KR101851946B1 - Bacillus subtilis Bs5C and a method for producing silk peptides using thereof - Google Patents

Abstract

The present invention relates to a novel Bacillus subtilis strain, and a protease produced from the strain. More specifically, the present invention relates to a novel Bacillus subtilis strain Bs5C (KCTC 18585P) isolated from cocoon, a method for mass-producing a protease from the strain, and a protease produced from the strain.

Description

Bacillus subtilis strain Bs5C (KCTC 18585P) and a method for producing silk peptide using the same (Bacillus subtilis Bs5C and a method for producing silk peptides using the same)

The present invention relates to a novel Bacillus subtilis strain and a protease produced from the strain. Specifically, the present invention relates to a novel Bacillus subtilis strain Bs5C (KCTC 18585P) isolated from a softened silkworm cocoon A method for mass-producing protease from the strain, and a protease produced from the strain. The strain is useful for the production of silk peptide by the enzymatic method by decomposing the silkworm cocoate by proteolytic enzyme produced from Bacillus subtilis Bs5C of the present invention and since the strain has high production ability of lipase and starch decomposing enzyme, For example, detergents, foods, feeds and the like.

Protease has various functions such as digestion, absorption and defense in vivo, and is divided into serine, cysteine, aspartic acid and metallo protease depending on the structure of the active site. In addition to treating human diseases such as thrombus dissolution, proteolytic enzymes are used as a component of detergent for clothes, as a component of a contact lens cleaner, as well as for modification of milk proteins, removal of rubber fibers from poultry, It is widely used for decomposition of feathers, improvement of feed, immersion of leather, hair removal, recovery of silver from x-ray film and regeneration of ultrafiltration membrane for concentration of protein solution.

Silk fibers made from silkworm cocoons have been extensively used in carpets, paintings and clothing. Recently, silk protein is similar to collagen, elastin, etc., and is used as a surgical suture or as a support in tissue engineering because of its strength against temperature, humidity and physical pressure.

Silk peptides that are poorly differentiated from silkworm cocoons have been steadily used to confirm the efficacy of natural silk peptide, which is a natural high-molecular-weight natural polymer substance made from 99% pure silkworm protein. Silk fibroin is known to increase the expression of osteoblast-forming genes such as alkaline phosphatase, collagen type-1 alpha-1, and transforming growth factor-beta1, and has been shown to be effective in diabetic and hypertensive as well as anti- .

However, about 25% of silkworm cocoons are made up of hydrophilic sericin proteins and about 75% are composed of hydrophobic fibroin proteins. Fibroin is a highly insoluble protein composed of glycine, alanine, serine, and more than 90% of total amino acids with a molecular weight of over 300,000. This protein structure forms a beta-sheet structure composed of hydrogen bonds with each other and is very stable. These chemical structures and supramolecular structures are not soluble in most solvents. Because of this feature, little research has been reported on the enzymatic degradation of fibroin.

Because it is not easy to enzymatically, it is generally necessary to decompose by strong acids or strong bases to make silk peptides. For example, silk fibroin peptides can be obtained by digesting fibroin with 6M HCl for 5 hours, neutralizing with NaOH, and desalting to remove it. Among the known enzymatic methods, fibroinase isolated from Bombyx mori is not suitable for commercial use of insoluble fibroin because its activity has been confirmed in liquid fibroin. In addition, it is known that Streptomyces has activity to greatly reduce silk. Even if it is cocoonase, it decomposes sericin and has no activity on fibroin decomposition. Then, Farah's reading (Variovorax paradoxus) had reported that a carbon and nitrogen source for the growth of the silk fibroin can be used for solids. Thus, the strain was able to degrade insoluble silk fibroin through proteolytic enzymes. However, this enzyme was very unstable, resulting in the loss of all of its activity after one day of storage at 4 ° C.

For example, Patent Publication No. 1019990074787 discloses an invention relating to the production of peptides obtained by digesting silk fibroin with proteolytic enzymes. The silk (silk) is enzymatically decomposed to obtain fibroin as a crude protein, which is then reacted with calcium chloride and ethanol and dialyzed A method of producing a powdered fibroin peptide by lyophilizing the protein by digesting it into flavorzyme and actinase, which are proteases, is disclosed in the obtained solution.

