CN115851509B - Bacillus subtilis strain for producing protease in distiller's yeast and application thereof - Google Patents
Bacillus subtilis strain for producing protease in distiller's yeast and application thereof Download PDFInfo
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- CN115851509B CN115851509B CN202211259384.XA CN202211259384A CN115851509B CN 115851509 B CN115851509 B CN 115851509B CN 202211259384 A CN202211259384 A CN 202211259384A CN 115851509 B CN115851509 B CN 115851509B
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- 238000012113 quantitative test Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000005932 reductive alkylation reaction Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229960002668 sodium chloride Drugs 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229930195727 α-lactose Natural products 0.000 description 1
Abstract
The invention discloses a bacillus subtilis strain for producing protease in distiller's yeast and application thereof. The bacillus subtilis (Bacillus subtilis) JQ-2 provided by the invention has a preservation registration number of CGMCC No.25660. The invention also protects application of the bacillus subtilis JQ-2 in preparation of protease and/or chymosin. The invention also provides a method for preparing protease, which comprises the following steps: fermenting and culturing bacillus subtilis JQ-2 to obtain fermentation liquor. The method further comprises the steps of: purifying the fermentation broth to obtain the JQ-2 protease. The invention also provides a method for preparing the hydrolyzed peptide, which comprises the following steps: sodium caseinate is used as a substrate, and JQ-2 protease is used for treatment to obtain the hydrolyzed peptide. The invention has application and popularization value for protease related industries.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a bacillus subtilis strain for producing protease in distiller's yeast and application thereof.
Background
Protease is the most important industrial enzyme preparation, can catalyze the hydrolysis of proteins and polypeptides, and is widely used in animal viscera, plant stems and leaves, fruits and microorganisms. Proteases are used in large amounts in cheese production, meat tenderization and vegetable protein modification.
The dehairing and softening of leather industry has largely utilized protease, which saves time and improves labor sanitation conditions. The protease can also be used for silk degumming, meat tenderization and wine clarification. Clinically, it can be used for treating dyspepsia with pepsin, bronchitis with acid protease, vasculitis with dread protease, and cleaning suppurative wound and intrathoracic serosal adhesion with trypsin and chymotrypsin. The enzyme-added washing powder is a new product in the detergent, contains alkaline protease and can remove blood stains and protein dirt on clothes.
Disclosure of Invention
The invention aims to provide a bacillus subtilis strain for producing protease in distiller's yeast and application thereof.
The bacillus subtilis (Bacillus subtilis) JQ-2 provided by the invention is preserved in China general microbiological culture collection center (CGMCC) of China Committee for culture Collection of microorganisms (CGMCC) on the 09 th month 07 of 2022, and has an address of CGMCC No.25660, namely, the institute of microbiological study, national academy of sciences, north Chen, west Lu No. 1, of the Korean region of Beijing.
The invention also protects application of the bacillus subtilis JQ-2 in preparation of protease and/or chymosin.
In the application, the protease and/or chymosin are obtained by fermenting and culturing bacillus subtilis JQ-2.
The fermentation culture of bacillus subtilis JQ-2 comprises the following steps: bacillus subtilis JQ-2 was inoculated into a fermentation medium and cultured with shaking at 30℃and 180r/min for 24 hours.
The fermentation culture of bacillus subtilis JQ-2 comprises the following steps: the bacillus subtilis JQ-2 seed solution is inoculated to a fermentation culture medium with an inoculum size of 5 percent, and is subjected to shaking culture for 24 hours at 30 ℃ and 180 r/min.
The fermentation culture of bacillus subtilis JQ-2 comprises the following steps: the bacillus subtilis JQ-2 seed solution is inoculated to a fermentation culture medium (the liquid loading amount of the culture medium is 20%) with an inoculum size of 5%, and is subjected to shaking culture at 30 ℃ and 180r/min for 24 hours.
Bacillus subtilisOD of bacterial JQ-2 seed solution 600nm The value was 1.3.
The preparation method of the bacillus subtilis JQ-2 seed liquid specifically comprises the following steps: inoculating bacillus subtilis JQ-2 into liquid LB culture medium, shake culturing at 30deg.C and 120r/min to OD 600nm The value was 1.3, and a seed solution was obtained.
The invention also protects the fermentation broth of the bacillus subtilis JQ-2.
The preparation method of the fermentation broth comprises the following steps: bacillus subtilis JQ-2 was inoculated into a fermentation medium and cultured with shaking at 30℃and 180r/min for 24 hours.
The preparation method of the fermentation broth comprises the following steps: the bacillus subtilis JQ-2 seed solution is inoculated to a fermentation culture medium with an inoculum size of 5 percent, and is subjected to shaking culture for 24 hours at 30 ℃ and 180 r/min.
The preparation method of the fermentation broth comprises the following steps: the bacillus subtilis JQ-2 seed solution is inoculated to a fermentation culture medium (the liquid loading amount of the culture medium is 20%) with an inoculum size of 5%, and is subjected to shaking culture at 30 ℃ and 180r/min for 24 hours.
OD of Bacillus subtilis JQ-2 seed solution 600nm The value was 1.3.
The preparation method of the bacillus subtilis JQ-2 seed liquid specifically comprises the following steps: inoculating bacillus subtilis JQ-2 into liquid LB culture medium, shake culturing at 30deg.C and 120r/min to OD 600nm The value was 1.3, and a seed solution was obtained.
The invention also protects the application of the fermentation broth in the preparation of protease and/or chymosin.
The invention also protects the use of the fermentation broth as protease and/or chymosin.
The invention also provides a method for preparing protease, which comprises the following steps: fermenting and culturing bacillus subtilis JQ-2 to obtain fermentation liquor.
The fermentation culture of bacillus subtilis JQ-2 comprises the following steps: bacillus subtilis JQ-2 was inoculated into a fermentation medium and cultured with shaking at 30℃and 180r/min for 24 hours.
The fermentation culture of bacillus subtilis JQ-2 comprises the following steps: the bacillus subtilis JQ-2 seed solution is inoculated to a fermentation culture medium with an inoculum size of 5 percent, and is subjected to shaking culture for 24 hours at 30 ℃ and 180 r/min.
The fermentation culture of bacillus subtilis JQ-2 comprises the following steps: the bacillus subtilis JQ-2 seed solution is inoculated to a fermentation culture medium (the liquid loading amount of the culture medium is 20%) with an inoculum size of 5%, and is subjected to shaking culture at 30 ℃ and 180r/min for 24 hours.
OD of Bacillus subtilis JQ-2 seed solution 600nm The value was 1.3.
The preparation method of the bacillus subtilis JQ-2 seed liquid specifically comprises the following steps: inoculating bacillus subtilis JQ-2 into liquid LB culture medium, shake culturing at 30deg.C and 120r/min to OD 600nm The value was 1.3, and a seed solution was obtained.
The method further comprises the steps of: purifying the fermentation broth to obtain the protease.
