (1-3) and (1-4) configuration.

Hyaluronic acid has various effects such as moisturizing effect, lubricating effect against physical friction, and protective effect against bacterial invasion, and excellent physical properties, and is widely used as a cosmetic additive, arthritis treatment agent, surgical assistant for ophthalmic surgery, and adhesion inhibitor after surgical surgery, as well as a material for cosmetics, medicines and quasi-drugs, and food. In addition, it is known to act as a mediator that controls the movement of physiologically active substances and induces cell differentiation and growth through a specific interaction with cells, and also acts as a support for proteins and glycoproteins that support tissues such as collagen, elastin, and chondroitin sulfate in the extracellular matrix. However, when hyaluronic acid is used in plants and cosmetics, hyaluronic acid with a large molecular weight has the disadvantage of not being easily absorbed into the body and the skin. Such hyaluronic acid is contained in large quantities in cow eyes, chicken combs, animal buffer tissue, placenta, cancer cells, and skin.

In the present invention, the enzyme is an enzyme that hydrolyzes hyaluronic acid. That is, the enzyme can be named hyaluronidase. The hyaluronidase is found in various organisms 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, a hydrolase that acts on a β-1,4-glucoside bond. 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 that acts on β-1,3-glucosidic bonds. It produces mainly tetrasaccharides and is found in leeches and hookworms. Hyaluronate lyase (EC 4.2.2.1) is a bacterial hyaluronidase that acts on β-1,4-glucosidic bonds and produces 4,5-unsaturated disaccharides through a β-elimination mechanism.

Hyaluronidases produced conventionally from microorganisms are mainly produced by gram-positive pathogenic bacteria such as Streptococcus , Staphylococcus , Peptostreptococcus , Clostridium , and Propionibacterium , as well as by Streptomyces , an actinomycete, Penicillium and Fistulina fungi, and marine microorganisms.

Enzymatic properties of hyaluronidases produced by microorganisms showed that hyaluronidase from Streptococcus dysgalactiae had a molecular weight of 80 kDa, and the optimal conditions for enzyme activity were pH 6 to pH 7 and 35 to 45°C. Hyaluronidase from Streptococcus koganiensis ATCC 31394 had a molecular weight of 21.6 kDa. Hyaluronidase from Streptococcus agalactiae had a molecular weight of 116 kDa. Hyaluronidase produced by Bacillus sp CGMCC5744 had a molecular weight of 123 kDa, and the optimal conditions for enzyme activity were pH 4 to pH 9 and 20 to 48°C. Hyaluronidase produced by Streptomyces actinocidus 77 had a molecular weight of 44 KDa, and the optimal conditions for enzyme activity were pH 6.5 to pH 7.0 and 50 to 60°C. Hyaluronidase produced by Penicillium spp. had a molecular weight of 31.7 KDa, and the optimal reaction temperature of the enzyme was pH 3.0 and 43°C.

The hyaluronidase of the present invention is produced from a novel soil microorganism, Penibacillus recidui KCTC15643BP. The strain can produce hyaluronidase in large quantities. The hyaluronidase of the present invention exhibits a mechanism for decomposing the β-1,4 bond of hyaluronic acid, similar to that of mammalian origin or microorganism origin. In particular, the Penibacillus recidui BSSK58 strain, which is a hyaluronidase producing strain of the present invention, can be safely used as a non-pathogenic microorganism.

According to one embodiment of the present invention, the molecular weight of the hyaluronidase obtained from the strain was confirmed to be about 161 kDa (Fig. 6). In addition, the hyaluronidase may include the sequence of SEQ ID NO: 2. In the present invention, the hyaluronidase purified from the Penicillium residueu BSSK58 strain may be collectively described as "BSSK58 hyaluronidase". In addition, the BSSK58 hyaluronidase may include a hyaluronidase precursor, a mature hyaluronidase, and a truncated form thereof having activity.

According to another embodiment of the present invention, the BSSK58 hyaluronidase can exhibit decomposition activity at pH 4.0 to pH 11.0. Preferably, it can be pH 9.0 (FIGS. 7A and 7B).

According to another embodiment of the present invention, the BSSK58 hyaluronidase can exhibit decomposition activity at about 25°C to about 60°C. Specifically, the hyaluronidase of the present invention can exhibit decomposition activity at about 30°C to about 55°C, about 40°C to about 50°C. Preferably, the temperature is about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, It can exhibit decomposition activity at about 49℃ or about 50℃. As the reaction temperature of hyaluronidase increased to 50℃, the decomposition activity of the enzyme increased, and the activity decreased rapidly at 55℃. Through this, it was confirmed that the optimal reaction temperature of BSSK58 hyaluronidase is 50℃ (Fig. 8a and Fig. 8b).