Although the chemical decomposition of cocoon cocoons is simple and its efficacy is known, the product produced by this method is called silk peptide. However, since it is actually produced as an amino acid, it is necessary to peptize the silk peptide to obtain its functionality. In addition, a purification process for removing the byproducts generated in the chemical process is required, and the waste water is generated during neutralization or desalination, thereby causing environmental pollution. The enzymatic degradation method to replace this is to prevent the generation of wastewater because the silk cocoon can be peptidized and neutralization or desalination is not required, and it has various advantages because it is eco-friendly. However, It is a trivial fact.

Accordingly, the present inventors have recognized the above problems and searched for various strains capable of degrading cocoon cocoons from cocoon cocoons, and found that among these strains, Bacillus subtilis (Generally Recognized As Safe) and Bacillus subtilis A strain producing proteolytic enzyme with degradative activity was isolated and identified. In addition, a method for mass production of proteolytic enzyme from the strain was established.

KR 1019990074787 A JP 2002125657 A JP 2005512567 T KR 1020060092671 A KR 1019940000114 A

 1. BioChip J., Vol. 8, 1 (2014), 1-7

The present invention provides a novel strain that simultaneously produces starcholytic enzymes and lipolytic enzymes, which are industrially valuable, as well as proteolytic enzymes.

It is another object of the present invention to provide a probiotic agent comprising the strain, a cell thereof, a culture solution or a fermented product.

It is another object of the present invention to provide a method for producing a silk peptide by producing an enzyme that decomposes silkworm cocoons at high efficiency, including fibroin by a proteolytic enzyme.

The present invention also provides a method for decomposing cocoon cocoons using the protease.

The present invention provides a strain of Bacillus subtilis Bs5C (Accession No. KCTC 18585P). The strain is derived from a cocoon and is characterized by producing a proteolytic enzyme having a cocoon decomposing activity. The silkworm cocoon repairing the silkworm, the silkworm silk, the silkworm, or a combination thereof, wherein the protein comprises fibroin or sericin.

Also, the strain of the present invention comprises the nucleotide sequence of SEQ ID NO: 1 and has an enzyme activity of any one of amylase, protease, and lipase.

The present invention provides, as yet another example, a probiotic comprising a Bacillus subtilis Bs5C strain (Accession No. KCTC 18585P), a bacterium thereof, a culture broth or a fermented product.

As another example, the present invention provides a method for producing a silk peptide from a cocoon using a protease produced by a strain of Bacillus subtilis Bs5C (Accession No. KCTC 18585P). Preferably, the proteolytic enzyme decomposes the cocoon at pH 5 to 9 and 40 ° C or lower.

The novel Bacillus subtilis Bs5C strain (KCTC 18585P) of the present invention is isolated from silkworm cocoons, and the proteolytic enzyme produced therefrom is excellent in cleavage activity of not only sericin but also fibroin of silkworm silkworm, so that the decomposition rate of silkworm cocoons is increased This makes it possible to mass-produce silk peptides by an enzymatic method.

Brief Description of the Drawings Fig. 1 shows Bacillus subtilis strains isolated from cocoon, and the degree of decomposition of cocoon by enzymes produced therefrom is shown.
FIG. 2 is a diagram showing the results of the analysis of the strain Bs5C having the best cocoon decomposing activity.
FIG. 3 is a diagram showing lipase activity, protease activity and amylase activity of Bacillus subtilis Bs5C strain. FIG.
FIG. 4 shows the growth curve and proteolytic enzyme activity of Bacillus subtilis Bs5C strain compared with Bacillus subtilis KCTC 3014, an industrial utilization strain for protein production.
5 (a) and 5 (b) show the results of comparing the cocoon-decomposing activity of the Bacillus subtilis Bs5C and KCTC 3014 cultures.
Fig. 6 shows the temperature-dependent activity and temperature stability of the proteolytic enzyme produced from Bacillus subtilis Bs5C.
Figure 7 shows the activity of the proteolytic enzyme produced from Bacillus subtilis Bs5C according to pH.
Fig. 8 shows the results of searching for the optimal temperature for the cocoon decomposition of the proteolytic enzyme produced from Bacillus subtilis Bs5C.
Fig. 9 shows the results of decomposition rate of silkworm cocoons according to the amount of proteolytic enzyme treatment produced from Bacillus subtilis Bs5C.
Fig. 10 shows the results of the cocoon decomposition rate under optimum conditions of the proteolytic enzyme produced from Bacillus subtilis Bs5C.
Fig. 11 shows the results of cocoon decomposing activity of purified proteolytic fractions of proteolytic enzymes.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, it should be understood that the present invention is not limited to the following embodiments, and various modifications and changes may be made by those skilled in the art without departing from the spirit and scope of the present invention.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

