CN115851509A - Bacillus subtilis strain producing protease in distiller's yeast and application thereof - Google Patents
Bacillus subtilis strain producing protease in distiller's yeast and application thereof Download PDFInfo
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- CN115851509A CN115851509A CN202211259384.XA CN202211259384A CN115851509A CN 115851509 A CN115851509 A CN 115851509A CN 202211259384 A CN202211259384 A CN 202211259384A CN 115851509 A CN115851509 A CN 115851509A
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- 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
- 230000035899 viability Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 238000005303 weighing Methods 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
Images
Abstract
The invention discloses a bacillus subtilis strain producing protease in distiller's yeast and application thereof. The preservation registration number of the Bacillus subtilis JQ-2 provided by the invention is CGMCC No.25660. The invention also protects the application of the bacillus subtilis JQ-2 in preparing protease and/or chymosin. The invention also provides a method for preparing the protease, which comprises the following steps: fermenting and culturing the bacillus subtilis JQ-2 to obtain fermentation liquor. The method further comprises the steps of: purifying the fermentation liquor to obtain the JQ-2 protease. The invention also provides a method for preparing the hydrolyzed peptide, which comprises the following steps: the hydrolyzed peptide is obtained by treating sodium caseinate as a substrate with JQ-2 protease. The invention has application and popularization values for related industries of protease.
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 protein and polypeptide hydrolysis, and is widely present in animal viscera, plant stems and leaves, fruits and microorganisms. Proteases are used in large quantities in cheese production, meat tenderization and vegetable protein modification.
The dehairing and softening of the leather industry has made use of a large amount of proteases, both to save time and to improve the labour hygiene conditions. The protease can also be used for degumming silk, tenderizing meat, and clarifying wine. The compound can be clinically used as a medicine, such as treatment of dyspepsia by pepsin, treatment of bronchitis by acidic protease, treatment of vasculitis by dread sexual protease, and cleaning of surgical suppurative wounds and treatment of pleural effusion adhesion by trypsin and chymotrypsin. The enzymatic washing powder is a new product in detergent, contains alkaline protease and can remove bloodstain and protein dirt on clothes.
Disclosure of Invention
The invention aims to provide a bacillus subtilis strain producing protease in distiller's yeast and application thereof.
The Bacillus subtilis JQ-2 provided by the invention has been preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 of West Lu No. 1 of the Beijing Korean district, the institute of microbiology of China academy of sciences) in 09.07.2022, and the preservation registration number is CGMCC No.25660.
The invention also protects the application of the bacillus subtilis JQ-2 in preparing protease and/or chymosin.
In the application, the protease and/or the chymosin are obtained by fermenting and culturing the bacillus subtilis JQ-2.
The fermentation culture of the bacillus subtilis JQ-2 comprises the following steps: inoculating the bacillus subtilis JQ-2 to a fermentation culture medium, and carrying out shaking culture at 30 ℃ and 180r/min for 24h.
The fermentation culture of the bacillus subtilis JQ-2 comprises the following steps: inoculating the bacillus subtilis JQ-2 seed solution to a fermentation culture medium in an inoculation amount of 5%, and performing shaking culture at 30 ℃ and 180r/min for 24h.
The fermentation culture of the bacillus subtilis JQ-2 comprises the following steps: inoculating the bacillus subtilis JQ-2 seed solution to a fermentation culture medium (the liquid loading of the culture medium is 20%) in an inoculation amount of 5%, and performing shaking culture at 30 ℃ and 180r/min for 24 hours.
OD of Bacillus subtilis JQ-2 seed liquid 600nm The value was 1.3.
The preparation method of the bacillus subtilis JQ-2 seed liquid comprises the following steps: inoculating Bacillus subtilis JQ-2 to liquid LB culture medium, performing shaking culture at 30 deg.C and 120r/min to OD 600nm The value was 1.3, and a seed liquid was obtained.
The invention also protects the fermentation liquor of the bacillus subtilis JQ-2.
The preparation method of the fermentation liquor can be as follows: inoculating the bacillus subtilis JQ-2 to a fermentation culture medium, and carrying out shaking culture at 30 ℃ and 180r/min for 24h.
The preparation method of the fermentation liquor can be as follows: inoculating the bacillus subtilis JQ-2 seed solution to a fermentation culture medium by the inoculation amount of 5%, and performing shake culture at 30 ℃ and 180r/min for 24h.
The preparation method of the fermentation liquor can be as follows: inoculating the bacillus subtilis JQ-2 seed solution to a fermentation culture medium (the liquid loading of the culture medium is 20%) in an inoculation amount of 5%, and performing shaking culture at 30 ℃ and 180r/min for 24 hours.
OD of Bacillus subtilis JQ-2 seed liquid 600nm The value was 1.3.
The preparation method of the bacillus subtilis JQ-2 seed liquid comprises the following steps: inoculating Bacillus subtilis JQ-2 to liquid LB culture medium, performing shaking culture at 30 deg.C and 120r/min to OD 600nm The value was 1.3, and a seed liquid was obtained.
The invention also protects the application of the fermentation liquor in the preparation of protease and/or chymosin.
The invention also protects the use of the fermentation broth as a protease and/or chymosin.
The invention also provides a method for preparing the protease, which comprises the following steps: fermenting and culturing the bacillus subtilis JQ-2 to obtain fermentation liquor.
The fermentation culture of the bacillus subtilis JQ-2 comprises the following steps: inoculating the bacillus subtilis JQ-2 to a fermentation culture medium, and carrying out shaking culture at 30 ℃ and 180r/min for 24h.
The fermentation culture of the bacillus subtilis JQ-2 comprises the following steps: inoculating the bacillus subtilis JQ-2 seed solution to a fermentation culture medium in an inoculation amount of 5%, and performing shaking culture at 30 ℃ and 180r/min for 24h.