According to another embodiment of the present invention, the activity of the BSSK58 hyaluronidase was inhibited by Ag ²⁺ , Fe ²⁺ , and Cu ²⁺ . On the other hand, Ca ²⁺ , Ni ²⁺ , Ba ²⁺ , Mg ²⁺ , and EDTA promoted its activity (Table 2).

According to another embodiment of the present invention, the BSSK58 hyaluronidase can specifically decompose hyaluronic acid. Specifically, the enzyme was reacted with a polysaccharide substrate solution such as hyaluronic acid, chondroitin sulfate (derived from bovine trachea or shark cartilage), heparin sodium, alginate, xanthan, or agarose, and the degree of decomposition of the polysaccharide was compared.

As a result, it was confirmed that hyaluronic acid, chondroitin sulfate (derived from bovine trachea or shark cartilage), heparin sodium, alginate, and xanthan gum were decomposed. In particular, the BSSK58 hyaluronidase was confirmed to have strong decomposition ability for hyaluronic acid and chondroitin sulfate (Table 1). That is, the BSSK58 hyaluronidase can decompose chondroitin sulfate derived from bovine trachea or shark cartilage and reduce it to a low molecular weight substance (Table 1).

Enzyme production method and low molecular weight hyaluronic acid/chondroitin sulfate production method

Another aspect of the present invention provides a method for producing an enzyme, comprising the steps of culturing a Penicillium recidui BSSK58 strain and purifying the enzyme from the culture solution.

At this time, the above Penicillium lactamase BSSK58 strain is as described above. In addition, the enzyme refers to BSSK58 hyaluronidase, which is as described above.

Another aspect of the present invention provides a method for producing low molecular weight hyaluronic acid and its salts, comprising the step of treating high molecular weight hyaluronic acid with the enzyme.

The above high molecular weight hyaluronic acid can be produced by extraction from biological tissue or fermentation from microorganisms according to a conventional method, and can be a commercially available high molecular weight hyaluronic acid. The high molecular weight hyaluronic acid can have a size of 1,200 kDa to 2,600 kDa, and preferably 1,200 kDa to 2,200 kDa. However, it is not limited thereto.

In the present invention, hyaluronic acid may include hyaluronate. The "hyaluronate" is a salt form of hyaluronic acid, and is produced by removing lipids, proteins, nucleic acids, etc. from hyaluronic acid, so its molecular size is smaller than that of hyaluronic acid. Examples thereof 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 the BSSK58 hyaluronidase may be performed by treating high-molecular-weight hyaluronic acid with 35 units/mL of BSSK58 hyaluronidase at 35°C, 200 rpm, in a 100 mM phosphate buffer solution at pH 8.0 for 1 hour, but is not limited thereto.

In addition, the low molecular weight hyaluronic acid may be about 300 Da to about 100 kDa. Or, the low molecular weight hyaluronic acid may be about 380 Da to about 50,000 Da. Or, the low molecular weight hyaluronic acid may be about 380 Da to about 10,000 Da. Or, the low molecular weight hyaluronic acid may be about 380 Da to about 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 about 400 Da.

Another aspect of the present invention provides a method for producing low molecular weight chondroitin and its salts, comprising the step of treating chondroitin sulfate with the enzyme.

The above chondroitin sulfate is a polysaccharide containing a polyanion. D-glucosan (GlcA) and N-acetyl-D-galactosamine (GalNAc) are bonded by a β-1,3 glycosidic bond to form a disaccharide unit, which are then bonded to each other by a β-1,4 glycosidic bond, and a sulfate group is then introduced to another position during the biosynthetic process. Chondroitin sulfate is widely present in various animal cartilages and connective tissues. In addition, the chondroitin sulfate may be about 50 kDa to about 100 kDa, and preferably, about 10 kDa to about 50 kDa, but is not limited thereto.

Specifically, in the method for producing low-molecular-weight chondroitin sulfate, the treatment of the BSSK58 hyaluronidase may be performed by treating chondroitin sulfate with 35 units/mL of BSSK58 hyaluronidase at 35°C, 200 rpm, in a 100 mM phosphate buffer solution at pH 8.0 for 1 hour, but is not limited thereto.

In addition, the low-molecular-weight chondroitin sulfate may be about 300 Da to about 20 kDa. Or the low-molecular-weight chondroitin sulfate may be about 400 Da to about 10 kDa. Or the low-molecular-weight chondroitin sulfate may be about 500 Da to about 5000 Da. Preferably, the produced low-molecular-weight chondroitin sulfate may be about 800 Da to about 930 Da. In addition, the produced low-molecular-weight chondroitin sulfate may be about 370 Da to about 470 Da.

