KR0127099B1 - Novel bacillus sp. and producing method of protease - Google Patents
Novel bacillus sp. and producing method of proteaseInfo
- Publication number
- KR0127099B1 KR0127099B1 KR1019940011237A KR19940011237A KR0127099B1 KR 0127099 B1 KR0127099 B1 KR 0127099B1 KR 1019940011237 A KR1019940011237 A KR 1019940011237A KR 19940011237 A KR19940011237 A KR 19940011237A KR 0127099 B1 KR0127099 B1 KR 0127099B1
- Authority
- KR
- South Korea
- Prior art keywords
- enzyme
- bacillus
- weight
- novel
- protease
- Prior art date
Links
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 31
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 title claims abstract description 12
- 239000004365 Protease Substances 0.000 title claims abstract description 11
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract 3
- 238000000034 method Methods 0.000 title claims description 3
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 abstract 2
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- 235000019796 monopotassium phosphate Nutrition 0.000 abstract 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 abstract 1
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 108010073771 Soybean Proteins Proteins 0.000 description 2
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- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
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- 108010011485 Aspartame Proteins 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229910020360 KCl—KOH Inorganic materials 0.000 description 1
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- 241000699670 Mus sp. Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
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- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
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- 238000006460 hydrolysis reaction Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
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- 231100000053 low toxicity Toxicity 0.000 description 1
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
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- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
- 229910052911 sodium silicate Inorganic materials 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/24—Non-sugar sweeteners
- A23V2250/248—Di-Peptides sweeteners
- A23V2250/2482—Aspartam
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Enzymes And Modification Thereof (AREA)
- Detergent Compositions (AREA)
Abstract
Description
제1도는 본 발명에 있어서 신규 단백질 가수분해효소의 최적 온도와 온도 안정성을 나타낸 그래프이고,1 is a graph showing the optimum temperature and temperature stability of the novel protease in the present invention,
제2도는 본 발명에 있어서 신규 단백질 가수분해효소의 최적 pH와 pH 안정성을 나타낸 그래프이다.2 is a graph showing the optimum pH and pH stability of the novel protease in the present invention.
본 발명은 신균주 바실러스 속(Bacillus sp.) NS-70(KCTC 8555P)와 이를 이용하여 발효 소요시간이 짧고, 특히 물에 친화력이 낮은 단백질 기질에서 잘 작용하여 식품 및 제약산업 등에 유용하게 사용할 수 있는 신규한 단백질 가수분해 효소를 제조하는 방법에 관한 것이다.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.
단백질 가수분해효소는 식품이 연화, 조미료 산업, 치이즈 숙성 및 아스파탐 제조 등의 식품공업, 효소세제, 환경처리제 등의 용도가 있는 환경공업, 인축용 소화제 등에 사용되는 제약공업 및 피혁가공공업 등 다방면의 산업에 사용되고 있을 뿐 아니라 단백질 가수분해효소는 효소전체 시장의 70%를 차지하는 큰 효소이기 때문에 이 분야에 대한 연구보고는 많은 편이다.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.
지금까지 단백질 가수분해효소를 생산하는 미생물은 바실러스(Bacillus) 속[Stark, W. 등 Eur, J. Biochem., 207, 781-791(1992)/Slomal. A. 등, J. Bacteriology., 172(2), 1024-1029(1990)], 써머스(Thermus)속 [Peek. K. 등, Eur. J. Biochem., 207, 1035-1044(1992)], 스트랩토마이세스(Srteptomyces)속 [Taguchi S. 등, Appl. Envir. Micorb., 59(12), 4338-4341(1993)], 피로코커스(Pyrococcus)속 [Blumentals, I. 등, Appl. Envir. Microb., 56(7), 1992-1998(1990)], 스태필로코커스(Staphylococcus)속 [Teufel, P. 등, Bacteriology, 175(13), 4218-4224(1993)] 등이 보고되고 있고, 단백질 가수분해 효소를 탐색하여 특허화한 예로는 스트랩토마이세스 속(일본특허울원 91-003169, 1991),락토바실로스 속(유럽특허출원 91-038622,1991),아스퍼질루스(Aspergillus)속 (일본특허출원 91-12787,1991) 플레우로투스(Pleurotus) 속(일본특허 91-180923, 1991), 호알칼리성 스트랩토마이세스 속(한국특허공고 제90-3924호, 1990) 등이 있다. 하지만, 보고되고 있는 단백질 가수분해효소는 측정 또는 탐색시 사용하는 효소의 기질이 스킴밀크(Skim milk), 카제인(Casein) 등 비교적 물에 잘 녹는 성질을 가지고 있는 물질을 선정함에 따라서, 세제용 단백질 분해효소 및 식품가공용 단백질 가수분해효소로 실제적으로 사용될때의 자연상태의 기질은 물에 잘 녹지 않은 상태로 존재하기 때문에 지금까지 개발된 단백질 가수분해효소는 물과 친화력이 낮은 자연상태로 존재하는 단백질 물질을 분해하는데는 한계를 가진다.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.
