KR0127099B1 - Novel bacillus sp. and producing method of protease - Google Patents

Abstract

The preparation method for the novel Bacillus sp. cell line NS-70 (KCTC 8555P) and the novel protease by using the bacillus sp. NS-70 includes the steps of: adding bacillus sp. cell line NS-70 (KCTC 8555P) to a culture containing polypeptone, yeast extract, beef extract, glycerol, KH2PO4, K2HPO4, MgSO4,7H2O and NaCl at pH 6 to 8 and fermenting the culture placed at 55 degrees centigrade for 6 hours in a culture containing phytonpeptone, K2HPO4, KH2HPO4, MgSO4,7H2O and NaCl at 50 degrees centigrade for 8-10 hours.

Description

Bacillus sp. NS-70 (KCTC 8555P) and a new method for preparing protease using the same strain Bacillus sp.

1 is a graph showing the optimum temperature and temperature stability of the novel protease in the present invention,

2 is a graph showing the optimum pH and pH stability of the novel protease in the present invention.

The present invention Bacillus sp. (Bacillus sp.) NS-70 (KCTC 8555P) and using the short fermentation time, using a particularly low protein affinity in water, can be useful in food and pharmaceutical industries, etc. To a novel proteolytic enzyme.

Protein hydrolase has many aspects such as pharmaceutical industry and leather processing industry, which are used for food softening, seasoning industry, food industry such as cheese aging and aspartame production, environmental industry with enzymatic detergent, environmental treatment agent, etc. In addition to being used in the industry, protein hydrolase is a large enzyme that accounts for 70% of the total enzyme market.

To date, microorganisms producing proteolytic enzymes are of the genus Bacillus [Stark, W. et al. Eur, J. Biochem., 207, 781-791 (1992) / Slomal. A. et al., J. Bacteriology., 172 (2), 1024-1029 (1990)], Genus Thermus [Peek. K. et al., Eur. J. Biochem., 207, 1035-1044 (1992)], genus Srteptomyces [Taguchi S. et al., Appl. Envir. Micorb., 59 (12), 4338-4341 (1993)], Genus Pyrococcus [Blumentals, I. et al., Appl. Envir. Microb., 56 (7), 1992-1998 (1990)], Staphylococcus genus [Teufel, P. et al., Bacteriology, 175 (13), 4218-4224 (1993), etc. Examples of patented proteolytic enzymes include the genus Straptomys (Japanese Patent Application No. 91-003169, 1991), genus Lactobacillus (European Patent Application 91-038622,1991), genus Aspergillus ( Japanese Patent Application No. 91-12787,1991) Pleurotus genus (Japanese Patent Nos. 91-180923, 1991), alkalophilic straptomyces genus (Korean Patent Publication No. 90-3924, 1990) and the like. However, the reported protein hydrolase is a detergent protein as the substrate of the enzyme used for measurement or search is relatively well soluble in water such as skim milk and casein. The protein hydrolase developed so far is a protein that has a low affinity with water because its natural substrate is insoluble in water when it is practically used as a degrading enzyme and a protein hydrolase for food processing. There is a limit to decomposing matter.

In addition, the production of protein hydrolases so far by using a fermentation tank has a disadvantage that the production cost is relatively high because it takes 30 to 48 hours in the case of bacteria, usually 3-5 days for fungi.

In order to overcome the problems of the conventional protease, the present inventors have been working nationwide to select proteolytic enzymes having a novel function that works well on a low water affinity protein substrate and has a short time required for fermentation and production. Using soil as a sample, we found microorganisms that produce proteolytic enzymes that are excellent in their ability to degrade defatted soybean powder, a protein that is stable to heat, has a short fermentation time, and has a low affinity for water. As a result of separating and purifying the protease, the present invention has been completed since the problems of the existing protease can be solved.

The present invention provides a novel protein hydrolase which is excellent in the ability to decompose proteins having low fermentation time and low affinity for water using NS-70 (KCTC 8555P) of the genus Bacillus. There is this.

Hereinafter, the present invention will be described in detail.

The present invention relates to a new strain Bacillus sP. NS-70 (KCTC 8555P) isolated from the soil.