Specifically, in one embodiment of the present specification, it was confirmed that when BSSK58 hyaluronidase was treated under the above conditions, high molecular weight hyaluronic acid was decomposed into low molecular weight hyaluronic acid after 6 hours of reaction (Fig. 11).

In addition, in another embodiment of the present specification, it was confirmed that when BSSK58 hyaluronidase was treated under the above conditions, chondroitin sulfate (derived from bovine trachea or shark cartilage) was decomposed into low-molecular-weight chondroitin sulfate after 6 hours of reaction (Figs. 12 to 13).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Phenibacillus Recidui Strain isolation and identification

To isolate the enzyme (hyaluronidase) producing strain from soil sample, it was isolated using minimal medium. The composition of minimal medium was peptone 0.1%, NaCl 0.5%, KH ₂ PO ₄ 0.02%, KCl 0.02%, Na ₂ HPO ₄ -7H ₂ O 0.14%, sodium hyaluronate 0.5%, agar 2.0%, and then autoclaved and used.

To select a strain that decomposes hyaluronic acid, a soil sample diluted in a minimal medium was smeared, and the grown colonies were transplanted to the minimal medium and cultured at 35°C for 2 to 3 days. After that, 10% CPC (cetylpyridinium chloride) was added to the surface of the medium, and the medium was left at room temperature, and colonies forming a clear zone, as shown in Fig. 2, were first selected. After that, strains with excellent enzyme activity were secondarily selected and identified. Thereafter, the 16S rRNA base sequence of the identified strain was analyzed (Solgent Co., Ltd., Republic of Korea). As a result, it was identified as Peanibacillus residui as shown in Fig. 1, and named Peanibacillus residui BSSK58.

Example 2. Safety confirmation of Penebacillus recidui BSSK58 strain

To confirm the safety of the selected Penicillium recidui BSSK58 strain, it was grown in serum medium and the production of hemolytic toxin (hemolysin) was confirmed. As a result, it was confirmed that it is a non-pathogenic bacterium that does not produce hemolytic toxin (Fig. 3). In addition, it was confirmed through full genome sequencing whether the Penicillium recidui BSSK58 strain has a toxin gene. As a result of verification using Virulencefinder version 2.0, seven virulence genes were confirmed as "No hit found", so it could be considered a non-pathogenic strain (Fig. 4).

Example 3. Enzyme production conditions of Penicillium residui BSSK58 strain

The medium for the optimal production conditions of the Penicillium residuei BSSK58 strain is a medium containing 1.13% tryptone, 0.3% soytone, 0.57% peptone, 0.5% NaCl, 0.25% K ₂ HPO ₄ , and 0.5% hyaluronate. Growth was performed at a culture temperature of 25 to 50°C, the optimal production temperature was 35 to 45°C, the initial pH of the medium was 6.0 to 8.5, and the optimal pH was 7.5 to 8.0 (Fig. 5a and Fig. 5b). In addition, it was confirmed that the optimal culture time was 1 to 4 days, and the optimal culture time was 2 to 3 days.

Example 4. Purification of BSSK58 hyaluronidase

In Example 3, the culture solution of the Penicillium residue BSSK58 strain was treated with ammonium sulfate to 70% (saturation concentration) to precipitate the enzyme protein. The precipitated hyaluronidase was recovered by centrifugation, and the active fraction was first collected and dissolved in a 10 mM sodium phosphate buffer solution (pH 7.2). Thereafter, the enzyme solution was dialyzed with the same buffer solution at 5°C for 1 day, and then purified once using an anion exchange resin (Q-sepharose FF, GE healthcare, USA). The active fraction was first collected and dialyzed against a solution containing 40% (w/v) ammonium sulfate in a 10 mM sodium phosphate buffer solution (pH 7.2). Afterwards, the enzyme active fraction was collected using HIC (hydrophobic interaction chromatography), that is, a butyl sepharose FF (GE healthcare, USA) column, dialyzed, and finally gel filtration was performed to obtain a solution containing pure hyaluronidase.