The fermentation culture of the bacillus subtilis JQ-2 comprises the following steps: inoculating the bacillus subtilis JQ-2 seed solution to a fermentation culture medium (the liquid loading of the culture medium is 20%) in an inoculation amount of 5%, and performing shaking culture at 30 ℃ and 180r/min for 24 hours.
OD of Bacillus subtilis JQ-2 seed liquid 600nm The value was 1.3.
The preparation method of the bacillus subtilis JQ-2 seed liquid comprises the following steps: inoculating Bacillus subtilis JQ-2 to liquid LB culture medium, performing shaking culture at 30 deg.C and 120r/min to OD 600nm The value was 1.3, and a seed liquid was obtained.
The method further comprises the steps of: and purifying the protease from the fermentation liquor.
The purification method sequentially comprises the following steps: ammonium sulfate precipitation and anion exchange column purification.
The ammonium sulfate precipitation is 70% saturation ammonium sulfate precipitation.
The ammonium sulfate precipitation is 70% saturation ammonium sulfate precipitation in an environment at 4 ℃.
The ammonium sulfate precipitation method specifically comprises the following steps: adding ammonium sulfate into the fermentation liquid to 70% saturation degree, magnetically stirring at 4 deg.C for 20min, and standing at 4 deg.C for 2 hr.
The parameters for anion exchange column purification were as follows:
anion chromatography column: the filler is DEAE-sepharose FF, and the column model is 2.6 multiplied by 40cm;
and (3) an elution process: the column was equilibrated with Tris-HCl buffer, then loaded, and then eluted with Tris-HCl buffer.
The elution flow rate may be specifically 2mL/min.
Collecting the solution after passing the column with retention time of 26-159min during the elution process.
The solution after passing the column with the retention volume of 52-318mL is collected during the elution process.
The preparation method of the sample liquid comprises the following steps: after completing the ammonium sulfate precipitation, centrifugally collecting the precipitate; dissolving the precipitate with PBS buffer solution, transferring into dialysis bag (molecular weight cut-off is 8000-14000 KDa), dialyzing in PBS buffer solution, collecting liquid phase in 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, namely the sample solution.
The preparation method of the sample liquid comprises the following steps: after completing the ammonium sulfate precipitation, centrifuging at 4 ℃ and 10000 Xg for 10min, and collecting the precipitate; dissolving the precipitate with PBS buffer solution, transferring into dialysis bag (molecular weight cut-off is 8000-14000 Da), dialyzing the dialysis bag in PBS buffer solution at 4 deg.C, collecting liquid phase in 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, namely the sample loading solution.
The protease has 11 segments; the 11 segments are respectively shown as a sequence 2 to a sequence 12 in a sequence table.
The protease is shown as a sequence 13 in a sequence table.
The fermentation medium (pH 7) described in any of the above: contains 5.4g/L potato extract powder, 1.8g/L malt extract, 10.3g/L 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 application of the JQ-2 protease as a protease.
Any one of the above proteases is a proteolytic enzyme.
Any one of the above proteases is a protein having proteolytic activity.
The invention also provides a method for preparing the hydrolyzed peptide, which comprises the following steps: the hydrolyzed peptide is obtained by treating sodium caseinate as a substrate with JQ-2 protease.
In the preparation method of the hydrolysis peptide, the ratio of the sodium caseinate to the 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, centrifuging, and collecting 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 37 deg.C for 24h, heating in 100 deg.C boiling water bath for 10min, centrifuging at 4 deg.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 hydrolyzed peptide prepared by the method also belongs to the protection scope of the invention.
The invention also protects the application of the hydrolyzed peptide, which is (e) or (f) or (g) as follows:
(e) Scavenging DPPH free radicals
(f) Inhibition of alpha-glucosidase
(g) Inhibit 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 the hydrolyzed peptide. The hydrolyzed peptide provided by the invention has excellent efficacy and performance. The invention has application and popularization values for related industries of protease.
Drawings
FIG. 1 is a photograph of Bacillus subtilis JQ-2 on a solid casein medium plate.
FIG. 2 is a photograph showing the cell morphology and 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 shows the results of evaluation of 6 factors affecting the enzyme production by Bacillus subtilis fermentation.
Fig. 6 is a three-dimensional response surface plot showing the effect of variables on proteolytic activity.
FIG. 7 is a graph of the results of the process of optimizing ammonium sulfate precipitation parameters.
FIG. 8 is a graph showing the results of the separation and purification process of JQ-2 protease.
FIG. 9 is an electrophoretogram during separation and purification 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 different substrates by JQ-2 protease.
FIG. 12 is a graph showing the results of kinetic analysis of JQ-2 protease.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way. The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Unless otherwise stated, the quantitative tests in the following examples were performed in triplicate, and the results were averaged. In the examples, the inoculation amount refers to the volume ratio of the seed solution to the culture medium, for example, 3% is inoculated when 3mL of the seed solution is inoculated to 100mL of the culture medium, and 5% is inoculated when 5mL of the seed solution is inoculated to 100mL of the culture medium. In the examples, the liquid content refers to the volume percentage of the culture medium in the culture container, for example, 50mL of the culture medium in a 250mL triangular flask, and the liquid content is 20%.
Example 1 method for detecting enzyme Activity
1. Determination of proteolytic Activity
Sodium caseinate: sigma-Aldrich, CAS-No 9005-46-3, C8654-500G.
Diluting the test solution to 10 times of the volume of the test solution by using water to obtain the test solution diluent.
1. Test group treatment method:
(1) taking 1mL of 1g/100mL sodium caseinate aqueous solution, adding 1mL of test solution diluent, uniformly mixing, reacting in a constant-temperature water bath at 40 ℃ for 10min, then adding 2mL of 0.4mol/L trichloroacetic acid aqueous solution to stop the reaction, then centrifuging at 10000r/min for 5min, and collecting supernatant.