또한, 지금까지의 단백질 가수분해효소를 발효조를 이용해서 생산할 경우 보통 곰팡이의 경우 3~5일 세균의 경우는 30~48시간 소요되어서 비교적 생산단가가 높은 단점을 갖는다.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.
이런 종래 단백질 가수분해효소의 문제점을 극복하기 위해 본 발명자들은 물에 친화력이 낮은 단백질 기질에서 잘 작용하고, 발효하여 생산하는데 소요되는 시간이 짧은 신규기능을 갖은 단백질 가수분해효소를 선발하기 위해서 전국의 토양을 시료로 하여 열에 안정하고, 발효시간이 짧고, 물에 친화력이 낮은 단백질인 탈지대두분(Defatted Soybean Powder) 분해하는 능력이 뛰어난 단백질 가수분해효소를 생산하는 미생물을 발견하고 이 미생물이 생산하는 단백질 가수분해효소를 분리정제하여 특성을 조사한 결과, 상기의 기존 단백질 가수분해효소가 갖는 제반 문제점을 해결할 수 있어서 본 발명을 완성하였다.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.
본 발명은 신균주 바실러스 속 NS-70(KCTC 8555P)와 이를 이용하여 발효소요시간이 짧고, 물에 친화력이 낮은 단백질의 분해능력이 우수한 신규한 단백질 가수분해효소를 제조하는 방법을 제공하는데 그 목적이 있다.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.
이하, 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.
본 발명은 토양에서 분리된 신균주 바실러스 속(Bacillus sP.) NS-70(KCTC 8555P)에 관한 것이다.The present invention relates to a new strain Bacillus sP. NS-70 (KCTC 8555P) isolated from the soil.
또한, 본 발명은 폴리펩톤 1중량%, 효모 추출물 0.5중량%, 소고기 추출물 0.2중량%, 글리세로 0.2중량%, KH2PO40.2중량%, K2HPO40.2중량%, MgSO4·7H2O 0.01중량% 및 NaCl 0.3중량%가 포함된 배지를 pH6내지 8로 조정한 후 바실러스 속(Bacillus sp.) NS-70(KCTC 8555P)을 접종하고 55℃에서 6시간 배양한액을 종자로 하여 수용성 녹말 1중량%, 휘톤펩톤(P hytonepepton) 0.5중량%, K2HPO40.2중량%, KH2PO40.2중량%, MgSO4·7H2O 0.01중량% 및 NaCl 0.3중량%를 포함하는 배지에서 50℃에서 8~10시간 발효시켜 제조하는 것을 특징으로 하는 신규한 단백질 가수분해효소의 제조방법을 포함한다.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.
이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다.Referring to the present invention in more detail as follows.