The purification method sequentially comprises the following steps: ammonium sulfate precipitation and anion exchange column purification.
The ammonium sulfate precipitate was a 70% saturation ammonium sulfate precipitate.
The ammonium sulfate precipitate is 70% saturated ammonium sulfate precipitate at 4deg.C.
The method for precipitating the ammonium sulfate comprises the following steps: ammonium sulfate was added to the fermentation broth to 70% saturation, followed by magnetic stirring at 4℃for 20min, and then standing at 4℃for 2h.
The parameters for anion exchange column purification are as follows:
anion chromatographic column: the packing material is DEAE-agarose gel FF, and the model of the column is 2.6X10 cm;
the elution process comprises the following steps: equilibrated with Tris-HCl buffer, then loaded and eluted with Tris-HCl buffer.
The elution flow rate may specifically be 2mL/min.
The post-column solution with retention time of 26-159min was collected during elution.
The post-column solution was collected during elution with a retention volume of 52-318 mL.
The preparation method of the sample loading liquid comprises the following steps: after the ammonium sulfate precipitation is completed, centrifugally collecting the precipitate; dissolving the precipitate with PBS buffer solution, transferring to dialysis bag (molecular weight cut-off is 8000-14000 kDa), dialyzing the dialysis bag in PBS buffer solution, collecting liquid phase in the dialysis bag, and lyophilizing to obtain lyophilized powder; dissolving the freeze-dried powder in Tris-HCl buffer solution, then filtering with a 0.45 mu m filter membrane, and collecting filtrate to obtain the sample loading solution.
The preparation method of the sample loading liquid comprises the following steps: after completion of ammonium sulfate precipitation, centrifuging at 4 ℃ and 10000 Xg for 10min, and collecting the precipitate; dissolving the precipitate with PBS buffer solution, transferring to dialysis bag (molecular weight cut-off is 8000-14000 Da), dialyzing the dialysis bag at 4deg.C in PBS buffer solution, collecting liquid phase in the dialysis bag, and lyophilizing to obtain lyophilized powder; dissolving the freeze-dried powder in Tris-HCl buffer solution to make the concentration of the freeze-dried powder be 10mg/mL, then filtering the solution by using a 0.45 mu m filter membrane, and collecting filtrate to obtain the sample loading solution.
The protease has 11 segments; the 11 segments are respectively shown as sequence 2 to sequence 12 in the sequence table.
The protease is shown as a sequence 13 in a sequence table.
Fermentation medium (pH 7) as described in any of the above: contains 5.4g/L of potato extract powder, 1.8g/L of malt extract, 10.3g/L of beef extract and the balance of water.
The invention also protects the protease (JQ-2 protease) prepared by any one of the methods.
The invention also protects the use of JQ-2 protease as protease.
Any of the above proteases are proteolytic enzymes.
Any of the above proteases are proteins having proteolytic activity.
The invention also provides a method for preparing the hydrolyzed peptide, which comprises the following steps: sodium caseinate is used as a substrate, and JQ-2 protease is used for treatment to obtain the hydrolyzed peptide.
In the preparation method of the hydrolyzed peptide, the mixture ratio of sodium caseinate and JQ-2 protease is as follows: 1g sodium caseinate: 1000U JQ-2 protease. 1000U refers to 1000U proteolytic activity.
The preparation method of the hydrolyzed peptide specifically comprises the following steps: adding JQ-2 protease into sodium caseinate solution, hydrolyzing, heating in boiling water bath, and centrifuging to collect supernatant to obtain hydrolyzed peptide solution.
The preparation method of the hydrolyzed peptide specifically comprises the following steps: adding JQ-2 protease into sodium caseinate solution, hydrolyzing at 37deg.C for 24 hr, heating in boiling water bath at 100deg.C for 10min, centrifuging at 4deg.C and 10000rpm for 15min, and collecting supernatant to obtain hydrolyzed peptide solution.
Sodium caseinate solution: sodium caseinate was dissolved in Tris-HCl buffer to a concentration of 10g/100mL.
The hydrolytic peptide prepared by the method also belongs to the protection scope of the invention.
The invention also protects the use of the hydrolyzed peptides as follows (e) or (f) or (g):
(e) Scavenging DPPH free radicals
(f) Inhibition of alpha-glucosidase
(g) Inhibiting Angiotensin Converting Enzyme (ACE).
The bacillus subtilis JQ-2 provided by the invention can be used for preparing protease and/or chymosin through fermentation. The protease provided by the invention can effectively degrade sodium caseinate to obtain hydrolyzed peptide. The hydrolyzed peptide provided by the invention has excellent efficacy and performance. The invention has application and popularization value for protease related industries.
Drawings
FIG. 1 is a photograph of Bacillus subtilis JQ-2 on a solid casein culture medium plate.
FIG. 2 is a photograph of cell morphology and a photograph of colony morphology of Bacillus subtilis JQ-2.
FIG. 3 shows the results of homology alignment and identification of Bacillus subtilis JQ-2.
FIG. 4 is a growth curve of Bacillus subtilis JQ-2.
FIG. 5 is a graph showing the results of evaluating 6 factors affecting the fermentation and enzyme production of Bacillus subtilis.
FIG. 6 is a three-dimensional response surface graph showing the effect of variables on proteolytic activity.
Fig. 7 is a graph of the results during optimization of the ammonium sulfate precipitation parameters.
FIG. 8 is a graph showing the results of the separation and purification of JQ-2 protease.
FIG. 9 is an electrophoresis chart of the separation and purification process of JQ-2 protease.
FIG. 10 is a graph showing the results of LC-MS protein mass spectrometry for identifying JQ-2 protease.
FIG. 11 is a graph showing the results of analysis of the degree of hydrolysis of various substrates by JQ-2 protease.
FIG. 12 is a graph showing the results of a kinetic analysis of JQ-2 protease.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged. In the examples, the inoculum size refers to the volume ratio of the seed solution to the medium, e.g. 3% for 3mL seed solution to 100mL medium and 5% for 5mL seed solution to 100mL medium. In the examples, the liquid loading means that the culture medium occupies a volume percentage of the capacity of the culture vessel, for example, 50mL of the culture medium in a 250mL triangular flask, and the liquid loading is 20%.
Example 1 enzyme Activity detection method
1. Determination of proteolytic Activity
Sodium caseinate: sigma-Aldrich, CAS-No 9005-46-3, C8654-500G.
And diluting the test solution to 10 times of the volume by using water to obtain the test solution diluent.
1. Test group treatment method:
(1) 1g/100mL of sodium caseinate aqueous solution is taken, 1mL of test solution diluent is added, uniformly mixed, reacted in a constant temperature water bath at 40 ℃ for 10min, then 2mL of 0.4mol/L trichloroacetic acid aqueous solution is added to terminate the reaction, and then 10000r/min is centrifuged for 5min, and the supernatant is collected.