Protein quantification was measured by the BCA method, and a commercially available kit (Thermo) was used, modifying the method provided by the manufacturer. BSA as a reference protein was diluted to 2 mg/mL to 15.6 μg/mL, and the analyte protein was appropriately diluted. 10 μL of the reference protein and the analyte protein were dispensed into a 96-well plate. Reagent A and reagent B of the protein analysis kit were mixed in a ratio of 50:1, and 200 μL was added to the plate where the protein was dispensed. After reaction at 37°C for 30 minutes, the absorbance was measured at 560 nm. A standard curve was drawn using the reference protein, and the concentration of the measured protein was calculated using a formula. The molecular weight of the finally purified hyaluronidase was measured by Native-PAGE electrophoresis, and the molecular weight of the enzyme was confirmed to be 161 kDa (Fig. 6).

Example 5. Amino acid sequence analysis of BSSK58 hyaluronidase

Among the total genomic DNA analysis data of the Penicillium residue BSSK58 strain, the amino acid sequence of hyaluronidase was confirmed by comparative analysis with the N-terminal 10 amino acid sequence ESPEPDNLLQ (SEQ ID NO: 4) obtained through Edman sequencing analysis of the protein isolated and purified in Example 4.

In addition, the full-length amino acid sequence of hyaluronidase was analyzed to confirm that it has the amino acid sequence of SEQ ID NO: 2. In addition, the analysis of amino acid sequence homology based on NCBI/BLAST software showed the following. The protein with the highest similarity to the amino acid sequence of SEQ ID NO: 2 had a similarity of 99.9% with Ig-like domain-containing protein, and the analysis of gene sequence homology based on NCBI/BLAST software showed the following. There was no homologous sequence to the nucleotide sequence of SEQ ID NO: 3, confirming that the gene encoding hyaluronidase having the nucleotide sequence of SEQ ID NO: 3 is a novel gene.

Example 6. Confirmation of optimal activity conditions for BSSK58 hyaluronidase

Example 6.1. Optimal pH of BSSK58 hyaluronidase

In order to investigate the optimal pH range of the enzyme for the hyaluronidase prepared in the above Example 4, a test method was conducted by preparing three 100 mM buffers, namely, sodium acetate buffer (pH 3 to pH 6), sodium phosphate buffer (pH 6 to pH 8), Tris-hydrochloric acid buffer (pH 7 to pH 9), and sodium carbonate buffer (pH 9 to pH 11), and then dissolving high molecular weight hyaluronic acid in these buffers to make 1% to prepare a substrate solution. The enzyme reaction was measured for enzyme activity in the same manner as in Experimental Example 1. As a result, it was confirmed that the active pH range was pH 4 to pH 11 and the optimal reaction activity pH range was pH 8 to pH 9 (Fig. 7a).

Example 6.2. Determination of the optimal pH buffer for BSSK58 hyaluronidase

In order to investigate the pH stability range of the hyaluronidase prepared in the above Example 4, a test method was conducted by preparing three 100 mM buffers, namely, sodium acetate buffer (pH 3 to pH 6), sodium phosphate buffer (pH 6 to pH 8), Tris-HCl buffer (pH 7 to pH 9), and sodium carbonate buffer (pH 9 to pH 11), and then dissolving 0.1 mg of freeze-dried hyaluronidase in 10 mL of each buffer. Then, after treatment in a refrigerator at 4°C for 24 hours, the enzyme reaction was measured for enzyme activity in the same manner as in Experimental Example 1. As a result, the pH range with stability was pH 4 to pH 11, and the buffer showing the highest stable pH was pH 9 of 100 mM sodium phosphate buffer (Fig. 7b).

Example 6.3. Optimal temperature of BSSK58 hyaluronidase

As a test method to investigate the optimal temperature range for the enzymatic reaction of hyaluronidase manufactured in the above Example 4, hyaluronidase was dissolved in a 100 mM sodium phosphate buffer and reacted at 25°C to 60°C, and the optimal temperature was 50°C (Fig. 8a).

Example 6.4. Enzyme thermal stability of BSSK58 hyaluronidase

In order to investigate the heat stability of the hyaluronidase manufactured in the above Example 4, the hyaluronidase was dissolved in a 100 mM sodium phosphate buffer, and then heat-treated at 50°C and 60°C for 1 to 5 hours. Then, the heat treatment was performed at 80°C for 10 to 50 minutes, and then cooled. The enzyme activity was measured using the same method as above. As a result, as shown in Fig. 8b, when the heat treatment was performed at 50°C for 1 hour, 60% of the enzyme activity remained, and when the heat treatment was performed at 60°C for 5 hours, about 40% of the enzyme activity remained, and at 80°C, almost all the activity was lost in 10 minutes. Therefore, when the enzyme reaction is stopped, if the heat treatment is performed at 80°C for 20 minutes, the enzyme activity is completely lost, so that the damage to the reaction product can be minimized.