(2) Taking 1mL of the supernatant obtained in the step (1), and adding 5mL of 0.4mol/L Na 2 CO 3 Reacting the aqueous solution with 1mL of forskolin-phenol reagent (Shanghai Michelin Biochemical technology Co., ltd.) at 40 deg.C for 20min, and measuring absorbance A at 680nm 680 。
2. Blank group processing method:
(1) adding 2mL of 0.4mol/L trichloroacetic acid aqueous solution into 1mL of test solution diluent, mixing, then adding 1mL of 1g/100mL of sodium caseinate aqueous solution, reacting in 40 ℃ constant temperature water bath for 10min, then centrifuging at 10000r/min for 5min, and collecting supernatant.
(2) Taking 1mL of the supernatant obtained in the step (1), and adding 5mL of 0.4mol/L Na 2 CO 3 The aqueous solution and 1mL of a folin-phenol reagent were reacted at 40 ℃ for 20min, and the absorbance, A ', at a wavelength of 680nm was measured' 680 。
3. Calculation of proteolytic Activity
The amount of enzyme required to catalyze the hydrolysis of sodium caseinate to produce 1. Mu.g of tyrosine within 1min at 40 ℃ is defined as 1U.
The Proteolytic Activity (PA) of the test solution is in units of: U/mL.
PA＝K×(A 680 -A’ 680 )×n×4÷10；
K＝105.52；
And n is the dilution multiple of the test solution diluent prepared by the test solution.
2. Determination of curd vitality (Arima time method)
Skim milk powder: a new zealand permanent natural group.
Diluting the test solution to 10 times of volume with water to obtain the test solution diluent.
Dissolving defatted milk powder in 0.01mol/L CaCl 2 The water solution is used to make the concentration of the skim milk powder be 10g/100ml, thus obtaining the skim milk solution. The fresh skim milk solution was allowed to stand at room temperature for 30min and then was dispensed into small test tubes, each of which was 5mL. And (3) placing the small test tube in a 35 ℃ constant-temperature water bath box, preserving the heat for 5min, then adding 0.5mL of test solution diluent in a 35 ℃ environment, shaking uniformly, starting timing, and stopping timing (timing unit is s) immediately when flocculent precipitates begin to appear.
The units of curd viability (MCA) of the test solution are: SU/mL.
MCA＝(2400×V 1 ×n)÷(V 2 ×t)；
t is timing time with the unit of s;
V 1 the volume of the skim milk solution is 5mL;
n is the dilution multiple of the test solution diluent prepared by the test solution;
V 2 the volume of the test solution dilution added was 0.5mL.
Example 2 acquisition and preservation of Bacillus subtilis JQ-2
1. Preliminary screening of strains from koji samples
The distiller's yeast samples are respectively: collecting wheat koji and bran koji from Shanxi Jincheng; daqu and Xiaoqu collected from Suzhou, jiangsu.
1. Separating single strain from sample
(1) Weighing 25g of distiller's yeast sample, adding into a conical flask containing 250mL of sterile normal saline, shaking, mixing, and performing gradient dilution with sterile normal saline to obtain 6 dilutions (10 dilutions) -1 -10 -6 Dilution). 100 mul of the dilution was spread on a solid LB medium plate and inverted in a 30 ℃ incubator for culture.
(2) And (2) respectively selecting a small amount of colonies with different shapes and sizes when the colonies are cultured in the step (1) on days 2, 3 and 4, inoculating the colonies on a solid medium plate, and performing streak purification for multiple times to obtain a plurality of single strains. Three solid media were set up: solid LB medium, solid YEPD medium and solid PDA medium. In the step, the growth condition of the strains is continuously observed, and each single strain selects an optimal culture medium from three solid culture media for subsequent culture of the single strain.
(3) And (3) after the step (2) is finished, picking the single colony to a corresponding liquid culture medium (the agar powder is removed from the corresponding liquid culture medium, namely the optimal culture medium screened in the step (2)), and culturing for 24 hours.
(4) After the step (3) is finished, the bacterial liquid and 50% glycerol are mixed uniformly in equal volume by using a freezing tube, and the mixture is stored at the temperature of-80 ℃.
2. Screening for protease producing strains
The test strains are respectively as follows: each individual strain obtained in step 1.
Test strains are selected and inoculated to a solid casein medium plate, cultured for 2d at 37 ℃, and then the diameter of a curdling ring, the diameter of a hydrolysis ring and the diameter of thalli are measured.
Solid casein medium: contains 2.5g/L of peptone, 10g/L of glucose, 1g/L of yeast extract powder, 10g/L of sodium caseinate, 20g/L of agar and the balance of water.
The activity of 11 strains is higher. The 11 strains were designated as: JQ-1, JQ-2, JQ-3, F1, F2, F3, F4, D1, D2, X1 and X2.JQ represents wheat koji isolated from Shanxi distillery, F represents bran koji isolated from Shanxi distillery, D represents Daqu isolated from Jiangsu Suzhou distillery, and X represents Xiaoqu isolated from Jiangsu Suzhou distillery. The "curd circle diameter/cell diameter" and "hydrolysis circle diameter/cell diameter" of the 11 strains are shown in Table 1.
TABLE 1
2. Further screening the target strain from the strains obtained by primary screening
Test strains: the strains obtained in step one were 11 strains (JQ-1, JQ-2, JQ-3, F1, F2, F3, F4, D1, D2, X1 and X2), respectively.
1. Activation of the Strain
Inoculating the test strain to liquid LB culture medium, performing shaking culture at 30 deg.C and 120r/min to OD 600nm The value was 1.3, and a seed liquid was obtained.
2. Fermentation of the Strain
Inoculating the seed solution obtained in the step 1 to a liquid LB culture medium with the inoculation amount of 3%, performing shaking culture at 30 ℃ and 120r/min for 24 hours, then centrifuging at 4 ℃ and 10000r/min for 10 minutes, and collecting supernatant, namely fermentation liquor.