본 발명은 토양에서 분리된 신균주 바실러스 속 NS-70(KCTC 8555P)와 이를 이용하여 제조한 단백질 가수분해효소 및 이의 제조방법에 관한 것으로서, 본 발명에서 선발된 신규 단백질 가수분해효소를 생산하는 신균주의 미생물학적 특성은 다음과 같다.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) 형태학적 특성1) Morphological characteristics
최적 배지조건에서 배양한 후 그램염색을 실시한 결과 전형적인 그램 양성균임을 확인할 수 있었고, 고체배지상에서 흰색 콜로니를 형성할 뿐 아니라, 콜로니를 형성하는 균체는 끈끈한 저밀상으로 뭉쳐서 존재 함을 알 수 있었다. 전자현미경을 이용해서 균체형태를 조사한 결과 간균(rod type)형태이었고, 포자를 가지고 있으며, 포자의 형태는 타원형(Ellipsoidal)으로 나타났다. 또한, 활동성에 있어서 배지상에서 머리카락 같은 생육(Hair-like Growth)을 보인다. 이와 같은 형태적 특성은 바실러스 속의 갖는 특성과 유사하다.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)배양학적 특성2) Culture Characteristics
배양학적 특성은 다음 표 1에 나타낸 바와 같이 스포란지움의 팽창(Sporangium swollen)이 일어나지 않고, 혐기적 상태에서도 어느정도 생육이 가능하고, 7%의 식염이 존재하는 배지에서 생육이 가능하고 생육 온도가 30~55℃에서 가능한 것으로 보아 이와 같은 특성도 바실러스 속이 갖고 있음을 알 수 있다.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 ℃.
[표1]분리된 미생물 바실러스 속 NS-70이 갖는 배양학적 특성Table 1 Culture Characteristics of Isolated Microorganism Bacillus genus NS-70
3)생리학적 특징3) physiological characteristics
분리된 미생물을 생리학적 특성은 다음 표 2에 나타난 바와 같이 일반적인 당이용성이 양호한 것으로 나타났고, 인돌(Indole)생산, 유레아(Urea) 분해능이 없고, 나이트레트(Nitrate)을 나이트리트(Nitrite)로 환원은 시키지만, 나아트리트를 질소로 환원은 시키지 못하는 것으로 나타났다. 이와 같은 생리학적 특성은 바실러스 리케니포미스(Bacillus licheniformis)가 갖는 특성과 아주 유사하다.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.
[표 2]분리된 미생물 바실러스 속 NS-70이 갖는 생리적 특성[Table 2] Physiological Characteristics of Isolated Microorganism Bacillus genus NS-70
4) 분리된 미생물의 세포벽 지방산 조성4) Cell Wall Fatty Acid Composition of Isolated Microorganisms
분리된 미생물의 세포벽을 분리하여 지방산의 유형별 분석을 한 결과 다음 표 3에 나타낸 바와 같이 나타났고, 이 결과를 균종류에 따라 세포벽 지방산의 프로파일(profile)이 준비된 미디시스템[Microbial Identification System, Clin. Microbiol. Rev. 5(3), 302-327(1992)]에서 조사한 결과 분리된 미생물은 바실러스 리케니포미스(Bacillus licheniformis)에 속하는 균주와 유사한 균임을 알앗다.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.
[표 3] 분리된 미생물 바실러스 속 NS-70의 세포벽 지방산 조성TABLE 3 Cell Wall Fatty Acid Composition of NS-70
5) 분리된 미생물의 동정 및 명명5) Identification and naming of isolated microorganisms
분리된 미생물을 형태학적, 배양학적, 생리학적 특성과 세포벽 지방산 성분의 분석결과 바실러스 리케니포미스(Bacillus licheniformis)에 속하는 균주와 유사한 것을 알았고, 상기한 바와 같이 본 발명에서 분리된 미생물은 물에 친화력이 낮은 단백질의 분해능이 큰 특성을 가지고 있으면서 호알칼리성 및 짧은 생산시간이 단백질 가수분해효소를 생산하는 신규한 특성을 가짐으로써 일반적으로 바실러스 리케니포미스가 가지는 특성과는 큰 차이가 있고 이에따라 본 발명자들은 본 발명의 신균주를 바실러스 리케니포미스(Bacillus lichemiformis)의 변종으로 판단하였고, 따라서 본 발명균주를 Badillus sp. NS-70이라 명명하고 1994년 2월 28일자로 한국과학기술연구원 부설 유전공학연구소의 유전자은행에 기탁하여 수탁번호 KCTC 8555P을 부여받았다.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.
또한 균주를 동정하기 위한 모든 실험방법과 동정기준은 버지스 매뉴얼[Peter H. A. sneath, Endospcre-forming Gram-positive Rods and Cocci, Bergey's Manual of systematic Bacteriology Vol. 2, 1104-1137(1986)]을 사용하였다.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).