Example  1. Isolation and identification of strains producing protease from cocoon

To obtain strain isolating cocoon (silk), select a softened silkworm cocoon and add 0.08% Triton X-100 to 100 times the total weight, mix well, dilute, and apply to LB plate medium containing 5% skimmed milk And then cultured at 30 ° C. Colonies were isolated from each colony, and the 16S rRNA gene was obtained by PCR using 27F and 1492R, the universal primers of the 16S rRNA gene from each single colony. Six strains (Bs1C, Bs2C, Bs3C, Bs4C, Bs5C, and Bs6C), which were identified as GRAS strain Bacillus subtilis , were selected through sequencing analysis of the obtained 16S rRNA gene. These strains were cultivated in LB liquid medium supplemented with 0.5% (w / v) cocoon cocoon for 4 days, and then centrifuged to remove the cells and residual cocoon, and the supernatant was recovered. The supernatant was filtered and used as an enzyme solution. The enzyme was treated with 20% (v / v) enzyme solution in 2% (w / v) silkworm cocoons and reacted at 40 ° C for 3 days. It can be confirmed that the silkworm is aggregated in the reaction solution for cocoon repairing, and it can be confirmed that the silkworm is loosened under the condition that the silkworm decomposing activity is present. A strain with cocoon decomposing activity was selected as the degree of visible decomposition and named Bacillus subtilis Bs5C (Fig. 1).

Selected B. subtilis Bs5C deposited microorganism patent deposit (Accession No. KCTC 18585P) to the BRC at the Korea Biotechnology Research Institute. The 16S rRNA gene sequence of this strain and the schematic diagram drawn therefrom (see FIG. 2) are shown.

Example 2. Isolated strain Bacillus subtilis Bs5C  Physiological property analysis

Using the API 20NE and API ZYM Bacillus subtilis (B. subtilis) results confirmed the characteristics of Bs5C, was with Esculine hydrolase, gelatinase, b-galactosidase activity, were able to use glucose, maltose and the malate, the trisodium citrate Weakly. esterase, esterase lipase (C8), lucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohyd -rolase, and a-glucosidase (see Table 1 and Table 2).

B. subtilis using API 20NE (a) - (Table 1) and API ZYM (b) - (Table 2) sp. Physiological characteristics of Bs5C>

Conventional tests Strain Bs5C      Nitrate reduction Negative      Indole production Negative      Glucose fermentation Negative      Arginine dihydrolase Negative      Urease Negative      Esculin hydrolysis Positive      Gelatinase Positive      β-galactosidase Positive Assimilation tests Strain Bs5C      Glucose assimilation Positive      Arabinose assimilation Negative      Mannose assimilation Negative      Mannitol assimilation Positive      N-Acetyl-glucosamine assimilation Negative      D-Maltose assimilation Positive      Potassium gluconate assimilation Negative      Capric acid assimilation Negative      Adipic acid assimilation Negative      Malate assimilation Positive      Trisodium citrate assimilation Weak positive      Phenylacetic acid assimilation Negative

Enzyme assayed for Strain Bs5C      Alkaline phosphatase Negative      Esterase (C4) Positive      Esterase lipase (C8) Positive      Lipase (C14) Negative      Leucine arylamidase Positive      Valine arylamidase Negative      Cystine arylamidase Negative      Trypsin Negative    alpha-chymotrypsin Negative      Acid phosphatase Positive      Naphthol-AS-BI-phosphohydrolase Positive    alpha -galactosidase Negative    β-galactosidase Negative    β-glucuronidase Negative    α-glucosidase Positive    β-glucosidase Negative      N-acetyl-β-glucosaminidase Negative    alpha-mannosidase Negative    alpha-fucosidase Negative