The fermentation broth was used as a test solution to examine the proteolytic activity and the curd activity, and the results 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
| Bacterial strains | Proteolytic Activity of fermentation broth (U/mL) | 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 ring and curd ring on solid casein culture medium plate (the picture is shown in figure 1, the dotted line marks the thallus diameter, the dotted line marks the hydrolysis ring diameter, and the straight line marks the curd ring diameter); the cells are in a short rod shape, single cells or multiple cells are connected end to form a chain shape (see the right graph of the figure 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 the left picture of figure 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 comparison identification is carried out in the NCBI website gene library, and the result is shown in figure 3.
As a result of comprehensive identification, the strain JQ-2 belongs to Bacillus subtilis, and is named as Bacillus subtilis JQ-2.
4. Preservation of JQ-2
Bacillus subtilis JQ-2 is preserved in China general microbiological culture Collection center (CGMCC, china academy of sciences microbiological research institute, no. 3 of Xilu No. 1 on North Chen of Chaoyang district, beijing) in 2022 at 07.09.3, and has a preservation registration number of CGMCC No.25660.
5. Growth curve of Bacillus subtilis JQ-2
Inoculating Bacillus subtilis JQ-2 to liquid LB culture medium, performing shaking culture at 30 deg.C and 120r/min, and sampling every 2 hr to detect OD 600nm The value is obtained. See fig. 4.
Example 3 optimization of culture conditions for Bacillus subtilis JQ-2
In this example, the pre-optimization medium was used as a control medium for the test medium to take into account the optimization effect. Optimization of pre-medium (natural pH): 5g/L of yeast extract powder, 10g/L of tryptone, 10g/L of sodium chloride and the balance of distilled water.
1. Preparation of seed liquid
Inoculating Bacillus subtilis JQ-2 to liquid LB culture medium, performing shaking culture at 30 deg.C and 120r/min to OD 600nm The value was 1.3, and a seed liquid was obtained.
2. Optimization of media composition
1. Effect of different carbon sources on proteolytic Activity
Test medium: a 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 of yeast extract powder, 10g/L of tryptone, 10g/L of sodium chloride and the balance of distilled water. The following carbon sources were set respectively: fructose (Beijing pinellia ternate science and technology development Co., ltd., product number a 3125), alpha-lactose (national drug group chemical reagent Co., ltd., product number 20111025), malt extract (Beijing Obormin biotechnology Co., ltd., product number 01-051), sucrose (Beijing pinellia ternate science and technology development Co., ltd., product number b 3075), or glucose (Beijing pinellia ternate science and technology development Co., ltd., product number b 2657).
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shaking culture at 30 deg.C and 120r/min for 24h, centrifuging at 4 deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the proteolytic activity assay using the fermentation broth as the test solution are shown in Table 3.
TABLE 3
| Proteolytic Activity of fermentation broth (U/mL) | |
| Carbon source culture medium for malt extract | 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. Effect of malt extract at different concentrations on proteolytic Activity
The test medium was obtained by adding malt extract to a basal medium. The concentration of the malt extract in the culture medium is respectively set to be 1.5g/L, 2g/L, 2.5g/L, 5g/L or 7.5g/L. Basal medium (natural pH): 5g/L of yeast extract powder, 10g/L of tryptone, 10g/L of sodium chloride and the balance of distilled water.
Inoculating the seed solution to a test culture medium by 3 percent of inoculation amount, performing shaking culture at 30 ℃ and 120r/min for 24h, then centrifuging at 4 ℃ and 10000r/min for 10min, and collecting supernatant to obtain fermentation liquor.
The fermentation broth was used as a test solution to examine proteolytic activity, and the results are shown in Table 4.
TABLE 4
| Proteolytic Activity of fermentation broth (U/mL) | |
| The concentration of malt extract in culture medium is 1.5g/L | 86.46±4.22 |
| The concentration of malt extract in culture medium is 2g/L | 103.4±3.97 |
| The concentration of malt extract in culture medium is 2.5g/L | 79.49±4.43 |
| The concentration of malt extract in culture medium is 5g/L | 33.53±1.48 |
| The concentration of malt extract in culture medium is 7.5g/L | 19.42±1.05 |
3. Effect of different Nitrogen sources on proteolytic Activity
Test medium: a 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): contains 5g/L yeast extract powder, 10g/L sodium chloride and the balance of distilled water. The following nitrogen sources were set respectively: sweet whey powder (Shanghai Yuan leaf science and technology Limited, cat. No. S27239-500 g), soybean peptone (Beijing Jin Ruilin science and technology development Limited, cat. No. hb 8275), skimmed milk powder (Beijing pinellia tuber science and technology development Limited, cat. No. p 2216), beef extract (Beijing Obo Boxing biotechnology Limited liability, cat. No. 01-009), or ammonium citrate (Beijing pinellia tuber science and technology development Limited, cat. No. b 2338).
Inoculating the seed solution to a test culture medium with an inoculum size of 3%, performing shaking culture at 30 deg.C and 120r/min for 24h, centrifuging at 4 deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The fermentation broth was used as a test solution to examine proteolytic activity, and the results are shown in Table 5.
TABLE 5
| Proteolytic Activity of fermentation broth (U/mL) | |
| Beef extract nitrogen source culture medium | 104.68±3.26 |
| Nitrogen source culture medium for sweet whey powder | 43.40±0.90 |
| Soybean peptone nitrogen source culture medium | 74.96±1.91 |
| Nitrogen source culture medium for skim milk powder | 43.87±1.26 |
| Ammonium citrate nitrogen source culture medium | 0.30±0.09 |
| Control Medium | 10.48±0.36 |
4. Effect of different concentrations of beef extract on proteolytic Activity
The test medium was prepared by adding beef extract to the basal medium. The concentration of the beef extract in the culture medium is respectively set to be 2.5g/L, 5g/L, 7.5g/L, 10g/L or 12.5g/L. Basal medium (natural pH): 5g/L of yeast extract 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 shaking culture at 30 deg.C and 120r/min for 24h, centrifuging at 4 deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The fermentation broth was used as a test solution to examine proteolytic activity, and the results are shown in Table 6.