본 발명에서 신규주 바실러스 NS-70(KCTDC 8555P)의 배양배지로는 폴리펩톤 1중량%, 효모 추출물 05.중량%, 소고기 추출물 0.2중량%,글리세롤 0.2중량%, KH2PO40.2중량%, K2HPO40.2중량%, MgSO4·7H2O 0.01중량% 및 NaCl 0.3중량%의 조성을 갖는 효소생산용 배지를 사용하여 50ℓ의 용량 발효조에서 공기유입량 1vvm. 교반속도 300rpm으로 50℃에서 8~10시간 발효시켜 신규한 단백질 가수분해효소를 제조한다.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.
이와 같이 제조된 본 발명의 신규한 단백질 가수분해효소는 후기 실시예에 구체적으로 나타내었으며 그 주요특성은 다음과 같다.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. 작용 : 단백질 가수분해1. Function: Protein Hydrolysis
2. 분자량 : 30,000 Dalton2. Molecular weight: 30,000 Dalton
3. 최적 pH : 123. Optimum pH: 12
4. 최적온도 : 65℃4. Optimum temperature: 65 ℃
5. 등전점 : 8.55. Isoelectric point: 8.5
또한, 상기에서 얻어진 본 발명의 신규주가 접종된 효소생산용 배양액을 원심분리한 후 배양 상등액을 30~80% 암모늄설페이트 처리 및 탈염과정을 거친 다음 DEAE-세파로스 컬럼 등을 사용하여 신규 단백질 가수분해효소를 순수 분리하여 그 특성을 조사하였다.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.
이와같이 본 발명에 의해 제조된 단백질 가수분해효소는 독성이 없고 탈지 대두분과 같은 물과 친화력이 낮은 단백질을 잘 분해하므로 콩단백질을 이용하여 독특한 맛을 내는 식품용 맛제제를 제조할 수 있고, 또한 최적 pH가 12이고, 특히 계면활성제에서 효소역가 잔존률이 놓아 세제 제조시에도 첨가하여 사용할 수 있다.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.
결국, 본 발명의 신규주 바실러스 속 NS-70(KCTC 8555P)에 의해 제조된 신규한 단백질 가수분해효소는 발효시간이 짧고 호알칼리성이며 물과 친화력이 낮은 단백질의 가수분해 능력이 우수하여 기존의 단백질 가수분해효소의 문제점을 해결할 수 있어 식품공업, 환경오염 또는 제약공업 등 다방면의 산업에 아주 유용하게 사용될 수 있다.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.
이하, 본 발명을 실시예에 의거 상세히 설명하면 다음과 같은 바, 본 발명의 실시예에 의해 한정되는 것은 아니다.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.
실시예 1Example 1
신균주 단백질 가수분해효소의 생산Production of Mycobacterial Proteinase
신규 바실러스 NS-70 균주의 효소생산배지로는 폴리펩톤(polypepton) 1중량%, 효모 추출물 0.5중량%, 소고기 추출물 0.2중량%, 글리세롤(Glycerol) 0.2중량%, KH2PO40.2중량%, K2HPO40.2중량%, MgSO4·7H2O 0.01중량% 및 NaCl 0.3중량%가 포함된 배지를 pH 7.2로 조정한 후 121℃ 15분간 멸균한 후 상기 신규 발명균주를 접종한 후 55℃에서 플라스크로 6시간 종배양한 액을 종자로 하여 수용성 녹말(Soluble starch) 1중량%, 휘톤펩톤(Phytonpepton) 0.5중량%, K2HPO40.2중량%, KH2PO40.2중량%, MgSO4·7H2O 0.01중량% 및 NaCl 0.3중량%의 조성을 갖는 효소생산용 배지를 사용하여 50ℓ의 용량 발효조(Fermentor)를 사용하여 공기유입량 1vvm, 교반속도 300rpm으로 50℃에서 8~10시간 발효하였을때, 최대 생산량 10,000~12,000IU/ml의 효소를 얻을 수 있었고, 이때 생산된 효소는 물에 친화력이 낮은 탈지대두분의 가수분해력이 뛰어났다.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.