(2) Taking 1mL of the supernatant obtained in the step (1), adding 5mL of 0.4mol/L Na 2 CO 3 The aqueous solution was reacted with 1mL of a forskolin-phenol reagent (Shanghai Meilin Biochemical Co., ltd.) at 40℃for 20 minutes to measure the absorbance at 680nm, A 680 。
2. The blank group treatment method comprises the following steps:
(1) taking 1mL of test solution diluent, adding 2mL of 0.4mol/L trichloroacetic acid aqueous solution, mixing, then adding 1mL of 1g/100mL sodium caseinate aqueous solution, reacting for 10min in a 40 ℃ constant temperature water bath, centrifuging for 5min at 10000r/min, and collecting supernatant.
(2) Taking 1mL of the supernatant obtained in the step (1), adding 5mL of 0.4mol/L Na 2 CO 3 The aqueous solution is reacted with 1mL of a furin-phenol reagent at 40 ℃ for 20min, and the absorbance at 680nm wavelength, A' 680 。
3. Calculation of proteolytic Activity
The amount of enzyme required to catalyze the hydrolysis of sodium caseinate to produce 1 μg of tyrosine in 1min at 40℃is defined as 1U.
The Proteolytic Activity (PA) of the test solution is given in units of: U/mL.
PA＝K×(A 680 -A’ 680 )×n×4÷10；
K＝105.52；
n is the dilution factor of the test solution diluent prepared from the test solution.
2. Determination of curd Activity (Arima time method)
Skim milk powder: new Zealand constant natural group.
And diluting the test solution to 10 times of the volume by using water to obtain the test solution diluent.
Dissolving skimmed milk powder in CaCl 0.01mol/L 2 The aqueous solution is the skim milk solution, and the concentration of the skim milk powder is 10g/100ml. Standing the newly prepared skim milk solution at room temperature for 30min, and then sub-packaging into small test tubes, wherein each small test tube is sub-packaged into 5mL. The small test tube was placed in a 35℃thermostat water bath for 5min, and then 0 was added in the 35℃environment.5mL of the test solution was diluted, the time was started after shaking, and the time was stopped immediately when the flocculent precipitate began to appear (time unit was s).
The unit of curd activity (MCA) of the test solution is: SU/mL.
MCA＝(2400×V 1 ×n)÷(V 2 ×t)；
t is timing time, and the unit is s;
V 1 the volume of the skim milk solution is 5mL;
n is the dilution factor of the test solution diluent prepared by the test solution;
V 2 the volume of the sample solution was 0.5mL.
Example 2 acquisition and preservation of Bacillus subtilis JQ-2
1. Preliminary screening of strains from distiller's yeast samples
The distiller's yeast samples are respectively: collecting wheat starter and bran starter in Shanxi jin city; collected from Daqu and Xiaoqu of Jiangsu Suzhou.
1. Isolation of single strains from samples
(1) 25g of distiller's yeast sample is weighed, added into a conical flask filled with 250mL of sterile physiological saline, mixed uniformly by shaking, and then subjected to gradient dilution by adopting the sterile physiological saline to obtain 6 dilutions (10) -1 -10 -6 Dilution). 100. Mu.L of the dilution was spread on a solid LB medium plate, and the plate was inverted to a constant temperature incubator at 30℃to perform cultivation.
(2) And (3) selecting a small number of colonies with different shapes and sizes on days 2, 3 and 4 of culture in the step (1), inoculating the colonies to a solid culture medium plate, and purifying by repeated streaking to obtain a plurality of single strains. Three solid media were set up separately: solid LB medium, solid YEPD medium and solid PDA medium. In this step, the growth conditions of the bacteria were continuously observed, and each single strain was selected from three solid media, respectively, to be used for the subsequent cultivation of the single strain.
(3) After the step (2) is completed, single colonies are picked up to corresponding liquid culture mediums (the corresponding liquid culture mediums are the optimal culture mediums screened in the step (2) are removed of agar powder), and the culture is carried out for 24 hours.
(4) After the step (3) is completed, the fungus liquid and 50% glycerol are uniformly mixed by using a freezing tube, and the mixture is preserved at the temperature of minus 80 ℃.
2. Screening protease-producing strains
The test strains were respectively: each single strain obtained in step 1.
The test strain was picked up and inoculated onto a solid casein medium plate, cultured at 37℃for 2 days, and then the diameter of the curd ring, the diameter of the hydrolytic ring and the diameter of the cell were measured.
Solid casein medium: contains peptone 2.5g/L, glucose 10g/L, yeast extract powder 1g/L, sodium caseinate 10g/L, agar 20g/L, and water in balance.
The activity of 11 strains is higher. The 11 strains were named: JQ-1, JQ-2, JQ-3, F1, F2, F3, F4, D1, D2, X1 and X2.JQ represents wheat starter isolated from shanxi winery, F represents bran starter isolated from shanxi winery, D represents Daqu isolated from Jiangsu su state winery, and X represents small starter isolated from Jiangsu state winery. The "curd ring diameter/cell diameter" and "hydrolysis ring diameter/cell diameter" of 11 strains are shown in Table 1.
TABLE 1
2. Further screening the target strain from the strain obtained by the preliminary screening
Test strain: 11 strains (JQ-1, JQ-2, JQ-3, F1, F2, F3, F4, D1, D2, X1 and X2) obtained in the first step, respectively.
1. Activation of strains
Inoculating the strain to liquid LB culture medium, shake culturing at 30deg.C and 120r/min to OD 600nm The value was 1.3, and a seed solution was obtained.
2. Fermentation of strains
Inoculating the seed liquid obtained in the step 1 into a liquid LB culture medium with an inoculum size of 3%, carrying out shaking culture for 24h at 30 ℃ and 120r/min, centrifuging for 10min at 4 ℃ and 10000r/min, and collecting the supernatant, namely the fermentation broth.
The results of the measurement of proteolytic activity and curd activity using the fermentation broth as a test solution are shown in Table 2.
The fermentation liquor of the strain JQ-2 has both proteolytic activity and curd activity and has the best effect.
TABLE 2
| Strain | Proteolytic activity (U/mL) of fermentation broth | Curd activity of fermentation broth (SU/mL) |
| JQ-2 | 10.48±0.36 | 62.72±1.05 |
| JQ-1 | 10.17±0.61 | - |
| JQ-3 | 9.99±0.79 | - |
| F1 | 8.17±2.18 | - |
| F2 | 7.46±0.68 | 56.67.±0.74 |
| F3 | 5.74±0.85 | 54.14±0.45 |
| F4 | 5.19±0.36 | 48.99±0.32 |
| D1 | 4.42±0.14 | - |
| D2 | 4.36±0.95 | - |
| X1 | 1.16±0.3 | - |
| X2 | 0.22±0.08 | - |
3. Identification of Strain JQ-2
Morphological characteristics: the strain has obvious hydrolysis circle and curd circle on the solid casein culture medium plate (the photo is shown in figure 1, the line-shaped dotted line marks the thallus diameter, the dot-shaped dotted line marks the hydrolysis circle diameter, and the straight line marks the curd circle diameter); the cells are in a short rod shape, single cells or multiple cells are connected end to end and are in a chain shape (see the right diagram of fig. 2); the colony is milky white, the surface of the colony is smooth and has spores, and the diameter of the colony is 1-6mm (see left graph of FIG. 2).