In addition, the present invention is polypeptone 1% by weight, yeast extract 0.5% by weight, beef extract 0.2% by weight, glycerol 0.2% by weight, KH ₂ PO ₄ 0.2% by weight, K ₂ HPO ₄ 0.2% by weight, MgSO ₄ · 7H ₂ After adjusting the medium containing 0.01% by weight of O and 0.3% by weight of NaCl to pH 6 to 8, inoculated with Bacillus sp. NS-70 (KCTC 8555P) and incubated for 6 hours at 55 ° C. Medium containing 1% by weight of water-soluble starch, 0.5% by weight of P hytonepepton, 0.2% by weight of K ₂ HPO _4, 0.2% by weight of KH ₂ PO ₄ , 0.01% by weight of MgSO ₄ .7H ₂ O and 0.3% by weight of NaCl. Including a method for producing a novel proteolytic enzyme, characterized in that prepared by fermentation at 50 ℃ for 8 to 10 hours.

Referring to the present invention in more detail as follows.

The present invention relates to a new strain Bacillus genus NS-70 (KCTC 8555P) isolated from the soil and a proteolytic enzyme prepared by using the same and a method for producing the same, a novel for producing a novel proteolytic enzyme selected from the present invention The microbiological characteristics of the strains are as follows.

1) Morphological characteristics

Gram staining after culturing under optimal media conditions confirmed that it was a typical Gram-positive bacterium, and not only white colonies were formed on the solid medium, but also the cells forming colonies were present in a sticky, low-density phase. As a result of examination of the cell morphology using an electron microscope, it was rod type, had spores, and the shape of spores was ellipsoidal. It also exhibits hair-like growth on the medium in activity. These morphological properties are similar to those of the genus Bacillus.

2) Culture Characteristics

As shown in Table 1, the swelling characteristics of Sporangium swollen do not occur, and growth is possible in an anaerobic state to some extent, and growth is possible in a medium containing 7% of salt. It can be seen that the Bacillus genus has such characteristics as it is possible at 30 ~ 55 ℃.

Table 1 Culture Characteristics of Isolated Microorganism Bacillus genus NS-70

3) physiological characteristics

The physiological characteristics of the isolated microorganisms were shown to be good in general sugar availability, as shown in Table 2, and there was no indole production, no urea resolution, and nitrate to nitrite. It was found to reduce but not to reduce nitrite to nitrogen. These physiological characteristics are very similar to those of Bacillus licheniformis.

[Table 2] Physiological Characteristics of Isolated Microorganism Bacillus genus NS-70

4) Cell Wall Fatty Acid Composition of Isolated Microorganisms

The cell wall of the isolated microorganism was separated and analyzed as a result of fatty acid type. As shown in Table 3, the result was a mid-system prepared with a profile of cell wall fatty acid according to the type of bacteria [Microbial Identification System, Clin. Microbiol. Rev. 5 (3), 302-327 (1992), isolated microorganisms are similar to the strain belonging to Bacillus licheniformis.

TABLE 3 Cell Wall Fatty Acid Composition of NS-70

5) Identification and naming of isolated microorganisms

As a result of analysis of morphological, culture, physiological characteristics and cell wall fatty acid components, the isolated microorganisms were found to be similar to strains belonging to Bacillus licheniformis. The low affinity of the protein has a high resolution, and the alkaline and short production time has a novel characteristic of producing protease, which is generally different from that of Bacillus rickenformis. The inventors have determined that the new strain of the present invention is a strain of Bacillus lichemiformis, and thus the strain of the present invention is Badillus sp. It was named NS-70 and was deposited on February 28, 1994 to the Gene Bank of the Institute of Genetic Engineering, affiliated with the Korea Institute of Science and Technology, and was given accession number KCTC 8555P.

In addition, all experimental methods and criteria for identifying strains are described in Peters H. A. sneath, Endospcre-forming Gram-positive Rods and Cocci, Bergey's Manual of systematic Bacteriology Vol. 2, 1104-1137 (1986).

As a culture medium of the new strain Bacillus NS-70 (KCTDC 8555P) in the present invention 1% by weight polypeptone, yeast extract 05.% by weight, beef extract 0.2% by weight, glycerol 0.2% by weight, KH ₂ PO ₄ 0.2% by weight, Air inflow 1 vvm in a 50 L capacity fermenter using an enzyme production medium having a composition of 0.2 wt% K ₂ HPO ₄ , 0.01 wt% MgSO ₄ · 7H ₂ O and 0.3 wt% NaCl. Fermentation is carried out at 50 ° C. for 8-10 hours at a stirring speed of 300 rpm to prepare a novel proteolytic enzyme.

The novel proteolytic enzyme of the present invention thus prepared is shown in detail in the later examples, and its main characteristics are as follows.