As a result of the test using API 20NE and API ZYM, B. subtilis Bs5C was found to produce various enzymes. Therefore, a plate assay was performed to compare the activity of lipase, protease, and amylase, which are widely used in industry, with B. subtilis KCTC 3014, a commercial strain of the same species. For the activation of lipase and protease, each strain was inoculated on a medium supplemented with 1% tributyrin or 5% casein, and cultured at 30 ° C. For the activity of amylase, the strains were inoculated in the same manner to the culture medium supplemented with 1% starch. After incubation at 30 ° C, iodine was added to the medium to determine the distance of the transparent circle after dyeing (see FIG. 3). As a result, B. subtilis Bs5C strains were broader than wild - type strains KCTC 3014 in all mediums, indicating that the activity of lipase, protease and amylase enzymes was excellent.

Example  3. Bacillus subtilis Bs5C  Proteolytic Enzyme Activity by Growth Curve and Culture Time

In order to determine the optimum conditions for the proteolytic enzyme production, the growth curve of B. subtilis Bs5C and the proteolytic activity thereof were measured. The medium used for the culture was a LB medium as the separation medium and a modified medium as the LB medium. The composition of the basic medium was as follows.

Basic medium: 10 g of Peptone, 10 g of YE, 10 g of glucose, 0.5 g of MgSo4, 0.5 g of CaCl2, 2 g of K2HPO4, 0.5 g / l of KH2PO4

B. subtilis Bs5C and KCTC 3014, which were preincubated in LB medium, were inoculated into LB medium and basic medium at an initial inoculation concentration of 0.1 and cultured at 30 ° C with shaking at 180 rpm. OD values were measured by sampling at 12, 24, 36, 48, 60, 72, 84 and 96 hours after inoculation and proteolytic enzyme activity was measured from the culture supernatant. As a result, when B. subtilis B. subtilis Bs5C and KCTC 3014 were cultured in LB, the highest OD value was observed at 6.2 hours and 6.5 hours after 12 hours of culture and then decreased thereafter. When cultured in the basic medium, The highest OD value was obtained at 4.2 and 7.6 after 24 hours. However, the proteolytic activity of each protease was 8.7 U at Bs 5 C at 72 hours after cultivation and was higher in B. subtilis Bs5C (7.6 U in KCTC 3014) (see FIG. 4).

Example  4. Bacillus subtilis Bs5C  Comparison of cocoon decomposing activity

Bacillus subtilis (B. subtilis) strain of hayeoteumeuro Bs5C is confirmed that protease activity is higher than KCTC 3014 used as a control, and to compare the cocoon-degrading activity by a protease. The two strains were cultured for 3 days at 30 ° C with shaking at 180 rpm in the primary culture medium. The culture supernatant was recovered by filtration, treated with sterilized silkworm cocoons, and the decomposition rate was observed after 4 days. (Fig. 5 (a) and Fig. 5 (b)) under conditions in which the cultures of B. subtilis Bs5C visibly silkworms were treated with the KCTC 3014 culture medium. The decomposition rate of the cocoons remaining after the decomposition was recovered through Miracloth, freeze-dried as much as possible, and lyophilized and weighed. As a result, the degradation rate of the culture medium of KCTC 3014 of the control group was about 11.9% under the treatment condition, but the degradation rate of B. subtilis Bs5C was about 26.4% under the treatment condition, and the protease of B. subtilis Bs5C decomposed cocoon It is confirmed that the efficiency is high.

Example  5. B. subtilis Bs5C  When the strain is cultured Carbon source  Effect on protease production (enzyme activity)

To investigate the effect of carbon source in the culture medium on proteolytic enzyme production when the culture medium of B. subtilis Bs5C strain was used as the enzyme, glycerol, sodium citrate , sucrose, and fructose at 1% concentration. As a result, glycerol and sucrose, which showed higher proteolytic activity in the whole growth period, were selected compared to 1% glucose (see Table 3).