Physiological and biochemical characteristics: gram positive bacteria.
16S rDNA identification: the 16SrDNA of the strain is shown as a sequence 1 in a sequence table; homology alignment identification was performed in NCBI website gene library, and the results are shown in FIG. 3.
As a result of comprehensive identification, the strain JQ-2 belongs to Bacillus subtilis (Bacillus subtilis), and thus was designated as Bacillus subtilis JQ-2.
4. Preservation of JQ-2
Bacillus subtilis (Bacillus subtilis) JQ-2 is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms (CGMCC, address: north Chenxi Lu No. 1, 3, university of China, academy of sciences of China) at a year 09 and 07, and a preservation registration number is CGMCC No.25660.
5. Growth curve of bacillus subtilis JQ-2
Inoculating bacillus subtilis JQ-2 into liquid LB culture medium, shake culturing at 30deg.C and 120r/min, sampling and detecting OD every 2 hr 600nm Values. See fig. 4.
EXAMPLE 3 optimization of Bacillus subtilis JQ-2 culture conditions
In this example, the pre-optimization medium was used as a control medium for the test medium to take into account the optimization effect. Pre-optimization medium (natural pH): 5g/L yeast extract powder, 10g/L tryptone, 10g/L sodium chloride and the balance of distilled water.
1. Preparation of seed liquid
Inoculating bacillus subtilis JQ-2 into liquid LB culture medium, shake culturing at 30deg.C and 120r/min to OD 600nm The value was 1.3, and a seed solution was obtained.
2. Optimization of Medium Components
1. Influence of different carbon sources on proteolytic Activity
Test medium: carbon source medium. Carbon source Medium was obtained by adding a carbon source to a basal medium (the concentration of the carbon source in the medium was 5 g/L). Basal medium (natural pH): 5g/L yeast extract powder, 10g/L tryptone, 10g/L sodium chloride and the balance of distilled water. The following carbon sources were set respectively: fructose (Beijing pinellia technologies development Co., ltd., product number a 3125), alpha-lactose (national pharmaceutical group chemical agent Co., ltd., product number 20111025), malt extract (Beijing obbo star biotechnology Co., ltd., product number 01-051), sucrose (Beijing pinellia technologies development Co., ltd., product number b 3075) or glucose (Beijing pinellia technologies development Co., ltd., product number b 2657).
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The fermentation broth was used as a test solution, and the proteolytic activity was measured, and the results are shown in Table 3.
TABLE 3 Table 3
| Proteolytic activity (U/mL) of fermentation broth | |
| Malt extract carbon culture medium | 33.53±1.48 |
| Fructose carbon source culture medium | 7.89±0.63 |
| Alpha-lactose carbon source culture medium | 1.34±0.53 |
| Sucrose carbon source culture medium | 5.50±0.32 |
| Glucose carbon source culture medium | 16.40±0.72 |
| Control medium | 10.48±0.36 |
2. Influence of malt extract of different concentrations on proteolytic Activity
The test medium is obtained by adding malt extract into basic medium. The concentration of malt extract in the culture medium was set to 1.5g/L, 2g/L, 2.5g/L, 5g/L or 7.5g/L, respectively. Basal medium (natural pH): 5g/L yeast extract powder, 10g/L tryptone, 10g/L sodium chloride and the balance of distilled water.
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the measurement of proteolytic activity using the fermentation broth as a test fluid are shown in Table 4.
TABLE 4 Table 4
| Proteolytic activity (U/mL) of fermentation broth | |
| The concentration of malt extract in the culture medium is 1.5g/L | 86.46±4.22 |
| The concentration of malt extract in the culture medium is 2g/L | 103.4±3.97 |
| The concentration of malt extract in the culture medium is 2.5g/L | 79.49±4.43 |
| The concentration of malt extract in the culture medium is 5g/L | 33.53±1.48 |
| The concentration of malt extract in the culture medium is 7.5g/L | 19.42±1.05 |
3. Influence of different nitrogen sources on proteolytic Activity
Test medium: nitrogen source medium. Nitrogen source Medium A nitrogen source was added to the basal medium (the concentration of the nitrogen source in the medium was 10 g/L). Basal medium (natural pH): 5g/L of yeast-containing soaked powder, 10g/L of sodium chloride and the balance of distilled water. The following nitrogen sources were set up: sweet whey powder (Shanghai Yuan leaf technology Co., ltd., product No. S27239-500 g), soybean peptone (Beijing Jin Ruilin technology development Co., ltd., product No. hb 8275), skim milk powder (Beijing pinellia technology development Co., ltd., product No. p 2216), beef extract (Beijing obbo star biotechnology Co., ltd., product No. 01-009) or ammonium citrate (Beijing pinellia technology development Co., ltd., product No. b 2338).
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the measurement of proteolytic activity using the fermentation broth as a test fluid are shown in Table 5.
TABLE 5
| Proteolytic activity (U/mL) of fermentation broth | |
| Beef extract nitrogen culture medium | 104.68±3.26 |
| Sweet whey powder nitrogen source culture medium | 43.40±0.90 |
| Soybean peptone nitrogen source culture medium | 74.96±1.91 |
| Skim milk powder nitrogen culture medium | 43.87±1.26 |
| Ammonium citrate nitrogen source culture medium | 0.30±0.09 |
| Control medium | 10.48±0.36 |
4. Effects of different concentrations of beef extract on proteolytic Activity
The test medium is obtained by adding beef extract into basal medium. The concentration of beef extract in the culture medium was set to 2.5g/L, 5g/L, 7.5g/L, 10g/L or 12.5g/L, respectively. Basal medium (natural pH): 5g/L of yeast-containing soaked powder, 10g/L of sodium chloride and the balance of distilled water.
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the measurement of proteolytic activity using the fermentation broth as a test fluid are shown in Table 6.
TABLE 6
| Proteolytic activity (U/mL) of fermentation broth | |
| The concentration of beef extract in the culture medium is 2.5g/L | 19.26±0.77 |
| The concentration of beef extract in the culture medium is 5g/L | 27.32±1.01 |
| The concentration of beef extract in the culture medium is 7.5g/L | 43.83±1.06 |
| The concentration of beef extract in the culture medium is 10g/L | 104.68±3.26 |
| The concentration of beef extract in the culture medium is 12.5g/L | 81.12±3.26 |
5. Effect of different concentrations of Potato Leaching powder on proteolytic Activity
The test medium is obtained by adding potato extract powder (product number is 01-141 of Beijing Oboc biotechnology liability company) into basic medium. The concentration of the potato extract powder in the culture medium is set to be 5g/L, 10g/L, 15g/L or 20g/L respectively. Basal medium (natural pH): 5g/L yeast extract powder, 10g/L tryptone, 10g/L sodium chloride and the balance of distilled water.