TABLE 6
| Proteolytic Activity of fermentation broth (U/mL) | |
| The concentration of the beef extract in the culture medium is 2.5g/L | 19.26±0.77 |
| The concentration of the beef extract in the culture medium is 5g/L | 27.32±1.01 |
| The concentration of the beef extract in the culture medium is 7.5g/L | 43.83±1.06 |
| The concentration of the beef extract in the culture medium is 10g/L | 104.68±3.26 |
| The concentration of the 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 was prepared by adding potato extract powder (product number 01-141, obo star Biotechnology, inc. of Beijing) to a basal 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 of yeast extract powder, 10g/L of tryptone, 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 shaking culture at 30 deg.C and 120r/min for 24h, centrifuging at 4 deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The fermentation broth was used as a test solution to examine proteolytic activity, and the results are shown in Table 7.
TABLE 7
| Proteolytic Activity of fermentation broth (U/mL) | |
| The concentration of potato extract powder in the culture medium is 5g/L | 24.26±0.32 |
| The concentration of potato extract powder in the culture medium is 10g/L | 65.42±1.42 |
| The concentration of potato extract powder in the culture medium is 15g/L | 36.38±1.36 |
| The concentration of potato extract powder in the culture medium is 20g/L | 25.69±0.75 |
6. Effect of Metal ion species on proteolytic Activity
Test medium: metal ion culture medium. The metal ion culture medium is obtained by adding the compound to a basic culture medium (the concentration of the compound in the culture medium is 10 g/L). Basal medium (natural pH): 5g/L of yeast extract powder, 10g/L of tryptone, 10g/L of sodium chloride and the balance of distilled water. The following compounds were each provided: 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 shaking culture at 30 deg.C and 120r/min for 24h, centrifuging at 4 deg.C and 10000r/min for 10min, and collecting supernatant to obtain fermentation broth.
The results of the proteolytic activity assay using the fermentation broth as the test solution are shown in Table 8.
TABLE 8
7. Effect of different concentrations of magnesium sulfate on proteolytic Activity
The test medium was prepared 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 of yeast extract powder, 10g/L of tryptone, 10g/L of sodium chloride and the balance of distilled water.
Inoculating the seed solution to a test culture medium by 3 percent of inoculation amount, performing shaking culture at 30 ℃ and 120r/min for 24h, then centrifuging at 4 ℃ and 10000r/min for 10min, and collecting supernatant to obtain fermentation liquor.
The fermentation broth was used as a test solution to examine proteolytic activity, and the results are shown in Table 9.
TABLE 9
| Proteolytic Activity of fermentation broth (U/mL) | |
| 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 was 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 was 17.5g/L | 11.99±0.77 |
8. Effect of different phosphate species on proteolytic Activity
Test medium: phosphate medium. The phosphate medium is obtained by adding phosphate to a basic medium. The concentration of phosphate in the medium was 1g/L. Basal medium (natural pH): 5g/L of yeast extract powder, 10g/L of tryptone, 10g/L of sodium chloride and the balance of distilled water. The following phosphates were set up respectively: dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.
Inoculating the seed solution to a test culture medium by 3 percent of inoculation amount, performing shaking culture at 30 ℃ and 120r/min for 24h, then centrifuging at 4 ℃ and 10000r/min for 10min, and collecting supernatant to obtain fermentation liquor.
The results of proteolytic activity measurements using the fermentation broth as the test solution are shown in Table 10.
| Proteolytic Activity of fermentation broth (U/mL) | |
| Dipotassium phosphate and 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 culture 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 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 of yeast extract powder, 10g/L of tryptone, 10g/L of sodium chloride and the balance of distilled water.
Inoculating the seed solution to a test culture medium by 3 percent of inoculation amount, performing shaking culture at 30 ℃ and 120r/min for 24h, then centrifuging at 4 ℃ and 10000r/min for 10min, and collecting supernatant to obtain fermentation liquor.
The results of proteolytic activity measurements using the fermentation broth as the test solution are shown in Table 11.
TABLE 11
| Proteolytic Activity of fermentation broth (U/mL) | |
| The concentration of sodium dihydrogen phosphate in the culture medium is 0.5g/L | 10.48±0.68 |
| The concentration of sodium dihydrogen phosphate in the culture medium is 1g/L | 15.42±0.41 |
| The concentration of sodium dihydrogen phosphate in the culture medium is 1.5g/L | 24.09±1.12 |
| The concentration of sodium dihydrogen phosphate in the culture medium is 2g/L | 11.30±1.25 |
| The concentration of sodium dihydrogen phosphate in the culture medium was 2.5g/L | 9.13±0.30 |
10. Effect of different inducer species on proteolytic Activity
Test medium: inducer medium. The inducer culture medium is obtained by adding an inducer to a basal culture medium. The concentration of the inducer in the medium was 5g/L. Basal medium (natural pH): 5g/L of yeast extract powder, 10g/L of tryptone, 10g/L of sodium chloride and the balance of distilled water. The following inducers were set up respectively: phytic acid, tween 80, tween 20, sodium acetate and methanol.
Inoculating the seed solution to a test culture medium by 3 percent of inoculation amount, performing shaking culture at 30 ℃ and 120r/min for 24h, then centrifuging at 4 ℃ and 10000r/min for 10min, and collecting supernatant to obtain fermentation liquor.
The fermentation broth was used as a test solution to examine proteolytic activity, and the results are shown in Table 12.
