KR20120131349A - Bacillus polyfermenticus I2000 strain having proteolysis ability and uses thereof - Google Patents

Abstract

PURPOSE: A Bacillus polyfermenticus I2000 with an ability of decomposing proteins is provided to enhance a thrombolytic activity and to be used in a health food or detergent. CONSTITUTION: A Bacillus polyfermenticus I2000(KCTC 11919BP) has an ability of decomposing proteins. A microorganism formulation for decomposing proteins contains the strain or a culture medium thereof as an active ingredient. A method for preparing the microorganism formulation comprises a step of culturing the strain. A probiotic composition contains the strain or a culture medium thereof as an active ingredient. A thrombolytic composition contains the strain or a culture medium thereof as an active ingredient. A detergent composition contains the strain or a culture medium thereof as an active ingredient.

Description

Bacillus polyfermenticus I2000 strain having proteolysis ability and uses approximately}

The present invention relates to Bacillus polyfermenticus I2000 having protein resolution, and more particularly, Bacillus polyfermenticus I2000 strain having the protein resolution, the protein comprising the strain or culture thereof as an active ingredient Degradation microbial preparation, a method for producing a protein degradation microbial preparation comprising the step of culturing the strain, a probiotic probiotic composition, a thrombi soluble composition and detergent composition comprising the strain or its culture as an active ingredient, It relates to a protease isolated from the strain and a gene encoding the protease.

Bacillus polyfermentus SCD ( Bacillus polyfermenticus SCD) is a bacterium isolated from Japan by Terakado in 1933. It is recently attracting attention as a probiotic probiotic for food. Bispan bacterium is an apolytic bacillus, which secretes 20 kinds of enzymes to recycle nutrients, and is an excellent source of vitamins B1, B2 and K. It enhances physiological activity in vivo, strengthens immune function and helps digestion.

In addition, it has recently been found to function to digest and absorb the three major nutrients of the body, carbohydrates, fats, proteins and fiber, and to inhibit proliferation by lysing pathogenic bacteria such as typhoid bacteria, paratyphoid bacteria, erythroid bacteria and cholera bacteria. In addition, in vitro and in vivo experiments have demonstrated the cholesterol-lowering and antioxidant effects of Bacillus polyfermentus SCD.

On the other hand, fibrinolytic enzyme (Fibrinolytic enzyme) is a kind of protease to dissolve fibrin (fibrin). Under normal conditions, fibrin production and degradation are properly regulated by thrombin and plasmin in vivo. However, when their balance is broken and blood clots adhere to and accumulate on blood vessel walls, blood vessels are narrowed, or further, they enter and close microvascular vessels such as cerebrovascular vessels along the bloodstream, causing various circulatory diseases. Since our body maintains metabolism by supplying nutrients and oxygen to each tissue through blood vessels, smooth flow of the circulatory system is very important for healthy living.

At present, thrombotic adult disease is increasing in Korea due to westernization of diet and excessive intake of fat. In Korea, the highest number of deaths due to circulatory diseases (hypertensive disease, cerebrovascular disease, ischemic heart disease) was 24.6%, followed by 21.7% of neoplasms (various cancers, leukemias, etc.) The death toll from accidents is reported to be 14.5%. In particular, the cerebrovascular disease of the circulatory disease is reported as the first cause of death in the 50s or older age bar This circulatory disease may be referred to as senile disease. Given the aging social structure and the fact that 60% of senile dementia patients are due to blood clots, the mortality rate from these circulatory diseases is unlikely to decrease. Therefore, the countermeasures against such circulatory diseases are an important problem that cannot be overlooked, and thus the importance and necessity for the development of thrombolytics have emerged.

Recently, researches on producing thrombolytic enzymes from traditional fermented foods have been conducted in Korea. Kim et al. Isolated thrombolytic enzymes from Bacillus sp. Strain CK 11-4 from Cheonggukjang, which is reported to be highly stable to heat and strong in thrombolytic ability. In addition, Kim et al. Reported the production and characterization of new thrombolytic enzymes using Bacillus sp. KA 38 isolated from salted fish.