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the measurement of proteolytic activity using the fermentation broth as a test fluid are shown in Table 7.
TABLE 7
| Proteolytic activity (U/mL) of fermentation broth | |
| The concentration of the potato leaching powder in the culture medium is 5g/L | 24.26±0.32 |
| The concentration of the potato extract powder in the culture medium is 10g/L | 65.42±1.42 |
| The concentration of the potato extract powder in the culture medium is 15g/L | 36.38±1.36 |
| The concentration of the potato leaching powder in the culture medium is 20g/L | 25.69±0.75 |
6. Influence of Metal ion species on proteolytic Activity
Test medium: metal ion medium. Metal ion Medium was obtained by adding a compound to a basal medium (the concentration of the compound in the medium was 10 g/L). Basal medium (natural pH): 5g/L yeast extract powder, 10g/L tryptone, 10g/L sodium chloride and the balance of distilled water. The following compounds were set up, respectively: sodium chloride, magnesium sulfate, copper sulfate, zinc sulfate, and manganese sulfate.
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the measurement of proteolytic activity using the fermentation broth as a test fluid are shown in Table 8.
TABLE 8
7. Effect of different concentrations of magnesium sulfate on proteolytic activity
The test medium was obtained by adding magnesium sulfate to the basal medium. The concentration of magnesium sulfate in the medium was set to 5g/L, 10g/L, 12.5g/L, 15g/L or 17.5g/L, respectively. Basal medium (natural pH): 5g/L yeast extract powder, 10g/L tryptone, 10g/L sodium chloride and the balance of distilled water.
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the measurement of proteolytic activity using the fermentation broth as a test fluid are shown in Table 9.
TABLE 9
| Proteolytic activity (U/mL) of fermentation broth | |
| The concentration of magnesium sulfate in the culture medium is 5g/L | 8.21±1.01 |
| The concentration of magnesium sulfate in the culture medium is 10g/L | 14.78±0.62 |
| The concentration of magnesium sulfate in the culture medium is 12.5g/L | 17.78±1.18 |
| The concentration of magnesium sulfate in the culture medium is 15g/L | 7.01±0.39 |
| The concentration of magnesium sulfate in the culture medium is 17.5g/L | 11.99±0.77 |
8. Influence of different phosphate species on proteolytic Activity
Test medium: phosphate medium. Phosphate medium is obtained by adding phosphate to basal medium. The concentration of phosphate in the medium was 1g/L. Basal medium (natural pH): 5g/L yeast extract powder, 10g/L tryptone, 10g/L sodium chloride and the balance of distilled water. The following phosphates were set up: dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate.
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the measurement of proteolytic activity using the fermentation broth as a test fluid are shown in Table 10.
Table 10
| Proteolytic activity (U/mL) of fermentation broth | |
| Dipotassium phosphate culture medium | 8.48±0.87 |
| Potassium dihydrogen phosphate culture medium | 13.38±0.78 |
| Sodium dihydrogen phosphate culture medium | 15.42±0.41 |
| Disodium hydrogen phosphate medium | 14.07±1.51 |
| Control medium | 10.48±0.36 |
9. Effect of varying concentrations of sodium dihydrogen phosphate on proteolytic Activity
The test medium was obtained by adding sodium dihydrogen phosphate to the basal medium. The concentration of sodium dihydrogen phosphate in the culture medium was set to 0.5g/L, 1g/L, 1.5g/L, 2g/L or 2.5g/L, respectively. Basal medium (natural pH): 5g/L yeast extract powder, 10g/L tryptone, 10g/L sodium chloride and the balance of distilled water.
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the measurement of proteolytic activity using the fermentation broth as a test fluid are shown in Table 11.
TABLE 11
| Proteolytic activity (U/mL) of fermentation broth | |
| The concentration of the sodium dihydrogen phosphate in the culture medium is 0.5g/L | 10.48±0.68 |
| The concentration of the sodium dihydrogen phosphate in the culture medium is 1g/L | 15.42±0.41 |
| The concentration of the sodium dihydrogen phosphate in the culture medium is 1.5g/L | 24.09±1.12 |
| The concentration of the sodium dihydrogen phosphate in the culture medium is 2g/L | 11.30±1.25 |
| The concentration of the sodium dihydrogen phosphate in the culture medium is 2.5g/L | 9.13±0.30 |
10. Effect of different inducer species on proteolytic Activity
Test medium: inducer media. The inducer culture medium is obtained by adding inducer into basal culture medium. The concentration of the inducer in the medium was 5g/L. Basal medium (natural pH): 5g/L yeast extract powder, 10g/L tryptone, 10g/L sodium chloride and the balance of distilled water. The following inducers were set separately: phytic acid, tween 80, tween 20, sodium acetate and methanol.
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the measurement of proteolytic activity using the fermentation broth as a test fluid are shown in Table 12.
Table 12
| Proteolytic activity (U/mL) of fermentation broth | |
| Phytic acid inducer culture medium | 0 |
| Tween 80 inducer culture medium | 26.49±2.33 |
| Tween 20 inducer culture medium | 2.81±0.17 |
| Sodium acetate inducer culture medium | 46.85±2.09 |
| Methanol inducer culture medium | 4.91±0.58 |
| Control medium | 10.48±0.36 |
11. Effect of sodium acetate at different concentrations on proteolytic Activity
The test medium was obtained by adding sodium acetate to the basal medium. The concentration of sodium acetate in the medium was set to 1g/L, 1.5g/L, 2g/L, 2.5g/L or 5g/L, respectively. Basal medium (natural pH): 5g/L yeast extract powder, 10g/L tryptone, 10g/L sodium chloride and the balance of distilled water.
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shake culture at 30deg.C and 120r/min for 24h, centrifuging at 4deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the measurement of proteolytic activity using the fermentation broth as a test fluid are shown in Table 13.
TABLE 13
12. Obtaining optimized post-fermentation culture medium
The influence of different carbon sources, nitrogen sources, metal ions, phosphate and inducers on the fermentation production of proteolytic enzymes by bacillus subtilis is analyzed through a single factor test, and 6 factors (carbon sources, nitrogen sources, potato extract powder, metal ions, phosphate and inducers) affecting the fermentation production of the proteolytic enzymes by bacillus subtilis are evaluated by using a Plackett-Burman test design, and are shown in figure 5. 3 factors with obvious influence, namely potato extract powder, malt extract and beef extract are screened out.