TABLE 12
| Proteolytic Activity of fermentation broth (U/mL) | |
| Culture medium of |
0 |
| Culture medium of |
26.49±2.33 |
| Culture medium of |
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 different concentrations of sodium acetate 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 of yeast extract powder, 10g/L of tryptone, 10g/L of sodium chloride and the balance of distilled water.
Inoculating the seed solution to a test culture medium by 3 percent of inoculation amount, performing shaking culture at 30 ℃ and 120r/min for 24h, then centrifuging at 4 ℃ and 10000r/min for 10min, and collecting supernatant to obtain fermentation liquor.
The results of detecting proteolytic activity using the fermentation broth as a test solution are shown in Table 13.
Watch 13
12. Obtaining an optimized post-fermentation medium
The influence of different carbon sources, nitrogen sources, metal ions, phosphates and inducers on the production of proteolytic enzyme by bacillus subtilis fermentation is analyzed through a single-factor test, and 6 factors (the carbon sources, the nitrogen sources, potato leaching powder, the metal ions, the phosphates and the inducers) influencing the production of enzyme by bacillus subtilis fermentation are evaluated by a Plackett-Burman test design, which is shown in figure 5. 3 factors with obvious influence, namely potato extract powder, malt extract and beef extract, are screened out.
And (3) determining the optimized fermentation medium for producing the proteolytic enzyme by fermenting the bacillus subtilis through central combination experimental design in the response surface. A three-dimensional response plot showing the effect of variables on proteolytic activity is shown in figure 6.
Optimized post-fermentation medium (natural pH): contains 5.4g/L potato extract powder, 1.8g/L malt extract, 10.3g/L beef extract and the balance of water.
Inoculating the seed solution to the optimized fermentation medium with an inoculum size of 3%, performing shaking culture at 30 ℃ and 120r/min for 24h, centrifuging at 4 ℃ and 10000r/min for 10min, and collecting supernatant to obtain fermentation liquor. And (3) taking the fermentation liquor as a test solution, and detecting the proteolytic activity. The proteolytic activity of the fermentation broth is 154.75 +/-1.76U/mL, which is close to the theoretical predicted value of 157.14U/mL.
3. Optimization of culture conditions
Optimizing a post-fermentation culture medium: contains 5.4g/L potato extract powder, 1.8g/L malt extract, 10.3g/L beef extract and the balance of water.
6 culture condition parameter factors are optimized. 6 culture condition parameter factors: fermentation time, fermentation temperature, rotating speed, pH value of optimized fermentation medium, inoculation amount and liquid loading amount.
The optimized parameters are as follows: the fermentation time is 24h, the fermentation temperature is 30 ℃, the rotating speed is 180r/min, the pH of the optimized fermentation medium is 7, the inoculum size is 5 percent, and the liquid loading amount is 20 percent.
4. Fermentation of bacillus subtilis to produce enzyme by using optimized post-fermentation culture medium and optimized post-culture conditions
Optimized post-fermentation medium (pH 7): contains 5.4g/L potato extract powder, 1.8g/L malt extract, 10.3g/L beef extract and the balance of water.
Inoculating the seed solution to an optimized fermentation medium (the liquid loading amount of the medium is 20%), performing shaking culture at 30 ℃ and 180r/min for 24h, centrifuging at 4 ℃ and 10000r/min for 10min, and collecting supernatant to obtain the fermentation solution.
And (3) taking the fermentation liquor as a test solution, and detecting the proteolytic activity, 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 potato extract powder, 1.8g/L malt extract, 10.3g/L beef extract and the balance of water.
Inoculating Bacillus subtilis JQ-2 to liquid LB culture medium, performing shaking culture at 30 deg.C and 120r/min to OD 600nm The value was 1.3, and a seed liquid was obtained. Inoculating the seed solution to a fermentation culture medium (the liquid loading amount of the culture medium is 20%), performing shaking culture at 30 ℃ and 180r/min for 24h, centrifuging at 4 ℃ and 10000r/min for 10min, and collecting supernatant to obtain fermentation liquid.
2. Optimizing ammonium sulfate precipitation parameters
Taking 27 parts of fermentation liquor prepared in the first step, dividing the fermentation liquor into 9 groups, and dividing each group into 3 parts of fermentation liquor. Slowly adding ammonium sulfate into the fermentation liquid at 4 deg.C to reach preset ammonium sulfate saturation (continuously magnetically stirring), magnetically stirring at 4 deg.C for 20min, standing at 4 deg.C for 2 hr, centrifuging at 4 deg.C and 10000 Xg for 10min, and collecting supernatant. The first group did not have ammonium sulfate added. The preset ammonium sulfate saturation of the second group to the ninth group is 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% in this order. The supernatant was used instead of the test broth to test the proteolytic activity. The results are shown in FIG. 7.
3. Separation and purification of JQ-2 protease
1. Ammonium sulfate precipitation
And (2) taking the fermentation liquor prepared in the step one, slowly adding ammonium sulfate into the fermentation liquor at the temperature of 4 ℃ to enable the fermentation liquor to reach 70% saturation (continuously performing magnetic stirring in the process), then performing magnetic stirring at the temperature of 4 ℃ for 20min, then standing at the temperature of 4 ℃ for 2h, then centrifuging at the temperature of 4 ℃ and 10000 Xg for 10min, and collecting precipitates.
2. Dialysis desalination treatment
(1) The precipitate obtained in step 1 was dissolved in PBS buffer (pH 7.2, 50mM, the same applies hereinafter), and then transferred to a dialysis bag (8000-14000 kDa molecular weight cut-off), which was then placed in the PBS buffer, and subjected to dialysis desalting at 4 ℃ (the PBS buffer was changed every 8 hours, and the conductivity of the PBS buffer was measured with a conductivity meter until the conductivity was unchanged).
(2) And (2) after the step (1) is finished, collecting the liquid phase in the dialysis bag, and freeze-drying to obtain freeze-dried powder.