<Low molecular weight carbon source is Bacillus subtilis sp. Bs5C on Growth and Proteinase Activity>

Bs5C Glucose Glycerol Sodium Citrate Sucrose Fructose OD600 Protease
activity OD600 Protease
activity OD600 Protease
activity OD600 Protease
activity OD600 Protease
activity 0h 0.10 0.10 0.10 0.10 0.10 12h 3.68 1.68 10.89 3.18 2.96 24h 4.15 1.90 4.65 2.80 2.95 36h 3.95 6.11 2.05 6.51 3.75 6.11 3.25 6.42 2.70 6.37 48h 3.65 6.34 2.55 8.08 2.70 7.36 3.05 8.14 2.25 7.38 60h 3.35 7.78 4.15 8.74 2.70 7.68 3.00 8.64 2.85 7.42 72h 2.95 8.72 5.20 10.12 2.60 11.26 2.90 9.46 2.80 9.4 84h 3.20 6.5 4.10 7.1 2.25 6.48 3.25 7.48 3.30 6.9 96h 3.50 6.5 4.00 6.98 2.05 6 3.20 7.5 3.50 6.72

As a polymeric carbon source, we can use the pre-classification. In addition, 4% concentration of potato starch, corn starch and wheat starch were added to the basic medium, and the culture was performed under the same conditions as above. As a result, the proteolytic activity was highest at 9.5 U at 72 hours in medium supplemented with potato starch.

&Lt; Polymer carbon source is Bacillus subtilis sp. Bs5C on Growth and Proteinase Activity>

Bs5C Glucose Glucose
+ wheat starch Glucose + corn starch Glucose
+ potato starch OD600 Protease
activity OD600 Protease
activity OD600 Protease
activity OD600 Protease
activity 0h 0.10 0.10 0.10 0.10 12h 3.68 10.48 7.51 6.50 24h 4.15 11.95 8.55 6.90 36h 3.95 6.11 11.70 6.68 7.95 6.54 7.25 6.56 48h 3.65 6.34 9.85 7.82 6.45 7.42 7.35 8.2 60h 3.35 7.78 9.35 8.06 6.80 7.94 7.15 8.08 72h 2.95 8.72 8.55 8.76 6.85 7.8 6.35 9.52 84h 3.20 6.5 9.55 7.04 6.05 6.7 6.65 7.14 96h 3.50 6.5 9.50 6.76 5.80 6.42 5.40 6.48

It was confirmed that the proteolytic activity was increased to 31 U, 25 U, or 26 U until 120 hours after the addition of potato starch, which is a carbon source, to the medium containing glycerol, sucrose or two carbon sources.

Example  6. B. subtilis Bs5C  Culture of strain City organic matter  Effect on protease production (enzyme activity)

In order to investigate the effect of organic nitrite in the culture medium on the proteolytic enzyme production when the culture medium of B. subtilis Bs5C was used as an enzyme, a low molecular weight carbon source such as glycerol, sucrose or two carbon sources And the proteolytic activity was measured. Peptone, silk peptide, casein, and soybean flour were added to the medium at 4% concentration. As a result, 4% silk peptide as a nitrogen source showed the highest protease activity. In detail, at 120 hours after cultivation, the maximum proteolytic activity was 111.8 U at 4% silk peptide as a nitrogen source and 0.5% glycerol, sucrose and 4% potato starch as carbon sources, Glycerol and 4% potato starch were the next highest in the culture condition, but within the error range (Table 5). Therefore, thereafter, 1% glycerol, 4% potato starch, 4% silk peptide, 1% Peptone, 1% YE, 0.05% MgSo4, 0.05% CaCl2, 0.2% K2HPO4, 0.05% KH2PO4 as a medium composition for proteolytic enzyme production Respectively.

<The organic matter is Bacillus subtilis sp. Effect of Bs5C on protease activity>