The optimized post-fermentation medium for producing the proteolytic enzyme by fermenting the bacillus subtilis is determined through a center combination test design in a response surface. A three-dimensional response surface graph showing the effect of variables on proteolytic activity is shown in FIG. 6.
Optimized post-fermentation medium (natural pH): contains 5.4g/L of potato extract powder, 1.8g/L of malt extract, 10.3g/L of beef extract and the balance of water.
Inoculating the seed solution to an optimized post-fermentation culture medium with an inoculum size of 3%, performing shake culture at 30 ℃ for 24 hours at 120r/min, centrifuging at 4 ℃ for 10 minutes at 10000r/min, and collecting the supernatant to obtain a fermentation broth. The fermentation broth was used as a test solution to detect proteolytic activity. The proteolytic activity of the fermentation broth is 154.75 +/-1.76U/mL and is close to a theoretical predicted value of 157.14U/mL.
3. Optimization of culture conditions
Optimizing the post-fermentation medium: contains 5.4g/L of potato extract powder, 1.8g/L of malt extract, 10.3g/L of beef extract and the balance of water.
The parameters of the 6 culture conditions are optimized. Parameters of 6 culture conditions: fermentation time, fermentation temperature, rotation speed, pH of optimized post-fermentation culture medium, inoculation amount and liquid loading amount.
The optimized parameters are as follows: fermentation time is 24 hours, fermentation temperature is 30 ℃, rotating speed is 180r/min, pH of post-fermentation culture medium is optimized to be 7, inoculum size is 5%, and liquid loading amount is 20%.
4. Bacillus subtilis fermentation enzyme production by using optimized post-fermentation culture medium and optimized post-culture conditions
Optimized post-fermentation medium (pH 7): contains 5.4g/L of potato extract powder, 1.8g/L of malt extract, 10.3g/L of beef extract and the balance of water.
Inoculating the seed solution to an optimized post-fermentation culture medium (the liquid loading amount of the culture medium is 20%) at 5%, performing shaking culture at 30 ℃ for 24 hours at 180r/min, centrifuging at 4 ℃ for 10 minutes at 10000r/min, and collecting the supernatant to obtain the fermentation broth.
And (3) taking the fermentation liquor as a test liquor, and detecting the proteolytic activity of the fermentation liquor, wherein the proteolytic activity of the fermentation liquor is 219.06U/mL.
EXAMPLE 4 isolation, purification and characterization of JQ-2 protease
1. Preparation of fermentation broth
Fermentation medium (pH 7): contains 5.4g/L of potato extract powder, 1.8g/L of malt extract, 10.3g/L of beef extract and the balance of water.
Inoculating bacillus subtilis JQ-2 into liquid LB culture medium, shake culturing at 30deg.C and 120r/min to OD 600nm The value was 1.3, and a seed solution was obtained. Inoculating the seed solution into the fermentation culture medium at an inoculum size of 5%The culture medium liquid loading amount is 20 percent, shaking culture is carried out for 24 hours at 30 ℃ and 180r/min, then centrifugation is carried out for 10 minutes at 4 ℃ and 10000r/min, and the supernatant is collected, thus obtaining the fermentation broth.
2. Optimizing ammonium sulfate precipitation parameters
27 parts of the fermentation broth prepared in the step one are taken and divided into 9 groups of 3 parts of fermentation broth. Slowly adding ammonium sulfate into the fermentation broth at 4deg.C to reach preset ammonium sulfate saturation (magnetic stirring is continued in the process), magnetic stirring at 4deg.C for 20min, standing at 4deg.C for 2 hr, centrifuging at 4deg.C 10000×g for 10min, and collecting supernatant. The first group was not added with ammonium sulfate. The second to ninth groups have preset ammonium sulfate saturation levels of 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% in this order. The proteolytic activity was measured by replacing the test broth with the supernatant. The results are shown in FIG. 7.
3. Separation and purification of JQ-2 protease
1. Ammonium sulfate precipitation
And (3) slowly adding ammonium sulfate into the fermentation liquor prepared in the step (I) in an environment of 4 ℃ to reach 70% saturation (continuous magnetic stirring in the process), magnetically stirring for 20min at 4 ℃, standing for 2h at 4 ℃, centrifuging for 10min at 4 ℃ at 10000 Xg, and collecting precipitate.
2. Dialysis desalination treatment
(1) The pellet obtained in step 1 was dissolved in PBS buffer (pH 7.2, 50mM, the same applies below), then transferred to a dialysis bag (8000-14000 kDa cut-off), and then the dialysis bag was placed in PBS buffer and desalted by dialysis in an environment of 4℃until the conductivity of the PBS buffer was unchanged by measuring the conductivity with a conductivity meter (PBS buffer was replaced every 8 hours).
(2) After the step (1) is completed, collecting liquid phase in the dialysis bag, and freeze-drying to obtain freeze-dried powder.
(3) Dissolving the freeze-dried powder obtained in the step (2) in Tris-HCl buffer (pH 7.2, 50mM, the same applies below) to obtain crude enzyme solution.
3. DEAE-sepharose FF purified protease
Dissolving the freeze-dried powder obtained in the step 2 in Tris-HCl buffer solution to make the concentration of the freeze-dried powder be 10mg/mL, filtering the freeze-dried powder with a 0.45 mu m filter membrane, and collecting filtrate.
The anion chromatographic column (packing is DEAE-agarose gel FF, column model is 2.6X140 cm), firstly, the column is equilibrated with Tris-HCl buffer solution, then 2mL of filtrate is loaded, and then elution is carried out (the solvent used for elution is Tris-HCl buffer solution or Tris-HCl buffer solution containing NaCl, the concentration gradient change process of NaCl is shown in figure 8, and the flow rate is 2 mL/min). The post-column solution was collected from the beginning of elution, with 1 tube per 1 min.
Each tube was sampled as a test solution, and the proteolytic activity was measured, and the results are shown in FIG. 8. The sampled samples from tube 26 to tube 159 have proteolytic activity, so the solutions from these tubes are combined and then concentrated by ultrafiltration with an ultrafiltration membrane having a molecular weight cut-off of 10kda (the buffer system is Tris-HCl buffer), and the resulting concentrate is the purified protein solution. As can be seen from FIG. 8, the 26 th to 159 th tubes are all eluted and collected post-column solutions of Tris-HCl buffer, and the corresponding elution time is 26-159min (corresponding retention volume is 52-318 mL).
4. SDS-PAGE detection
SDS-PAGE (12.5% separation gel and 4.5% concentration gel) was performed on the crude enzyme solution prepared in step 2 and the purified protein solution prepared in step 3, respectively, as shown in FIG. 9 (lane 1 is crude enzyme solution, and lane 2 is purified protein solution).
The purified protein solution showed electrophoretically pure single protein, so that the protein in the purified protein solution was designated as JQ-2 protease, and the purified protein solution was designated as JQ-2 protease solution.