(3) And (3) dissolving the freeze-dried powder obtained in the step (2) in Tris-HCl buffer solution (pH7.2, 50mM, the same below) to obtain crude enzyme solution.
3. DEAE-sepharose FF purified protease
And (3) dissolving the freeze-dried powder obtained in the step (2) in Tris-HCl buffer solution to enable the concentration of the freeze-dried powder to be 10mg/mL, then filtering the solution by using a 0.45-micrometer filter membrane, and collecting filtrate.
An anion chromatographic column (the filler is DEAE-sepharose FF, the model of the column is 2.6 multiplied by 40 cm), is firstly equilibrated by 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 start of elution, and 1 tube was collected every 1 min.
Each tube was sampled as a test solution to examine proteolytic activity, and the results are shown in FIG. 8. The samples from 26 th to 159 th tubes were proteolytically active, so the solutions from these tubes were combined and concentrated by ultrafiltration with an ultrafiltration membrane having a molecular weight cut-off of 10kda (Tris-HCl buffer as the buffer system) to obtain a concentrated solution, i.e., a purified protein solution. As can be seen from FIG. 8, from the 26 th to 159 th tubes, the collected post-column solution eluted with Tris-HCl buffer corresponds to the elution time of 26-159min (corresponding to the retention volume of 52-318 mL).
4. SDS-PAGE detection
The crude enzyme solution prepared in step 2 and the purified protein solution prepared in step 3 were subjected to SDS-PAGE (12.5% separation gel and 4.5% concentrated gel), respectively, as shown in FIG. 9 (lane 1 is crude enzyme solution, and lane 2 is purified protein solution).
The purified protein solution shows single protein with electrophoresis purity, so the protein in the purified protein solution is named as JQ-2 protease, and the purified protein solution is named as JQ-2 protease solution.
5. Proteolytic Activity assay
And (3) respectively taking the fermentation liquor prepared in the step one, the crude enzyme solution 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 by adopting a Bradford method on the other hand.
Specific activity (U/mg) = proteolytic activity/total protein concentration.
The results are shown in Table 14.
TABLE 14
| Purification step | Total protein (mg) | Total activity (U) | Specific activity (U/mg) | Recovery (%) | Multiple of purification |
| 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. LC-MS protein mass spectrum identification of JQ-2 protease
Recovering the target band of the purified protein solution from the SDS-PAGE gel of step three 4 and cutting into 1mm 3 Transferred to 1.5mL microtubes and digested with trypsin. The method comprises the following steps: and (3) decoloring colloidal particles: using 50% ACN-50% 4 HCO 3 Decolorizing the solution, placing in 100 μ L/tube for 10-30min, and sucking off. And (3) dehydrating colloidal particles: adding 100% ACN 80-100 μ L, standing for 30min until the colloidal particles are white to dough, discarding ACN, and standing at room temperature for drying. Reductive alkylation: adding 10mmol/L DTT into 100 μ L/tube, reducing in 56 deg.C water bath for 1 hr, sucking out, and discarding; adding 55mmol/L IAA according to 100 μ L/tube, reacting at room temperature in dark for 1h, sucking out, and discarding; adding destaining solution (50% ACN-50% 4 HCO 3 ) Washing, removing by suction, adding 100% (v/v) ACN 80-100 μ L, standing for 30min until the colloidal particles are white and in the form of dough, removing ACN, and standing at room temperature for drying. Enzyme digestion: the enzyme (25 mmol/L NH) was taken at a concentration of 15 ng/. Mu.L 4 HCO 3 Diluting), adding enzyme solution according to the amount of 7-10 μ L/tube, incubating in 4 deg.C refrigerator for 40min, taking out, and supplementing5-10μL 25mmol/L NH 4 HCO 3 The solution was sealed and digested in a 37 ℃ water bath for 16h. Peptide fragment extraction: adding 100 μ L/tube of extractive solution (5% TFA-50% ACN-45% water), water bathing at 37 deg.C for 1h, sonicating for 5min, centrifuging for 5min, transferring the extractive solution into another new EP tube, repeating the extraction once, combining the extractive solutions, and vacuum centrifuging for drying. Mass spectrometry was performed and the results are shown in FIG. 10. Ionization fragments of the trypsin hydrolyzed bacillus subtilis JQ-2 protease are shown in a table 15, and amino acid sequences of the fragments are sequentially shown as a sequence 2 to a sequence 12 in a sequence table.
TABLE 15 ionized fragments of Bacillus subtilis JQ-2 protease after trypsin hydrolysis
| Amino acid sequence | Length of | Molecular weight | Starting position | End 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 |
| |
8 | 856.5494 | 146 | 153 |
| |
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 degree of hydrolysis of different substrates by JQ-2 protease
The test substrates were: sodium caseinate or whey protein.
Test protein solution: and (3) diluting the JQ-2 protease solution prepared in the third step with a 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 test protein solution. Tris-HCl buffer was used as a0 concentration control for the test protein solutions.
(1) Dissolving a test substrate in Tris-HCl buffer solution to ensure that the concentration of the test substrate is 1mg/mL, thus obtaining a substrate solution.
(2) 1mL of the substrate solution was mixed with 100. Mu.L of the test protein solution, incubated at 37 ℃ for 30min, then incubated in a water bath at 100 ℃ for 5min, and then sampled for SDS-PAGE.
The results are shown in FIG. 11. The degree of degradation of sodium caseinate increased with increasing concentration of JQ-2 protease. As the JQ-2 protease concentration increased, no significant degradation of the whey protein occurred.
6. JQ-2 protease kinetic analysis
And (4) taking the JQ-2 protease solution prepared in the third step as a test solution, and detecting the proteolytic activity.
The process is essentially the same as in step one of example 1. The only difference is that: the gradient concentration solution was used instead of 1g/100mL of the aqueous solution of sodium caseinate. The gradient concentration solution was as follows: sodium caseinate was dissolved in Tris-HCl buffer to concentrations 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 were calculated.