Example 6.5. Analysis of enzyme reaction rate constant of hyaluronidase

The enzyme reaction was performed in the same manner as in Experimental Example 1 above, and the enzyme reaction rate constant was analyzed. The optimal substrate concentration for hyaluronidase enzyme is that there is no inhibition up to 1%, and the optimal substrate concentration for chondroitinase enzyme activity is that there is no inhibition up to 10%. Concentrations higher than that are difficult to dissolve due to high viscosity, and thus were omitted.

As a result of analyzing the enzyme reaction rate constant of hyaluronidase, the Km value was 28.38 mM/mL, indicating affinity for the substrate, and the Vm was 5.78 mM/mL/min, indicating the enzyme reaction rate (Figure 9).

In addition, in the case of bovine tracheal-derived chondroitin sulfate, the Km value was 4.93 mM/mL/min, indicating affinity for the substrate, and the Vm was 1.12 mM/mL/min, indicating enzymatic reaction rate, and in the case of shark cartilage-derived chondroitin sulfate, the Km value was 8.06 mM/mL/min, indicating affinity for the substrate, and the Vm was 6.78 mM/mL/min, indicating enzymatic reaction rate (Fig. 10a and Fig. 10b).

Experimental method 1. Hyaluronidase activity test (titer)

Experimental method 1.1. Function and principle

This test method is a method for measuring the activity of hyaluronidase obtained from a culture of Penicillium residue BSSK58 strain. The activity test was performed by hydrolyzing a high molecular weight hyaluronic acid (sodium hyaluronate) substrate at pH 8.0 and a temperature of 35℃ for 60 minutes, producing N-acetyl glucosamine, which was then reacted with p- DMAB ( p -dimethylaminobenzaldehyde) color developing solution and measuring the absorbance using the absorbance measurement method.

Experimental Method 1.2. Preparation of Test Solution

A certain amount of the sample was taken and diluted to contain 30 to 70 units per mL to prepare a test solution. The solution was then diluted using a buffer solution. The enzyme blank test solution was prepared by inactivating the sample by heating it in a water bath for 10 minutes.

Experimental method 1.3. Test manipulation

In advance, 2 mL of the substrate solution was precisely placed in a 15 mL tube and incubated in a 30°C constant temperature water bath 30 minutes before the test. 0.2 mL of the test solution and the enzyme blank test solution were each added to the prepared substrate solution, placed in a 35°C constant temperature water bath, and reacted at 100 rpm for exactly 60 minutes. Then, 0.44 mL of potassium tetraborate tetrahydrate solution was added and mixed. Then, it was heated in a water bath for 5 minutes and cooled in ice water for 5 minutes. 0.3 mL of the reaction solution was added to 1.5 mL of the p -DMAB color developing solution prepared in advance in a 2 mL tube, mixed well, and reacted in a 37°C constant temperature water bath for 20 minutes. After centrifuging the reaction solution at 10,000 × g for 10 minutes, the supernatant was collected and the absorbance was measured at 1 cm of liquid layer and 585 nm within 20 minutes, and the concentration (μmol) of N-acetylglucosamine was obtained from the calibration curve.

Experimental Method 1.4. Preparation of Calibration Curve

The pre-dried, constant-weight N-acetylglucosamine was precisely weighed and diluted in phosphate buffer to prepare 3,000 μmol. Using the prepared solution, standard solutions of 25, 50, 100, 200, and 300 μmol were respectively prepared and 2 mL each was precisely added to 15 mL tubes to prepare them. 0.4 mL of potassium tetraborate tetrahydrate solution was added to the prepared standard solution, mixed, heated in a water bath for 5 minutes, and cooled in ice water for 5 minutes. 0.3 mL of the reaction solution was added to 1.5 mL of the pre-prepared p -DMAB color developing reagent in a 2 mL tube, mixed well, and reacted in a constant-temperature water bath at 35°C for 20 minutes. The reaction solution was centrifuged at 10,000 × g for 10 minutes, the supernatant was collected, and the absorbance was measured at 1 cm of liquid layer and 585 nm within 20 minutes to create a calibration curve for the concentration (μmol) of N-acetylglucosamine.

The enzyme activity was calculated according to the following formula:

[Mathematical Formula 1]

Enzyme Units (μmol/mL) = (A x D)/(T x S x W)

A: Concentration of N-acetylglucosamine in the test solution obtained from the calibration curve (μmol)

At this time, the concentration of N-acetylglucosamine in the test solution is calculated by subtracting the absorbance of the enzyme blank test solution from the absorbance of the test solution.