5. Proteolysis activity assay
And (3) respectively taking the fermentation liquor prepared in the step (I), the crude enzyme liquor prepared in the step (2) and the JQ-2 protease solution prepared in the step (3) as test solutions, and detecting the proteolytic activity on one hand and the total protein concentration on the other hand by adopting a Bradford method.
Specific activity (U/mg) =proteolytic activity/total protein concentration.
The results are shown in Table 14.
TABLE 14
| Purification step | Total protein (mg) | Total vitality (U) | Specific activity (U/mg) | Recovery (%) | Purification times |
| Fermentation liquor | 424.5 | 4387.67 | 10.34 | 100 | 1 |
| Crude enzyme solution | 63.70 | 3477.23 | 54.59 | 79.25 | 5.28 |
| Purified protein liquid | 3.12 | 1958.69 | 627.79 | 56.33 | 60.71 |
4. Identification of JQ-2 protease by LC-MS protein mass spectrum
Recovering the target band of the purified protein solution from the SDS-PAGE gel of step three, 4 and cutting into 1mm 3 Transfer to 1.5mL microtubes and digest with trypsin. The method comprises the following steps: and (3) decoloring colloidal particles: using 50% ACN-50%50mmol/L NH 4 HCO 3 The solution was decolorized, 100. Mu.L/tube, left to stand for 10-30min and aspirated for disposal. Dewatering the colloidal particles: adding 100% ACN 80-100 μl, standing for 30min until the colloidal particles are white to bulk, discarding ACN, and standing at room temperature for drying. Reductive alkylation: 10mmol/L DTT is added according to 100 mu L/pipe and reduced for 1h in 56 ℃ water bath, sucked out and discarded; 55mmol/L IAA was added at 100. Mu.L/tube and reacted in the dark at room temperature for 1h, sucked out and discarded; adding decolorizing solution (50% ACN-50%50mm NH) 4 HCO 3 ) Washing once, sucking out, discarding, adding 100% ACN 80-100 μl, standing for 30min until the colloidal particles are white rope to form a lump, discarding ACN, standing at room temperature, and drying. And (3) enzyme cutting: the enzyme was used at a concentration of 15 ng/. Mu.L (25 mmol/L NH 4 HCO 3 Dilution), adding enzyme solution according to the amount of 7-10 mu L/pipe, placing into a refrigerator at 4 ℃ for incubation for 40min, taking out, and adding 5-10 mu L25 mmol/L NH 4 HCO 3 The solution is sealed and placed in a water bath at 37 ℃ for enzyme digestion for 16h. Extracting peptide: adding 100 μl/tube of extractive solution (5% TFA-50% ACN-45% water), standing in water bath at 37deg.C for 1 hr, ultrasonic treating for 5min, centrifuging for 5min, transferring the extractive solution into another new EP tube, repeating extraction once, mixing extractive solutions, and vacuum centrifuging for drying. Mass spectrometry was performed and the results are shown in fig. 10. The ionized fragments of the bacillus subtilis JQ-2 protease after trypsin hydrolysis are shown in Table 15, and the amino acid sequences of the fragments are shown in sequence 2 to sequence 12 in a sequence table.
TABLE 15 ionizing fragments of Bacillus subtilis JQ-2 protease after trypsin hydrolysis
| Amino acid sequence | Length of | Molecular weight | Initial position | Termination position |
| HAYSTISQLSEAIGPR | 16 | 1728.88 | 47 | 62 |
| GDLTYYEK | 8 | 987.4549 | 154 | 161 |
| VLKSVPSDKEIR | 12 | 1369.793 | 276 | 287 |
| FNIPDRLEGTLSSAGR | 16 | 1731.89 | 92 | 107 |
| ESLVPMTPNLSGNK | 14 | 1485.75 | 180 | 193 |
| VGIPVVGIK | 9 | 880.5746 | 194 | 202 |
| GKIALISR | 8 | 856.5494 | 146 | 153 |
| LQQAGDLVTAAVYEAVKK | 18 | 1903.042 | 415 | 432 |
| NAEAAGAKAVIIYNNK | 16 | 1645.879 | 164 | 179 |
| LKAFTNQTSQNIIGIK | 16 | 1774.994 | 218 | 234 |
| KEDGEALTQQKEATLK | 16 | 1787.927 | 201 | 217 |
5. Analysis of the degree of hydrolysis of different substrates by JQ-2 protease
The test substrates were respectively: sodium caseinate or whey protein.
Test protein solution: and (3) diluting the JQ-2 protease solution prepared in the step (III) with Tris-HCl buffer solution to ensure that the protein concentration is 0.3125mg/mL, 0.625mg/mL, 1.25mg/mL, 2.5mg/mL, 5mg/mL, 10mg/mL or 20mg/mL respectively, thus obtaining the protein solution to be tested. Tris-HCl buffer was used as a0 concentration control for the protein solution tested.
(1) The test substrate is dissolved in Tris-HCl buffer solution to make the concentration of the test substrate be 1mg/mL, and the test substrate is the substrate solution.
(2) 1mL of the substrate solution was mixed with 100. Mu.L of the protein solution to be tested, incubated at 37℃for 30min, then water-bathed in 100℃water for 5min, and then sampled for SDS-PAGE.
The results are shown in FIG. 11. As JQ-2 protease concentration increases, the extent of sodium caseinate degradation increases. With increasing JQ-2 protease concentration, no significant degradation of whey protein occurred.
6. JQ-2 protease kinetic analysis
And (3) taking the JQ-2 protease solution prepared in the step (III) as a test solution, and detecting the proteolytic activity.
The process is essentially the same as step one of example 1. The only differences are: 1g/100mL of the sodium caseinate aqueous solution is replaced by a gradient concentration solution. The gradient concentration solution was as follows: sodium caseinate was dissolved in Tris-HCl buffer to a concentration of 0.5g/L, 1g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L or 5g/L, respectively.
The Michaelis constant Km and the maximum reaction rate Vmax are calculated.
The 1/V versus 1/[ S ] graph of JQ-2 protease hydrolyzed sodium caseinate is shown in FIG. 12. When the substrate concentration is low, a low value indicates a higher enzyme affinity for the substrate. Km and Vmax values were 10.8g/L and 35.84U/mL, respectively.
Example 5 preparation of a hydrolyzed peptide Using JQ-2 protease and verification of the function of the hydrolyzed peptide
1. Preparation of hydrolyzed peptides
Sodium caseinate solution: sodium caseinate was dissolved in Tris-HCl buffer to a concentration of 10g/100mL.
The JQ-2 protease solution obtained in the step three of example 4 was freeze-dried to obtain a powdered JQ-2 protease.
Adding powdered JQ-2 protease into sodium caseinate solution (in the initial system, the ratio of sodium caseinate to JQ-2 protease is 1g sodium caseinate: 1000U JQ-2 protease; 1000U refers to 1000U proteolytic activity), hydrolyzing at 37deg.C for 24 hr, heating in boiling water bath at 100deg.C for 10min, centrifuging at 4deg.C and 10000rpm for 15min, and collecting supernatant to obtain hydrolyzed peptide solution.