1. Function: Protein Hydrolysis

2. Molecular weight: 30,000 Dalton

3. Optimum pH: 12

4. Optimum temperature: 65 ℃

5. Isoelectric point: 8.5

In addition, after centrifuging the culture medium for enzyme production inoculated with the new strain of the present invention obtained above, the supernatant of the culture supernatant was subjected to 30-80% ammonium sulfate treatment and desalting, followed by a novel protein hydrolysis using a DEAE-Sepharose column. The enzyme was purified purely and its properties were investigated.

As described above, the proteolytic enzyme prepared by the present invention decomposes proteins with low toxicity and low affinity to water such as skim soybean flour, so that it is possible to prepare a food flavoring agent having a unique taste using soy protein. The pH is 12, in particular, the enzyme titer residual ratio in the surfactant can be added and used in the preparation of the detergent.

As a result, the novel proteolytic enzyme prepared by the new strain Bacillus genus NS-70 (KCTC 8555P) of the present invention has a short fermentation time, an alkaline property, and excellent hydrolysis ability of a protein having low affinity with water. It can solve the problem of hydrolase, so it can be very useful for various industries such as food industry, environmental pollution or pharmaceutical industry.

Hereinafter, the present invention will be described in detail with reference to the following Examples, but is not limited by the embodiments of the present invention.

Example 1

Production of Mycobacterial Proteinase

The enzyme production medium of the new Bacillus NS-70 strain was 1% by weight of polypepton, 0.5% by weight of yeast extract, 0.2% by weight of beef extract, 0.2% by weight of glycerol, 0.2% by weight of KH ₂ PO ₄ , K ₂ HPO ₄ 0.2% by weight, 0.01% by weight of MgSO ₄ · 7H ₂ O and 0.3% by weight of NaCl was adjusted to pH 7.2, sterilized for 15 minutes at 121 ℃ and then inoculated with the new invention strain at 55 ℃ Seed was cultured in a flask for 6 hours and seeded with 1% by weight of water-soluble starch, 0.5% by weight of Phytonpepton, 0.2% by weight of K ₂ HPO _4, 0.2% by weight of KH ₂ PO ₄ , MgSO _4. When fermentation was carried out at 50 ° C. at a temperature of 1vvm and a stirring speed of 300 rpm using a 50 L capacity fermenter using an enzyme production medium having a composition of 0.01% by weight of 7H ₂ O and 0.3% by weight of NaCl, A maximum yield of 10,000 to 12,000 IU / ml of enzyme was obtained, and the enzyme produced was made from the degreased soybean powder with low affinity for water. This can bunhaeryeok was excellent.

Example 2

Characteristics of Novel Proteinases

After centrifuging 12,000 g of the new microorganism culture solution obtained in Example 1 for 15 minutes, the culture supernatant was treated with 30-80% ammonium sulfate and desalted, DEAE-Sepharose CL-6B column, Sephacryl S-200 column, and CM-Sepharose column. Using pure Phenyl Sepharose CL-4B column (above, manufactured by Sigma, USA), pure protein was isolated and the properties of the novel protease were examined according to the following experimental example.

Enzyme activity was measured by adding 0.5 ml of enzyme solution to 3 ml of 0.6% Hammarsten casein solution dissolved in 10 mM Glycine-KCl-KOH buffer (pH 10.0) and reacting at 55 ° C for 30 minutes. 3.2 ml of the solution was added and left at room temperature for 20 minutes to precipitate unreacted protein, which was then filtered through Whatman No. 5 filter paper and the absorbance of the filtrate was measured at 275 mm. When using skim soybean meal, skim soybean meal was used instead of hammersten casein, and precipitated with TCA solution, 1 ml 0.55M Na ₂ CO ₃ 2.5ml supernatant centrifuged at 12,000 rpm for 5 minutes, and 0.5 ml of Folin reagent were added. After color development at 37 ° C. for 30 minutes, the absorbance was measured at 75 mm. The enzyme activity unit was the amount of enzyme that changes the absorbance per minute by 0.1 at 275 mm when using Hammersten casein.

Experimental Example 1

Determination of the molecular weight and isoelectric point (pl) of the enzyme

SDS-PAGE electrophoresis of purely isolated enzyme with standard protein showed molecular weight of about 30,000 Dalton, and isoelectric point was also measured using Isoelectric forcusing device (Phast systen, pharmacia). Was found to be 8.5.