D _: Dilution ratio of enzyme

T: Enzyme reaction time (min)

S: Amount of substrate used in the reaction (mL)

W: Amount of sample used in the reaction (mL)

Definition of activity: 1 Hyaluronidase unit (unit/mL) is the amount of enzyme that produces 1 μmol of N-acetylglucosamine per minute under the above test conditions.

At this time, the solution is as follows.

Substrate solution : 0.25 g of high molecular weight hyaluronic acid (sodium hyaluronate) was dissolved in 100 mL of phosphate buffer solution in a shaking constant temperature water bath at 30°C to prepare a substrate solution.

Phosphate buffer solution (100 mM sodium phosphate buffer (containing 150 mM NaCl), pH 8.0)

Potassium tetraborate tetrahydrate solution (0.8 M potassium tetraborate tetrahydrate, pH 10.0): Add 700 mL of distilled water to 244.4 g of potassium tetraborate tetrahydrate (Na ₂ B ₄ K ₂ O ₇ 4H ₂ O, MW: 305.5), heat to dissolve, and after 24 hours, when it is completely dissolved, add distilled water to make 1 L, and store in a dark place. Preheat in a constant temperature water bath at 35℃ before use.

p -DMAB color developing solution ( p -dimethylaminobenzaldehyde): 12.5 mL of 10 M HCl was mixed with 80 mL of acetic acid, and 10 g of p -dimethylaminobenzaldehyde was added to the solution to make 100 mL with acetic acid, and dissolved to prepare a stock solution of the color developing reagent. 90 mL of acetic acid was added to 10 mL of the stock solution of the color developing reagent and used as a p -DMAB color developing solution. The color developing solution was prepared on the day of the test and used in a light-shielded manner.

Example 7. Degradation ability of hyaluronidase on various polysaccharides

The following experiment was conducted to determine the catalytic decomposition ability of chondroitin sulfate (derived from shark cartilage, sigma), chondroitin sulfate (derived from bovine trachea, sigma), heparin sodium (sigma), alginate (sigma), xanthan (sigma), or agarose (sigma) using the hyaluronidase prepared in Example 4.

Chondroitin sulfate (from shark cartilage), chondroitin sulfate (from bovine trachea), heparin sodium, alginate, xanthan gum, and agarose were dissolved in 250 mM sodium phosphate buffer at 10 mg/mL to prepare a substrate solution. The hyaluronidase enzyme solution was prepared at 350 units/mL. The reaction was performed by adding 600 μL of distilled water, 200 μL of sodium phosphate buffer, and 100 μL of enzyme solution to 100 μL of the substrate solution, and stirring at 150 rpm at 35°C for 12 hours. To stop the enzyme reaction, the mixture was treated at 100°C for 10 minutes and then cooled in ice water for 10 minutes. Then, to remove the precipitate, centrifugation was performed at 15,000 g and 4°C for 15 minutes to obtain only the supernatant.

The results of measuring the absorbance using the reaction solution containing the enzyme solution inactivated at an absorbance of 232 nm as a control group are shown in Table 1. Chondroitin sulfate (derived from shark cartilage), chondroitin sulfate (derived from bovine trachea), sodium heparin, alginate, and xanthan gum were catalytically decomposed, but agarose was not decomposed.

substrate Decomposition by hyaluronidase High molecular weight hyaluronic acid +++ Chondroitin sulfate (shark cartilage, sigma) +++ Chondroitin sulfate (bovine trachea, sigma) +++ Heparin sodium (sigma) + Alginate (sigma) + Xanthan (sigma) ++ Agarose (sigma) -

Example 8. Effect of hyaluronidase on metal ions and chelating agents

The activity change of hyaluronidase manufactured in Example 4 in response to metal ions was observed. Hyaluronidase was dissolved in distilled water at a concentration of 35 μ/mL. The concentration of various metal ions was adjusted to 10 mM. The hyaluronidase solution and the 10 mM metal ion solution were mixed in a 1:1 ratio and stirred at 35°C for 1 hour at 150 rpm, and then the activity was measured using the hyaluronidase titer measurement method. As a result, as shown in Table 2, the enzyme activity was inhibited by silver ions, iron ions, and copper ions, whereas calcium ions, Enzyme activity was stimulated by nickel ions, barium ions, magnesium, and EDTA.