The present invention is derived from the above requirements, the present invention is Bacillus polyfermenticus I2000 ( Bacillus) isolated from Meju polyfermenticus I2000) completed the present invention by confirming that fibrin-like protein resolution is much better than conventional Bacillus polyfermenticus SCN11 (Mo. et al., 2010 World J Microbiol Biotechnol 26: 1099-1105).

In order to solve the above problems, the present invention provides a Bacillus polyfermenticus I2000 strain (KCTC 11919BP) having a protein resolution.

The present invention also provides a microbial preparation for protein degradation comprising the strain or its culture as an active ingredient.

In addition, the present invention provides a method for producing a microbial preparation for protein degradation comprising the step of culturing the strain.

The present invention also provides a probiotic probiotic composition, a blood clotting composition and a detergent composition comprising the strain or its culture as an active ingredient.

The present invention also provides a protease isolated from the strain and a gene encoding the protease.

Since the Bacillus polypermanticus I2000 strain of the present invention is very excellent in protein resolution and thrombogenic solubility, the Bacillus polypermanticus I2000 strain and its culture medium are very useful for detergents or blood clot soluble health foods that require protein degradation. Can be.

1 is a result of observing the morphology of Bacillus polyfermentus I2000 strain using a scanning electron microscope.
Figure 2 shows the results of biochemically identifying the Bacillus polyperientus I2000 strain using the API 50CHB kit system.
Figure 3 is a result of comparing the proteolytic enzyme activity of Bacillus polypermanticus I2000 strain in skim milk medium (from the top left to the right, the first five strains control, the remaining seven strains Bacillus polypermanticus I2000).
4 is a result of comparing the fibrin degrading ability of Bacillus polyfermentus I2000 (1: Bacillus polyfermentus I2000, 2: Bacillus polypermanticus SCN11, 3: Bacillus certilis BD170).

In order to achieve the object of the present invention, the present invention has a protein resolution Bacillus polyfermenticus I2000 ( Bacillus polyfermenticus I2000) strain.

The Bacillus polypermanticus I2000 strain isolates Bacillus strains from Meju prepared in the Sunchang region of Jeollabuk-do, and confirmed the characteristics of Bacillus polypermanticus I2000 by identifying the characteristics of the Bacillus strain having excellent protease titer.

The Bacillus polypermanticus I2000 strain exhibited acid resistance and bile resistance, and was confirmed to be resistant to some antibiotics, and the fibrin resolution was measured by conventional Bacillus polypermanticus SCN11 (Mo. et al., 2010 World J Microbiol). Biotechnol 26: 1099-1105) was confirmed to be 6 times higher.

Bacillus polyfermenticus I2000 ( Bacillus polyfermenticus I2000) was deposited with the Korea Institute of Bioscience and Biotechnology Center on April 19, 2011 (Accession No .: KCTC 11919BP).

The present invention also provides a microbial preparation for protein degradation comprising the strain or its culture as an active ingredient.

The microbial preparation for protein degradation of the present invention can be prepared using Bacillus polyperientus I2000 strain as an active ingredient. The microbial preparation for protein degradation according to the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oily dispersion, paste, dust, scattering material or granules, but is not limited thereto.

In addition, the present invention provides a method for producing a microbial preparation for protein degradation comprising the step of culturing the strain. The method of culturing the strain of the present invention may be cultured according to methods commonly used in the art.

The present invention also provides a probiotic bioactive composition comprising the strain or its culture as an active ingredient. Since the strain of the present invention has a relatively high acid resistance of about 76% or more and about 23 to 28% of bile resistance, it has been confirmed to have very excellent properties as probiotic live bacteria.

In addition, the present invention provides a composition for dissolving a blood clot comprising the strain or its culture as an active ingredient.