The 1/V and 1/[ S ] curves of JQ-2 protease hydrolysis of sodium caseinate are shown in FIG. 12. When the substrate concentration is low, a low value indicates a high affinity of the enzyme for the substrate. Km and Vmax values were 10.8g/L and 35.84U/mL, respectively.
Example 5 preparation of 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 prepared in step three of example 4 was freeze-dried to obtain a powdery JQ-2 protease.
Adding powdery JQ-2 protease into a sodium caseinate solution (in an initial system, the ratio of the sodium caseinate to the JQ-2 protease is 1g of sodium caseinate: 1000U of JQ-2 protease; 1000U refers to 1000U of proteolytic activity), hydrolyzing for 24h at 37 ℃, heating in a boiling water bath at 100 ℃ for 10min, centrifuging for 15min at 4 ℃ and 10000rpm, and collecting supernatant, namely the hydrolyzed peptide solution.
2. In vitro bioactivity assay for hydrolyzed peptides
1. Ability to scavenge DPPH free radicals
1mL of the hydrolyzed peptide solution was mixed with 2mL of a DPPH solution (DPPH solution: methanol as a solvent, DPPH concentration 0.2 mmol/L), and the mixture was reacted at room temperature in the dark for 1 hour, and then centrifuged at 10000r/min for 10min, and the supernatant was collected to measure the absorbance at a wavelength of 517 nm.
The blank group was replaced with an equal volume of methanol for the DPPH solution and the control group with an equal volume of sterile water for the hydrolyzed peptide solution.
Clearance (%) = [1- (A1-a)/A0 ] × 100%;
in the formula: a is the absorbance of the blank group; a0 is the absorbance of the control group; a1 is the absorbance of the sample set.
The DPPH free radical clearance 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 at 37 ℃ for 10min in a 96-well plate, 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 reaction was terminated with an aqueous solution.
Group A: the hydrolyzed peptide solution was replaced with an equal volume of sterile water, otherwise as in group C.
Group B: the aqueous alpha-glucosidase solution was replaced with an equal volume of sterile water and the hydrolyzed peptide solution was replaced with an equal volume of sterile water and p-nitrophenyl-alpha-D-glucopyranoside (normal phosphate buffer added), otherwise as in group C was removed.
Group D: p-nitrophenyl-alpha-D-glucopyranoside (phosphate buffer) was removed, and the rest was the same as in group C.
The absorbance at a wavelength of 405nm was measured.
The α -glucosidase inhibition ratio (%) = [1- (C-D)/(a-B) ] × 100%.
The alpha-glucosidase inhibition rate of the hydrolyzed peptide solution is 83.33 percent
3. Ability to inhibit ACE
ACE, known collectively as angiotensin converting enzyme, sigma, cat # a6778.
N- [3- (2-Furyl) acryloyl ] -L-phenylalanyl-glycyl-glycine (N- [3- (2-Furyl) acryloyloyl ] -Phe-Gly-Gly, FAPGG) as a substrate, and FAGG shows an absorbance change at 340nm when hydrolyzed under the catalysis of ACE.
Phosphate buffer solution: a phosphate buffer solution of 100mmol/L and pH 8.3.
Taking the hydrolyzed peptide solution, and diluting the hydrolyzed peptide solution to 2 times of volume by using phosphate buffer solution containing 300mmol/L NaCl to obtain the inhibitor solution. Dissolving FAPGG by using phosphate buffer solution to ensure that the concentration of the FAGGG is 1.6mmol/L, thus obtaining the FAGGG solution. 0.1U of ACE is taken and dissolved in 1mL of phosphate buffer solution, and the ACE solution is obtained.
Sample group: mixing 85 μ L of inhibitor solution and 15 μ L of ACE solution uniformly, adding 100 μ L of FAPGG solution, and immediately measuring absorbance at 340nm wavelength, and recording as A0; incubating at 37 deg.C for 60min, and measuring absorbance at 340nm wavelength to obtain A1; Δ a = A0-A1.
Control group: the inhibitor solution was replaced with an equal volume of phosphate buffer, and the sample sets were otherwise identical.
ACE inhibitory activity (%) = (1- Δ a sample group/. DELTA.a control group) × 100%.
The ACE inhibitory activity of the hydrolyzed peptide solution was 92.19%.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is made possible within the scope of the claims attached below.
Claims (10)
1. Bacillus subtilis JQ-2 with the preservation registration number of CGMCC No.25660.
2. A fermentation broth of the Bacillus subtilis of claim 1.
3. A method of preparing a protease, comprising the steps of: fermenting and culturing the Bacillus subtilis of claim 1 to obtain a fermentation broth.
4. The method of claim 3, wherein: the method further comprises the steps of: and purifying the protease from the fermentation liquor.
5. The method of claim 4, wherein: the protease has 11 segments; the 11 segments are respectively shown as a sequence 2 to a sequence 12 in a sequence table.
6. A protease produced by the method of any one of claims 3 to 5.
7. The application is (a) or (b) or (c) or (d) as follows:
(a) Use of the bacillus subtilis of claim 1 for the preparation of a protease and/or chymosin;
(b) Use of a fermentation broth according to claim 2 for the preparation of a protease and/or chymosin;
(c) Use of the fermentation broth according to claim 2 as protease and/or chymosin.
(d) Use of the protease of claim 6 as a protease.
8. A method of preparing a hydrolyzed peptide comprising the steps of: hydrolyzed peptides are obtained by treating with the protease of claim 6 using sodium caseinate as a substrate.
9. A hydrolyzed peptide prepared according to the method of claim 8.
10. The use of the hydrolyzed peptide of claim 9 in (e) or (f) or (g) as follows:
(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 |
|---|---|---|---|---|
| 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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