Sample Relative activity(%) Control group 100 Silver ion (Ag ²⁺ ) 6 Nickel ion (Ni ²⁺ ) 122 Iron ion (Fe2 ⁺ ) 46 Barium ion (Ba2 ⁺ ) 128 Calcium ion (Ca2 ⁺ ) 173 Zinc ion (Zn ²⁺ ) 85 Copper ion (Cu2 ⁺ ) 27 Manganese ion (Mn ²⁺ ) 100 Magnesium ion (Mg ²⁺ ) 128 Potassium ion (K ⁺ ) 95 Sodium ion (Na ⁺ ) 100 EDTA 116

Example 9. Preparation of low-molecular-weight hyaluronic acid and low-molecular-weight chondroitin sulfate

The hyaluronidase manufactured in Example 4 was used at 35 unit/mL, and the substrate concentration for the enzymatic reaction was 1% for high molecular weight hyaluronic acid and 10% for chondroitin sulfate, and 10% (v/v) of the enzyme solution was added to the substrate solution, and the reaction was performed at 35°C with stirring at 200 rpm. The decomposition process of high molecular weight hyaluronic acid and chondroitin sulfate according to the hyaluronidase reaction time is shown in Figs. 11 to 13. At this time, HPLC analysis was performed using a GPC (OHpak SB-804 HQ, SHodex, Japan) column at an ultraviolet absorbance of 210 nm, a 10 mM sodium phosphate buffer solution was used as the mobile phase, and the flow rate was measured at 0.7 mL/min.

Example 10. Expression of recombinant hyaluronidase

Example 10.1. Chromosomal DNA extraction of Penebacillus recidui strain BSSK58

The chromosomal DNA of the Penicillium residui BSSK58 strain in Example 1 was extracted using TRIzol ^TM (Sigma, USA). 1 mL of the culture solution of the Penicillium residui BSSK58 strain was centrifuged at 6,000 rpm for 5 minutes, the supernatant was removed, 1 mL of Trizol reagent was added, the suspension was reacted at room temperature for 5 minutes, 0.2 mL of chloroform was added, the mixture was reacted at room temperature for 2 minutes, centrifuged at 4°C and 12,000×g for 15 minutes, and the aqueous phase on the middle layer was removed. 0.3 mL of 100% ethanol was added, mixed, and the mixture was reacted at room temperature for 2 minutes, centrifuged at 4°C and 2,000×g for 5 minutes, and the supernatant was removed.

After that, the pellet was resuspended in 1 mL of 0.1 M sodium citrate (pH 8.5) containing 10% ethanol, reacted at room temperature for 30 minutes, centrifuged at 4°C, 2,000×g for 5 minutes, and the supernatant was removed. Again, the pellet was resuspended in 1 mL of 0.1 M sodium citrate (pH 8.5) containing 10% ethanol, reacted at room temperature for 30 minutes, centrifuged at 4°C, 2,000×g for 5 minutes, and the supernatant was removed. The pellet was resuspended in 1.5 mL of 75% ethanol, reacted at room temperature for 20 minutes, centrifuged at 4°C, 2,000×g for 5 minutes, and the supernatant was removed. The pellet was dried for 5 minutes, dissolved in 0.3 mL of 8 mM NaOH, centrifuged at 4°C, 12,000 × g for 10 minutes, and the supernatant was transferred to a new tube and neutralized with HEPES buffer.

Example 10.2. Amplification of hyaluronidase gene

The hyaluronidase gene was amplified using PCR (polymerase chain reaction). The DNA extracted in the above Example 10.1 was used as a template, and the primers in Table 3 were designed using the whole genome sequencing (WGS) results of the above Example 5. The designed primers were custom synthesized (Macrogen, Korea) and used. The PCR reaction was performed in a total of 50 μL, and 1 μL of template DNA, 2 μL each of 10 pmol/μL forward and reverse primers, 25 μL of 2Х PCR master mix (HighQu GmbH, Germany), and 20 μL of sterile distilled water were mixed for the reaction. The PCR reaction conditions were as follows: 95°C for 10 minutes, followed by 30 cycles of 95°C for 1 minute, 58°C for 1 minute, and 72°C for 4 minutes, and the reaction was terminated at 72°C for 10 minutes and then 4°C. As a result, the amplified gene after PCR was confirmed as a band corresponding to 3,000 bp or more (Fig. 14).

Primer Base sequence Forward SK58_Nhe_I_5 5'-TTT GCTAGC ATGATTCCAAGGAAATGGTTTTCCG-3' (SEQ ID NO: 5) Reverse SK58_Xho_I_3 5'-TTTT CTCGAG ATCACCGTTCTTCGGACCG-3' (SEQ ID NO: 6)

Example 10.3. Production of recombinant vector

In order to express the hyaluronidase gene obtained through PCR reaction in recombinant E. coli, a recombinant vector was constructed. The amplified hyaluronidase gene in Example 10.2 was TA cloned into pLUG-Prime ^® (Intron Bio, Korea), and then treated with Nhe I and Xho I restriction enzymes, and the hyaluronidase gene was isolated and purified. pET24a (Merck, Germany), an expression vector for E. coli, was also treated with the same restriction enzymes, and then isolated and purified. The hyaluronidase gene treated with the restriction enzyme was ligated using DNA ligase to construct the pET24a/BSSK58 recombinant vector (Fig. 15).