Since the protease produced by the strain of the present invention is particularly excellent in thrombolytic ability, it can be provided as a thrombolytic health food using the same as an active ingredient. For example, the thrombolytic health food may be ingested for the prevention and treatment of circulatory diseases including coronary artery disease, thrombosis, cerebrovascular disease, hypertensive disease, ischemic heart disease, and the like.

When using the strain or its culture obtained in the step of culturing the strain of the present invention as a food additive, the strain or its culture may be added as it is, or used with other food or food ingredients, and may be appropriately used according to a conventional method. Can be. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment).

There is no particular limitation on the kind of the food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes.

In addition, the present invention provides a detergent composition comprising the strain or its culture as an active ingredient. In addition to proteolytic enzymes and lipolytic enzymes, laundry detergent enzymes have fiber degrading enzymes that remove the lint of clothing to remove the fading effect of lint, but enzymes widely used around the world are proteolytic enzymes. Since these enzymes are also added to detergents for automatic dishwashers and serve to increase their cleaning power, the proteolytic enzymes isolated from the strains of the present invention or cultures or cultures thereof can be used as detergents.

The present invention also provides a protease isolated from the strain of the present invention having an amino acid sequence of SEQ ID NO: 2 and a gene encoding the protease. The gene may preferably have a nucleotide sequence of SEQ ID NO: 1. As a result of determining the nucleotide sequence of the cloned gene from the strain of the present invention, as a serine protease-based proteolytic enzyme, the homology with more than 99% homology with the bacilli sutilis and the bactilisin (aprE) gene derived from Bacillus amyloliquefaciens It turned out to have a gene.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One. Bacillus Polyperientus I2000  ( B. polyfermenticus I2000 Separation and Microbiological Identification

The researchers to determine the characteristics of the Bacillus strains were isolated from the produced Meju, the protease activity of which excellent Bacillus (Bacillus) strain Jeollabukdo Doi area Bacillus poly flops mentee kusu (Bacillus polyfermenticus ) was identified as CBG and its characteristics are as follows.

As a result of gram staining, the isolated strains were Gram-positive bacteria and spores were produced at 60 hours of incubation. The form of the strain was bacillus, was observed in the size of about 0.8 × 3 ~ 4μm (Fig. 1).

In addition, recent results confirm the API 50CHB kit system that is used to identify the genus Bacillus in ATB computer database operated by the manufacturer, depending on the method used, standing Bacillus subtilis with 99.5% similarity (B. subtilis) or Bacillus amyl Lori query It was consistent with B. amyloliquefacience and was found to have lactose resolution (FIG. 2).

In order to determine 16S DNA nucleotide sequences, primers were prepared for 16S and 23S DNA, and the 16S-ITS-23S region was cloned by PCR, and the nucleotide sequence was determined. As a result, the Bacillus polypermanticus 16S DNA sequence ( Genebank registration number: AY149473.2) was found to be 100% consistent.

Thus, the strain isolated by the present inventors turned out to be Bacillus polyperientus and named Bacillus polyperientus I2000.

Example  2. Bacillus Polyperientus I2000 As live bacteria

The resistance of Bacillus polyperientus I2000 to artificial gastric juice and artificial bile acid, proteolytic enzyme activity, and antibiotic resistance were investigated. In the gastrointestinal resistance test, the survival rate in artificial gastric juice was more than 76% in the gastric juice adjusted to pH 2.5, and the survival rate in artificial gastric juice was very high. Was confirmed to have a survival rate of 23%, it was confirmed that the probiotic probiotic has very excellent characteristics.