Bs5C
BS (-glucose)
+ 4% potato starch) NONE PEPTONE SILK PEPTIDE SKIM MILK SOYBEAN FLOUR Glycerol Sucrose Glycerol
+ Sucrose Glycerol Sucrose Glycerol
+ Sucrose Glycerol Sucrose Glycerol
+ Sucrose Glycerol Sucrose Glycerol
+ Sucrose Glycerol Sucrose Glycerol
+ Sucrose 24h 5.1 5.6 5.6 12.4 12.2 12.2 9.8 10.0 10.2 5.5 8.2 8.0 9.1 8.9 8.6 48h 6.0 5.5 5.6 17.3 17.1 17.6 24.9 18.3 22.0 7.2 8.9 9.3 19.5 17.9 19.4 72h 12.9 11.9 11.5 36.7 36.2 35.1 32.5 26.9 31.5 15.3 15.9 17.6 25.5 25.8 28.3 96h 16.7 17.3 17.7 74.0 66.3 63.8 50.3 36.7 48.4 18.8 24.1 24.7 44.0 41.0 45.0 120h 30.5 25.0 26.0 85.8 60.3 78.0 99.5 77.8 111.8 31.0 41.5 45.0 93.0 87.3 86.0

Example  7. B. subtilis Bs5C  Detection of active conditions of proteolytic enzymes produced from strains

B. In order to examine the temperature stability of a protease produced from Bs5C subtilis strain, B. Subtilis Bs5C cultures were stored at 30, 40, 50, 60, and 70 ℃ for enzyme activity. At 30 ° C and 40 ° C, the enzyme activity was maintained at 90% or more based on the activity of the refrigerated storage enzyme until 1 hour, but then decreased to about 70% (see FIG. 6). The enzyme activity was maintained at 90% or more for 10 minutes at 50 ℃, but decreased rapidly after 30 minutes. At 60 ° C, the activity decreased sharply at 5 minutes, and after 20 minutes, the activity was less than 10% (FIG. 6). The results show that the proteolytic enzyme is stable at a temperature of 40 ° C or below at a constant level.

In order to confirm the pH condition with the highest enzyme activity, when the protein enzyme activity was measured after adjusting each reaction buffer to pH 5, 6, 7, 8, 9 and 10, the highest activity was observed at pH 8 (see FIG. 7) .

The optimal temperature and pH of the protease produced from B. subtilis Bs5C strain were confirmed, and the optimum temperature for the decomposition reaction of cocoon decomposition was determined from this culture. The culture solution used was pH 7.8, so it was used without any adjustment. 1 g of silkworm cocoon was treated with 20 ml of B. subtilis Bs5C culture to obtain final volume of 50 ml, followed by reaction at 30, 40, 50, 60 and 70 ℃ for 4 days. The reaction product was centrifuged to remove the aqueous layer, and the dry weight of the remaining cocoon was measured to calculate the decomposition rate. As a result, when the reaction was carried out at 40 ° C., the dry weight decreased by about 40%, showing the highest efficiency (see FIG. 8).

Example  8. B. subtilis Bs5C  Of the enzyme produced from the strain On the amount of treatment  Cocoon decomposition followed by

When the silkworm was over 1% (w / v) of the total reaction solution, it was immobilized in the culture solution. Sterile silkworm cocoa was prepared so as to have a concentration of 1% at 4, 10, 20, 30, 40, 50, 60, and 70% The reaction was carried out to measure the dry weight. As a result, the degradation rates of the cultures were 10% and 20%, respectively, and the decomposition rates were 19.6% and 18.8%, respectively. The decomposition rates were 30.0%, 30.9% and 30.9%, respectively, and the maximum degradation rate per culture treated concentration was 50% (see FIG. 9) .

Example  9. Bacillus subtilis sp . From Bs5C  Cocoon decomposition rate in the optimum condition of the produced proteolytic enzyme

As a result of the previous experiment, it was confirmed that the optimum condition for cocoon decomposition was 1% of cocoon cocoons and 50% of B. subtilis Bs5C culture, Cocoon digestion efficiency was investigated. The decomposition rate of cocoon was determined by centrifuging the reaction product treated with B. subtilis Bs5C culture to determine the remaining cocoon particles and measuring their dry weight. The results showed that B. subtilis After the treatment of the Bs5C culture medium, it was confirmed that the cocoons of the cocoons were loosened considerably, and the dry weight of the residue showed that the B. subtilis Bs5C culture broth had a decomposition rate of about 35% . In addition, since it is possible to reduce the raw material cost by decomposing the residue remaining after the decomposition, the remaining cocoon remaining after the decomposition is precipitated in the enzyme solution again from the cocoon under the same conditions to attempt the second reaction. As a result of measuring the weight, it was confirmed that about 45.7-45.9% was decomposed by the Bs5C culture medium compared to the initial cocoon input weight (see FIG. 10). This result shows that subsequent yields can be increased through repeated decomposition of the residue.