Example 10.4. Expression and purification of recombinant proteins

Recombinant hyaluronidase was expressed using BL21(DE3) E. coli cells. E. coli BL21(DE3) was transformed with the recombinant vector of Example 10.3 and cultured in LB (Luria-bertani) liquid medium containing kanamycin until the OD600 value was 0.6 or higher, and IPTG (Isopropyl-β-D-thio-Galactoside) was added to 1 mM. After culturing overnight at 30°C, the transformed E. coli cells were recovered by centrifugation and the supernatant was removed. The recombinant hyaluronidase protein expressed in the transformed E. coli cells was purified using affinity chromatography.

The harvested cells were resuspended in phosphate buffer solution (20 mM sodium phosphate, 500 mM NaCl, 20 mM Imidazole (pH 7.2)) and sonicated 20 times to disrupt the cells. The disrupted cells were centrifuged at 13,000 rpm for 20 minutes, the supernatant was recovered, and filtered through a 0.45 μM syringe filter (Sartorius, Germany). The cell disruption solution was applied to a HiScreen ^TM Ni FF column (GE Healthcare, UK) equilibrated with phosphate buffer solution to allow the recombinant protein to attach to the column, and the phosphate buffer solution was applied to remove unbound protein, and then the elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM Imidazole (pH 7.2)) was applied in a gradient to elute the recombinant hyaluronidase. As a result of checking the purified protein by SDS-PAGE, a protein of approximately 170 kDa was confirmed (Fig. 16).

Example 10.5. Measurement of hyaluronidase activity of recombinant proteins

The transformed E. coli cells of Example 10.4 above were recovered by centrifugation, suspended in phosphate buffered saline (PBS, pH 7.2), and the cells were disrupted. The disrupted cells were centrifuged at 13,000 rpm for 20 minutes, and the supernatant was recovered. The enzyme activity was tested by adding 0.1 mL of the supernatant to 1 mL of a 1% substrate high molecular weight hyaluronic acid solution, and reacting at 35°C for 1 hour at 150 rpm. After the reaction was completed, the enzyme activity was inactivated at 80°C for 20 minutes, and the absorbance was measured at 232 nm.

As a result, it was confirmed that the activity of the enzyme obtained from the transformed cells was higher than that of the supernatant (without hyaluronidase) from the non-transformed cells (Fig. 17). The experimental results confirmed that the hyaluronidase produced in E. coli exhibited the same activity as the hyaluronidase isolated from the BSSK58 strain.

Korea Research Institute of Bioscience and Biotechnology KCTC15643BP 20231016

Attach electronic file of sequence list

Claims (13)

Paenibacillus residui strain BSSK58 (Accession number: KCTC15643BP). In the first paragraph,
A Phenybacillus residue BSSK58 strain, characterized in that the 16s rRNA of the strain comprises sequence number 1. In the first paragraph,
A Penibacillus residue BSSK58 strain, characterized in that the strain is non-hemolytic. An enzyme comprising an amino acid represented by sequence number 2. In paragraph 4,
The above enzyme is an enzyme characterized in that it decomposes hyaluronic acid. In paragraph 4,
The above enzyme is an enzyme obtained from Penicillium recidui strain BSSK58 (accession number: KCTC15643BP). In paragraph 4,
An enzyme characterized in that the enzyme exhibits decomposition activity at 25°C to 60°C. In paragraph 4,
An enzyme characterized in that the enzyme exhibits decomposition activity at pH 4.0 to pH 11.0. In paragraph 4,
An enzyme characterized in that the decomposition activity of the enzyme is inhibited by any one selected from silver ions, iron ions, and copper ions. In paragraph 4,
The above enzyme is characterized in that it decomposes chondroitin sulfate, heparin sodium, alginate or xanthan. A step of culturing Penicillium residui strain BSSK58 (Accession number: KCTC15643BP); or
A method for producing an enzyme, comprising the step of purifying the enzyme of claim 4 from a culture solution. A method for producing low molecular weight hyaluronic acid and its salt, comprising the step of treating high molecular weight hyaluronic acid with the enzyme of claim 4. A method for producing low-molecular-weight chondroitin and its salts, comprising the step of treating high-molecular-weight chondroitin sulfate with the enzyme of claim 4.