실시예 2Example 2
신규 단백질 가수분해효소의 특성Characteristics of Novel Proteinases
상기 실시예 1에서 얻어진 신규 미생물의 배양액 12,000g을 15분간 원심분리한 후 배양상등액을 30~80% 암모늄설페이트 처리 및 탈염, DEAE-Sepharose CL-6B column, Sephacryl S-200 column, CM-Sepharose column, Phenyl Sepharose CL-4B column(이상, Sigma사 제품, 미국)을 사용하여 신규 단배질 가수분해효소를 순수 분리한 후 아래의 실험예에 따라서 신규 단백질 가수분해효소의 특성을 조사하였다.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.
효소의 활성측정은 10mM Glycine-KCl-KOH 완충액(pH 10.0)에 용해시킨 0.6% 함머스텐 카제인(Hammarsten casein)용액 3ml에 효소용액 0.5ml을 첨가하여 55℃에서 30분간 반응시킨 후 0.44M TCA 용액 3.2ml을 첨가하여 실온에서 20분간 방치함으로써 반응하지 않은 단백질을 침전시킨 후 화트만(Whatman) No.5 여과지로 여과한 후에 275mm에서 여과액의 흡광도를 측정하였다. 탈지 대두박을 사용할시는 함머스텐 카제인 대신 탈지 대두박을 사용하였고 TCA용액으로 침전시킨 후 12,000rpm에서 5분간 원심분리한 상등액 1ml 0.55M Na2CO32.5ml, 폴린시약(Folin reagent) 0.5ml을 넣고서 37℃에서 30분간 발색시킨 후 75mm에서 흡광도를 측정하였다. 효소활성 단위는 함머스텐 카제인을 사용했을시 275mm에서 분당 흡광도를 0.1만큼 변화시키는 효소의 량으로 하였다.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.
실험예 1Experimental Example 1
효소의 분자량 및 등전점(pl)의 결정Determination of the molecular weight and isoelectric point (pl) of the enzyme
순수 분리된 효소를 표준단백질과 함께 SDS-PAGE 전기영동을 행한 결과 분자량이 30,000Dalton 정도로 나타났으며, 등전점은 Isoelectric forcusing 장치(Phast systen, pharmacia)를 활용하여 역시 표준단백질을 비교하여 측정한 결과 등전점이 8.5임을 알 수 있었다.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.
실험예 2Experimental Example 2
효소의 특성시험Characteristic test of enzyme
순수 분리된 효소의 특성중 열특성을 조사한 결과 65℃에서 최적 온도임을 알 수 있어서 식품가공용 또는 세제용으로 사용하기 적합한 온도로 나타났고 열에 대한 안정성도 65℃에서 10분간 처리하였을때 82%이 잔존활성을 가지고 있었고, 10mM의 Cacl2첨가에 의해서 열안정성이 증가되는 것으로 나타났다(제1도). pH의 영향을 조사한 결과 최적 pH는 12로 나타나 강알칼리에서 활성이 높음을 알았고 pH 6~13의 높은 알칼리 범위에서 안정한 것으로 나타났다(제2도). pH의 측면에서 본 발명 효소는 강알칼리에 잘 적응하기 때문에 세제 및 식품용으로 사용하기 적합한 효소로 평가된다. 효소의 특성중 저해제의 영향을 알아보기위해 PMSF(Phenylmethylsulfonylfluride)에 조사한 결과, 대부분의 효소활성의 없어지는 것으로 보다 본 발명의 효소는 활성부위에 세린(Serine) 잔기를 가진 단백질 가수분해효소(Serine Protease)임을 알 수 있었다. 그리고 효소에 대한 금속이온의 영향을조사한 결과 Hg2+과 Cu2+에는 강력하게 효소역가가 약 1.7배가량 증가하는 특성을 보인 반면 에스디에스(SDS)을 1% 농도로 처리했을때는 약 92%의 효소역가를 보전하는 성질로 보아 세제용이나 알칼리 상태에서 식품의 분해에 적합한 효소임을 알았다.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.