① Gastric juice resistance (acid resistance) of Bacillus polyperientus strain

For gastric juice resistance experiments, artificial gastric fluid (autoclaved LB liquid medium adjusted to pH 4 with 1N HCl, cooled, and then added 1,000 units pepsin) was prepared, and the standard strain and Bacillus polyfermentus strain used in the experiment were 37 Strains grown at ℃ were used, 0.2 ml of strain solution was mixed with 9.8 ml of artificial gastric juice or LB medium, and then cultured at 37 ℃ for 1 hour. The number of surviving strains in artificial gastric juice was measured by the continuous dilution method. As a result, bacteria in Bacillus have endogenous spores, so the survival rate in gastric juice is generally higher than that of other bacteria. Cous was found to have a relatively high acid resistance with a survival rate of 76% or more (Table 1).

Gastric juice resistance (acid resistance) of Bacillus polyperientus strains After 1hr of artificial gastric juice
(Bacterial number / ml) After LB 1hr
(Bacterial number / ml)  Survival rate (%) B. polyfermenticus I2000 1.6 x 10 ⁶ 2.1 x 10 ⁶ 76 B. subtilis control 1.4 x 10 ⁶ 1.8 x 10 ⁶ 77

② Comparison of Bile Resistance of Bacillus polyperientus strain

For gastric juice resistance experiments, artificial bile solution (autoclaved LB liquid medium, ox-bile powder was added to 0.3% and sterilized again through a 0.25 μm membrane syringe filter) was prepared, and the standard strain and Bacillus poly were used in the experiment. The strain of Permanticus was used for 24 hours at 37 ° C., and 0.2 ml of the strain solution was mixed in 9.8 ml of artificial bile solution or LB medium and cultured at 37 ° C. for 1 hour. The number of viable strains in artificial bile was measured by the serial dilution method. As a result, bacteria in Bacillus have endogenous spores, so the viability of intestinal bile is higher than that of other bacteria. In the case of Permanticus, bile resistance was confirmed to show a survival rate of about 23-28% (Table 2).

Comparison of Bile Resistance of Bacillus polyperientus Strains After 4hr of artificial bile
(Bacterial number / ml) After LB 4hr
(Bacterial number / ml) Survival rate (%) B. polyfermenticus I2000 7.2 x 10 ⁴ 3.1 x 10 ⁵ 23 B. subtilis control 8.3 x 10 ⁴ 3.0 x 10 ⁵ 28

③ Comparison of antibiotic resistance of Bacillus polyperientus strain

In order to determine the resistance to various antibiotics, antibiotics such as Rifampicin, Tetracyclin, Kanamycin, and Ampicillin were used.The standard strains used in the experiment and Bacillus were used. Polyperiticus strains were inoculated with strains grown at 37 ° C. for 24 hours, and successive half-dilution methods starting at concentrations up to 200 μg / ml in LB liquid medium containing the respective antibiotics. As a result of determining the minimum inhibitory concentration (MIC) for each strain, the bacteria in Bacillus showed little resistance to rifampicin as a whole, and relatively high resistance to ampicillin.

Comparison of Antibiotic Resistance of Bacillus Polyfermentus Strains Strain B. polyfermenticus I2000 B. subtilis control Rifampicin (μg / ml) 0.3 0.3 Tetracyclin (μg / ml) 18.7 9.4 Kanamycin (µg / ml) 5 10 Ampicillin (μg / ml) 50 50

Example  3. Bacillus Polyperientus I2000 Of proteolytic enzymes in

① Comparison of Proteolytic Enzyme Performance of Bacillus Polyperientus I2000 Strain

In general, Bacillus strains have high protein resolution. As a result of comparing the relative activity of the protease of the Bacillus polypermanticus I2000 strain used in the present invention with the control strain in skim milk medium, the Bacillus polypermanticus I2000 strain showed higher activity than the other Bacillus strains. (FIG. 3).

② Comparison of Fibrin Degradation Capacity of Bacillus Polyfermentus I2000

In general, enzymes with high protein resolution are known to have excellent thrombolytic ability. In order to compare the fibrin degradability of the Bacillus polyfermentus I2000 strain used in the present invention with the existing Bacillus polyfermentus and Bacillus certilis strains, the same amount of culture solution was administered in an artificially synthesized fibrin medium to compare the activity. It was. As a result, the Bacillus polyperientus I2000 strain exhibited a high activity of fibrin decomposing ability 6 times or more compared with other Bacillus strains (Table 4 and Figure 4).