Experimental Example 2

Characteristic test of enzyme

As a result of investigating the thermal properties of the purely separated enzyme, it was found that the optimum temperature at 65 ℃ was suitable for use in food processing or detergent, and the stability against heat remained 82% when treated at 65 ℃ for 10 minutes. It was active and appeared to increase thermal stability by adding 10 mM Cacl ₂ (FIG. 1). As a result of investigating the effect of pH, the optimum pH was found to be 12, indicating that the activity was high in strong alkali and stable in the high alkali range of pH 6-13 (FIG. 2). In terms of pH, the enzyme of the present invention is well adapted to strong alkalis, so it is evaluated as an enzyme suitable for use in detergents and foods. Investigation of PMSF (Phenylmethylsulfonylfluride) to investigate the effect of inhibitors in the properties of enzymes revealed that most of the enzyme activity was lost. The enzyme of the present invention is a protease (Serine Protease) having a serine residue in the active site. Was found. In addition, the effects of metal ions on the enzyme showed that the enzyme titer was strongly increased by about 1.7 times in Hg ²⁺ and Cu ²⁺ , whereas when SDS was treated at 1% concentration, it was about 92%. It was found that the enzyme was suitable for the decomposition of foods in detergent or alkali state by the nature of preserving the enzyme titer.

Experimental Example 3

Virulence Studies of the Novel Bacillus sp. NS-70

In order to determine whether the enzyme produced by the novel Baillus sp. NS-70 has a high industrial applicability in the detergent or food industry, the proteolytic enzyme of the present invention has a virulence to killing when used directly. Toxicity tests were performed using mice. Injecting a new Bacillus sp. NS-70 in 50ml rats intraperitoneally with 0.5ml of the culture medium produced according to the method of Example 1 and monitoring the survival rate for 14 days, the representative pathogenic bacteria treated with control Staphylococcus asreus died within 6 hours after administration, whereas the new Baillus sp. NS-70 did not die at all after 14 days of healthy growth. The microorganisms of the present invention are considered to have no acute toxicity and pathogenicity, and thus, the microorganisms of the invention are expected to be applied as industrial enzymes such as food and environment.

Preparation Example 2

Preparation of Flavor Preparations Using Novel Protein Hydrolysis

The proteolytic enzyme of the present invention is not only toxic as shown in the result obtained in Example 3, but also utilizes the characteristic of degrading a protein having low affinity with water such as skim soy flour, a unique flavor component using soy protein. We decided that there would be many ways to develop Dilute soybean meal in 10 times of water, adjust the temperature to 50 ° C, add yeast extract to 1% level, treat enzyme of the present invention at 50 ° C for 1 hour, and then add 2% glucose. Add and steam for 30 minutes at 100 ° C. After adding 8% alcohol and 1% salt to the cooked liquid, the sensory test was carried out using the solid product spray-dried (Spray Dry) as a finished product. As a result, it was judged that 80% of the sensory inspectors had a good taste with Jimmy.

Preparation Example 2

Detergent enzyme production

The proteolytic enzyme of the present invention uses an optimum pH of 12 as shown in Example 2 and Experimental Example 2, and particularly, a high enzyme titer retention rate in surfactants SDS and Tween 20 and 80. Was developed as an enzyme for detergent. 15% Sodium LAS, 8% Tallowether sulfate, Sodium CMC 0.5, a composition for heavy duty proposed in the US as a non-phosphate enzyme cleaner %, Sodium silicate 15%, Sodium carbonate 20%, Fluorescent 0.3%, Enzyme detergent of the present invention 1.25% and Sodium sulfate (39.95%) after making the enzyme detergent and general enzyme detergent As a result of comparing washing power and washing time with general laundry, the same washing power was found when 80% of the amount of general enzyme detergent was used.

Claims (5)

Bacillus sp. NS-70 (KCTC 8555P) isolated from soil. Bacillus sp. NS after adjusting the medium containing polypeptone, yeast extract, beef extract, glycerol, KH ₂ PO ₄ , K ₂ HPO ₄ , MgSO ₄ · 7H ₂ O and NaCl to pH 6-8 Water-soluble starch, Phytonpe-pton, K ₂ HPO ₄ , KH ₂ PO ₄ , MgSO ₄ · 7H ₂ O and NaCl were inoculated with -70 (KCTC 8555P) and incubated for 6 hours at 55 ° C. Method for producing a novel proteolytic enzyme, characterized in that prepared by fermentation at 50 ℃ 8 ~ 10 hours in a medium containing. A novel proteolytic enzyme prepared by the method of claim 2 having the following properties. Molecular Weight: 30,000 Dalton Optimal pH: 12 Optimum temperature: 65 ℃ Isoelectric point: 8.5 Use of the proteolytic enzyme of claim 3 in the preparation of a taste preparation for food. Use of the protease of claim 3 in the manufacture of detergents.