2. In vitro bioactivity assay of hydrolyzed peptides
1. Ability to scavenge DPPH free radical
1mL of the hydrolyzed peptide solution was mixed with 2mL of DPPH solution (DPPH solution: methanol as solvent, DPPH concentration of 0.2 mmol/L), reacted at room temperature in the absence of light for 1 hour, centrifuged at 10000r/min for 10 minutes, and the supernatant was taken to determine absorbance at 517nm wavelength.
The blank group replaced the DPPH solution with an equal volume of methanol, and the control group replaced the hydrolyzed peptide solution with an equal volume of sterile water.
Clearance (%) = [1- (A1-a)/A0 ] ×100%;
wherein: a is absorbance of a blank group; a0 is absorbance of the control group; a1 is the absorbance of the sample group.
The DPPH radical scavenging rate of the hydrolyzed peptide solution was 91.34%.
2. Ability to inhibit alpha-glucosidase
Group C: mu.L of an aqueous solution of alpha-glucosidase (1.0U/mL), 20. Mu.L of a hydrolyzed peptide solution, and 165. Mu.L of a phosphate buffer (pH 6.8, 0.1 mmol/L) were mixed, reacted in a 96-well plate at 37℃for 10min, 10. Mu.L of a phosphate buffer (pH 6.9, 0.1 mol/L) containing 0.95mmol/L of p-nitrophenyl-alpha-D-glucopyranoside was added, reacted at 37℃for 10min, and 100. Mu.L of 1mol/L Na was added 2 CO 3 The aqueous solution terminated the reaction.
Group A: equal volumes of sterile water were used instead of the hydrolyzed peptide solution, otherwise identical for group C.
Group B: equal volumes of sterile water were used instead of the aqueous α -glucosidase solution and equal volumes of sterile water were used instead of the hydrolyzed peptide solution and p-nitrophenyl- α -D-glucopyranoside (phosphate buffer normally added) was removed, otherwise identical for group C.
Group D: p-nitrophenyl-alpha-D-glucopyranoside (phosphate buffer) was removed, otherwise identical to group C.
The absorbance at 405nm was measured.
Alpha-glucosidase inhibition rate (%) = [1- (C-D)/(a-B) ]x100%.
The inhibition rate of alpha-glucosidase of the hydrolyzed peptide solution is 83.33 percent
3. ACE inhibition ability
ACE, collectively referred to as angiotensin converting enzyme, sigma, cat No. a6778.
N- [3- (2-Furyl) acryloyl ] -L-phenylalanyl-glycyl-glycine (N- [3- (2-Furyl) acylyl ] -Phe-Gly-Gly, FAPGG) as substrate, and when FAPGG is hydrolyzed by ACE catalysis, absorbance change occurs at 340 nm.
Phosphate buffer: phosphate buffer at pH8.3, 100 mmol/L.
And (3) taking the hydrolyzed peptide solution, and diluting the hydrolyzed peptide solution to 2 times of volume by using a phosphate buffer solution containing 300mmol/L NaCl, thus obtaining the inhibitor solution. Dissolving FAPGG in phosphate buffer to obtain FAPGG solution with concentration of 1.6 mmol/L. 0.1U of ACE is taken and dissolved in 1mL of phosphate buffer, namely ACE solution.
Sample group: 85 μl of inhibitor solution was mixed with 15 μl of ACE solution, 100 μl of FAPGG solution was added, and absorbance was measured immediately at 340nm wavelength and designated as A0; after incubation at 37℃for 60min, the absorbance was again measured at 340nm wavelength and designated A1; Δa=a0-A1.
Control group: equal volumes of phosphate buffer were used instead of inhibitor solution, otherwise identical sample sets.
ACE inhibitory activity (%) = (1- Δa sample group/Δa control group) ×100%.
The ACE inhibitory activity of the hydrolyzed peptide solution was 92.19%.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. The bacillus subtilis (Bacillus subtilis) JQ-2 has a preservation registration number of CGMCC No.25660.
2. The fermentation broth of bacillus subtilis of claim 1.
3. A method of preparing a protease comprising the steps of: fermenting and culturing the bacillus subtilis according to claim 1 to obtain a fermentation broth.
4. A method as claimed in claim 3, wherein: the method further comprises the steps of: purifying the fermentation broth to obtain the protease.
5. The method of claim 4, wherein: the protease has 11 segments; the 11 segments are respectively shown as sequence 2 to sequence 12 in the sequence table.
6. A protease obtainable by the process of any one of claims 3 to 5.
7. The application is as follows (a) or (b) or (c) or (d):
(a) Use of the bacillus subtilis according to claim 1 for the preparation of proteases and/or chymosins;
(b) Use of the fermentation broth of claim 2 for the preparation of proteases and/or chymosins;
(c) Use of the fermentation broth of claim 2 as protease and/or chymosin.
(d) The use of the protease of claim 6 as a protease.
8. A method of preparing a hydrolyzed peptide comprising the steps of: treatment with the protease of claim 6 with sodium caseinate as substrate gives a hydrolysed peptide.
9. The hydrolyzed peptide produced by the method of claim 8.
10. The use of a hydrolyzed peptide according to claim 9, as follows (e) or (f) or (g):
(e) Scavenging DPPH free radicals
(f) Inhibition of alpha-glucosidase
(g) Inhibiting angiotensin converting enzyme.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| GB2024830A (en) * | 1978-07-04 | 1980-01-16 | Novo Industri As | Protease Product of Reduced Allergenicity |
| CN101892182A (en) * | 2010-06-07 | 2010-11-24 | 中国农业大学 | Bacillus licheniformis and application thereof in promotion of cellulose degradation |
| KR20220039618A (en) * | 2020-09-21 | 2022-03-29 | 국민바이오 주식회사 | Development of bioconversion process of bioconverted functional soybean powder using enzymes from Bacillus and its use |
| CN114680337A (en) * | 2022-03-21 | 2022-07-01 | 杭州娃哈哈科技有限公司 | Application of lactobacillus helveticus in preparing fermented product for reducing blood pressure |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2024830A (en) * | 1978-07-04 | 1980-01-16 | Novo Industri As | Protease Product of Reduced Allergenicity |
| CN101892182A (en) * | 2010-06-07 | 2010-11-24 | 中国农业大学 | Bacillus licheniformis and application thereof in promotion of cellulose degradation |
| KR20220039618A (en) * | 2020-09-21 | 2022-03-29 | 국민바이오 주식회사 | Development of bioconversion process of bioconverted functional soybean powder using enzymes from Bacillus and its use |
| CN114680337A (en) * | 2022-03-21 | 2022-07-01 | 杭州娃哈哈科技有限公司 | Application of lactobacillus helveticus in preparing fermented product for reducing blood pressure |
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