Fibrin Degradability Comparison of Bacillus Polyfermentus I2000 Strain Fibrin resolution B. polyfermenticus I2000 36 U / ml B. polyfermenticus SCN11 6.25 U / ml B. subtilis BD170 4.2 U / ml

③ Cloning and activity analysis of proteolytic enzymes from Bacillus polyperientus I2000 strain

In order to clone proteolytic enzymes similar to nattokinase, a representative protease of Bacillus polypermanticus strains, primers were prepared from a common site of protease, followed by PCR to isolate 400bp fragments. The whole gene was cloned using this as a probe. As a result of determining the nucleotide sequence of the cloned gene, it was found to be a gene that has 99% or more homology with the seric protease family of protease from Bacillus sertilis and Bacillus amyloliquefaciens. SEQ ID NO: 1 is the nucleotide sequence of the isolated protease, and SEQ ID NO: 2 is the amino acid sequence of the expected protein encoded by the gene. The homology between the expected protein and nattokinase, a representative antithrombotic enzyme of the Bacillus strain, was found to have 86% homology with nattokinase.

Korea Biotechnology Research Institute KCTC11919 20110419

<110> INTRA Inc. <120> Bacillus polyfermenticus I2000 strain having proteolysis ability          and uses <130> PN11057 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 2072 <212> DNA <213> Bacillus polyfermenticus <400> 1 gcatgccgtt atgaagagaa cggataagat catcagcata ctgaaaattg gtttcatctg 60 ttcctcctct ttcatttttc cgcaattata tcattgacaa tataagtatc aatgatattc 120 attatcatta tttttataaa atggtttcac agcttttctc cgtcaagaaa gccgaagact 180 gattcatgcc ggccgcttta tttcattccg ctttcgtttc catcagactt ctgcattcaa 240 caaaaggtga catttatcct gtttttgccg cagcttccaa aaatggaatc aaaccgttcg 300 acccagcaaa caagagagcg atcgcggctg tgtacaaata ctcatgtcct tccatcggtt 360 ttttccatta aaatttaaat atttcgggtt cctattaaac gaaagagaga tgatatacct 420 aaatagaaat aaaacaaact gaaaaaaatt gggtctacta aaatattatt ccatgctata 480 caattaatcc acagaataat ctgtctattg gttgttctgc aaatgaaaaa aaggagagga 540 taaagagtga gaggcaaaaa ggtatggatc agtttgctgt ttgctttagc gttaatcttt 600 acgatggcgt tcggcagcac gtctcctgcc caggcggcag ggaaatcaaa cggggaaaag 660 aaatacattg tcggatttaa acagacaatg agcacgatga gcgccgctaa gaaaaaagat 720 gtcatttctg aaaaaggcgg gaaagtgcaa aagcaattca aatatgtaga cgcagcttca 780 gctacattaa atgaaaaagc cgtaaaagag ctgaaaaaag accctagcgt cgcttacgtt 840 gaagaagatc acgttgcaca ggcgtacgcg cagtccgtgc cttacggcgt atcacagatt 900 aaagcccctg ctctgcactc tcaaggcttc accggatcaa atgttaaagt agcggttatc 960 gacagcggta tcgattcttc tcatcctgat ttaaaggtag caggcggggc cagcatggtt 1020 ccatctgaaa caaatccttt ccaagacaac aactctcacg gaactcacgt tgccggtaca 1080 gttgcggctc ttaataactc agtcggtgta ttaggcgttg cgccaagcgc atctctttac 1140 gctgtaaaag ttctcggcgc tgacggttcc ggccagtaca gctggatcat taacggaatt 1200 gagtgggcga tcgcaaacaa tatggacgtt attaacatga gcctcggcgg accttctggt 1260 tctgcagcgt taaaagcggc agttgacaaa gccgttgctt ccggcgtcgt agtcgtagcg 1320 gcagccggta acgaaggcac ttccggcggc tcaagcacag tgggctaccc tggtaaatac 1380 ccttctgtca ttgcggtagg ggcagttaac agcagcaacc aacgagcatc attctcaagc 1440 gtaggttctg agcttgatgt catggcacca ggcgtctcta tccaaagcac gcttcctgga 1500 aacaaatacg gcgcgtacaa tggtacgtca atggcatctc cgcacgttgc cggagcggct 1560 gctttgattc tttctaagca cccgaactgg acaaacactc aagtccgcag cagtttagaa 1620 aacaccacta caaaacttgg tgatgctttc tactacggaa aagggctgat caacgtacag 1680 gcggcagctc agtaaaacat aaaaaaaccg gcgtcgggca tggccccgcc ggttttttta 1740 ttatttttct tcctccgcat