Example  10. Bacillus subtilis sp . Bs5C  Isolation and purification of cocoon decomposing enzyme from culture

Anion exchange (HiTrap Q HP) was used to purify proteolytic enzymes with cocoon decomposing activity from B. subtilis Bs5C. Washing buffer was 20mM Tris and 20mM Tris buffer containing 1M NaCl was used as an elution buffer to gradually increase the concentration to 0-100%. The separated proteins were separated by 5% of each fraction, and proteins isolated from each fraction were identified by SDS-PAGE. At the same time, each fraction was subjected to cocoon treatment and reacted at 40 ° C to determine whether the cocoin- (See FIG. 11). As a result, it was confirmed that the cocoons were decomposed in the fraction 5 - treated group. As a result, it was estimated that the fraction 5 had a protein having cocoon decomposing activity.

To further clarify the enzymes exhibiting cocoon decomposing activity, the protein bands appearing in fraction 5 were individually sectioned and then identified and characterized by mass spectrometry (Orbitrap Elite hybrid mass spectrometry (Thermo), Easy-nLC 1000 Respectively. The result was a protein band at about 33kDa appears in fraction 5 it appears that with a Mascot search results 32.7kDa of Neutral protease NprE (of Bacillus subtilis subsp . Amylosacchariticus) and protein sequence similarity of about 52%, with the result B. It was confirmed that the cleavage activity of subtilis Bs5C was due to proteolytic enzymes similar to Neutral protease NprE.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Accordingly, the scope of the present invention should not be construed as being limited to the above-described embodiments, but should be determined by equivalents to the appended claims, as well as the following claims.

Korea Biotechnology Research Institute KCTC18585P 20170713

<110> KRIBB <120> Bacillus subtilis Bs5C and a method for producing silk peptides          using <130> M17-4776 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1411 <212> RNA <213> Bacillus subtilis Bs5C <400> 1 ctcaggacga acgctggcgg cgtgcctaat acatgcaagt cgagcggaca gatgggagct 60 tgctccctga tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact 120 gggataactc cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaaa 180 cataaaaggt ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt 240 gaggtaacgg ctcaccaagg cgacgatgcg tagccgacct gagagggtga tcggccacac 300 tgggactgag acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg 360 gacgaaagtc tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc 420 tgttgttagg gaagaacaag taccgttcga atagggcggt accttgacgg tacctaacca 480 gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc 540 cggaattatt gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc 600 ggctcaaccg gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga 660 attccacgtg tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac 720 tctctggtct gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac 780 cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt 840 gctgcagcta acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa 900 aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga 960 agaaccttac caggtcttga catcctctga caatcctaga gataggacgt ccccttcggg 1020 ggcagagtga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag 1080 tcccgcaacg agcgcaaccc ttgatcttag ttgccagcat tcagttgggc actctaaggt 1140 gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg 1200 acctgggcta cacacgtgct acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa 1260 gccaatccca caaatctgtt ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc 1320 ggccttgtac 1380 acaccgcccg tcacaccacg agagtttgta a 1411

Claims (9)

Bacillus subtilis Bs5C strain (Accession No. KCTC 18585P), which produces a proteolytic enzyme having cocoon decomposing activity, wherein the protein comprises fibroin and sericin,
delete The method according to claim 1, wherein said strain is selected from the group consisting of cocoon cocoon, silkworm, subtilis or combinations thereof
delete The strain according to claim 1, wherein the strain is derived from a cocoon.
The strain according to claim 1, wherein the strain comprises a 16S rRNA sequence of SEQ ID NO: 1 and has an enzyme activity of any one of amylase, protease and lipase
A probiotic agent comprising the strain Bacillus subtilis Bs5C according to claim 1 (accession number KCTC 18585P), a cell, a culture or a fermentation product thereof.
A silkworm characterized by decomposing cocoon cocoons at pH 5 to 9 and 30 to 40 占 폚 using a protease produced by a strain of Bacillus subtilis Bs5C according to claim 1 (accession number KCTC 18585P) A method for producing a silk peptide from cocoons.




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