실험예 3Experimental Example 3
신규 바실러스 속(bacillus sp.) NS-70의 병독성 실험Virulence Studies of the Novel Bacillus sp. NS-70
본 발명의 단백질 가수분해효소는 신규 바실러스 속(Badillus sp.) NS-70에 의해서 생산된 효소가 세제 또는 식품산업에서 산업적 응용성이 클 것으로 판단하여 직접적으로 이용시 인축에 병독성이 있는지를 판단하기 위해서 쥐를 이용하여 독성시험을 실시하였다. 신규 바실러스 속(Badillus sp.) NS-70을 상기 실시예 1의 방법에 따라 생산한 배양액 0.5ml씩 50마리 쥐의 복강내에 주사하고 14일간관찰하면서 생존률을 조사한 결과, 대조구로 처리한 대표적인 병원성균인 스태필로코커스 아우에우스(Staphylococcus asreus)의 경우는 투여한 후 6시간 이내 모두 죽었는데 비해서, 신규 바실러스 속(Badillus sp.) NS-70은 14일이 지나도 전혀 죽지않고 건강하게 생육하는 것으로 보아 본 발명 미생물은 급성 독성 및 병원성이 전혀 없는 것으로 판단되어 식품 및 환경등 산업적 효소로서 응용이 클것으로 판단한다.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.
제조예 2Preparation Example 2
신규 단배질 가수분해를 이용한 맛제제 제조Preparation of Flavor Preparations Using Novel Protein Hydrolysis
본 발명의 단백질 가수분해효소는 상기 실시예 3에서 얻어진 결과와 같이 독성이 없는 것으로 나타날 뿐 아니라 탈지대두분과 같은 물과 친화력이 낮은 단백질을 잘 분해하는 특성을 활용하여 콩단백질을 이용해서 독특한 맛성분을 개발할 수 있는 방법이 많을 것을 판단하였다. 탈지대두분을 10배의 물에 희석하고 50℃로 온도를 맞추고, 여기에 효모엑기스(Yeast Extract)을 1% 수준으로 첨가하고 본 발명의 효소를 50℃ 에서 1시간 처리한 후 포도당 2%를 첨가하여 100℃에서 30분간 증자한다. 증자한 액에 주정 8%, 소금 1%를 첨가한 후 스프레이 드라이(Spray Dry)한 고형분을 완성품으로 하여 관능검사를 한 결과 관능 검사원의 80%가 지미와 감칠맛이 있어 양호하다고 판단하였다.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.
제조예 2Preparation Example 2
세제용 효소제조Detergent enzyme production
본 발명의 단백질 가수 분해효소는 상기 실시예 2와 실험예 2에서의 결과와 같이 최적 pH가 12이고 특히, 계면활성제인 에스디에스(SDS) 및 트윈 20 및 80에서 효소역가 잔존률이 높은 것을 이용하여 세제용 효소로 개발하였다. 무인계(Non-phosphate) 효소세제로 미국에서 제안되는 헤미듀티(Heavy Duty)용 조성인 소디움라스(Sodium LAS) 15%, 탈로이스 설페이트(Tallowether sulfate) 8%, 소디움 시엠시(Sodium CMC) 0.5%, 소디움 실리케이트(Sodium silicate) 15%, 소디움 카본에이트(Sodium carbonate) 20%, 형광제 0.3%, 본 발명 효소 1.25% 및 소디움 설페이트(Sodium sulfate) 39.95%로 효소세제를 만든 후 일반 효소세제와 일반 세탁물로 세척력 및 세척시간을 비교한 결과 일반 효소세제량의 80% 사용시 동일한 세척력을 가지는 것으로 나타나서 세제용 효소로서 효과가 상당이 좋은 것으로 나타났다.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.
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KR100750659B1 (en) * | 2005-02-18 | 2007-08-20 | 인하대학교 산학협력단 | Detergent and oxidant-stable haloalkalophilic protease and a gene coding it |
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KR100465852B1 (en) * | 2002-06-29 | 2005-01-13 | 인하대학교 산학협력단 | Bacillus sp. I-52 which produces highly active alkaline protease |
KR100750659B1 (en) * | 2005-02-18 | 2007-08-20 | 인하대학교 산학협력단 | Detergent and oxidant-stable haloalkalophilic protease and a gene coding it |
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