gttcaatccg ctccatgatc gacgggtggc tccctctgaa 1800 aattttgacg agaaacggcg ggttgacccg gctcagtccc gtatcggcca agtcctgaaa 1860 cgtctcaatc gccgcttccc ggtttccgat cagctcgatg ccgtagcggt cggcggcgtt 1920 ttcctgatac ctggagacgg cattcgtaat cggatcagaa gcaaaactga gaacggatat 1980 gaggagcaat aacagcggga gggcggccag atcttcaggc ccctttatat gaagcatcgg 2040 ccgaagaatt tggacagccg cttataaagc tt 2072 <210> 2 <211> 382 <212> PRT <213> Bacillus polyfermenticus <400> 2 Met Arg Gly Lys Lys Val Trp Ile Ser Leu Leu Phe Ala Leu Ala Leu   1 5 10 15 Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Pro Ala Gln Ala Ala Gly              20 25 30 Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gly Phe Lys Gln Thr Met          35 40 45 Ser Thr Met Ser Ala Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly      50 55 60 Gly Lys Val Gln Lys Gln Phe Lys Tyr Val Asp Ala Ala Ser Ala Thr  65 70 75 80 Leu Asn Glu Lys Ala Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala                  85 90 95 Tyr Val Glu Glu Asp His Val Ala Gln Ala Tyr Ala Gln Ser Val Pro             100 105 110 Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Phe         115 120 125 Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser     130 135 140 Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala Ser Met Val Pro Ser 145 150 155 160 Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His Gly Thr His Val Ala                 165 170 175 Gly Thr Val Ala Ala Leu Asn Asn Ser Val Gly Val Leu Gly Val Ala             180 185 190 Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser         195 200 205 Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn     210 215 220 Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala 225 230 235 240 Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Val Val Val                 245 250 255 Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Gly Ser Ser Thr Val             260 265 270 Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala Val Gly Ala Val Asn         275 280 285 Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val Gly Ser Glu Leu Asp     290 295 300 Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr Leu Pro Gly Asn Lys 305 310 315 320 Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly                 325 330 335 Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln             340 345 350 Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Ly Lys Leu Gly Asp Ala Phe         355 360 365 Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln     370 375 380

Claims (9)

Bacillus polyfermenticus I2000 strain (KCTC 11919BP) with protein resolution. A microbial preparation for protein degradation comprising the strain of claim 1 or a culture thereof as an active ingredient. Method for producing a microbial agent for protein degradation comprising the step of culturing the strain of claim 1. Probiotic bioactive composition comprising the strain of claim 1 or a culture thereof as an active ingredient. Thrombolysis soluble composition comprising the strain of claim 1 or a culture thereof as an active ingredient. Detergent composition comprising the strain of claim 1 or a culture thereof as an active ingredient. A protease isolated from the strain of claim 1 and having the amino acid sequence of SEQ ID NO. Gene encoding the protease of claim 7. The gene according to claim 8, which has a nucleotide sequence of SEQ ID NO: 1.

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