CN117487783A - Bacillus bailii (Bacillus velezensis) CYSZG-3 for producing neutral protease and application thereof - Google Patents
Bacillus bailii (Bacillus velezensis) CYSZG-3 for producing neutral protease and application thereof Download PDFInfo
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 59
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- 102000035092 Neutral proteases Human genes 0.000 title claims abstract description 26
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- 238000004519 manufacturing process Methods 0.000 claims abstract description 15
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 230000007935 neutral effect Effects 0.000 claims abstract description 7
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
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- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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Abstract
Bacillus belicus for producing neutral proteinaseBacillus velezensis) The invention relates to CYSZG-3 and application thereof, which belongs to the field of bioengineering, and provides bacillus bailii for overcoming the defect of the category of enzyme-producing strains in the current enzyme preparation fieldBacillus velezensis) CYSZG-3, the Bacillus bailiiBacillus velezensis) The CYSZG-3 can produce neutral protease through a fermentation process, and the enzyme yield of the direct fermentation broth is about 270U/mL, so that the biological safety risk is avoided. A neutral protease producing strain (bacillus bailii,Bacillus velezensis) The invention belongs to the field of enzyme preparation production and application thereofThe invention provides a neutral protease production strain, which aims to overcome the shortage of germ germplasm resources in the field of neutral protease production and reduce the production cost of neutral protease. By controlling the formula of the culture medium and the fermentation conditions, the strain can be used for producing neutral protease, and the enzyme yield of the direct fermentation broth is 270U/mL.
Description
Technical Field
The invention relates to a neutral protease-producing strain and application thereof, in particular to bacillus belicus (Bacillus velezensis) CYSZG-3 for producing neutral protease and related application thereof.
Background
Protease is used as a biocatalyst to decompose protein macromolecular substances into polypeptides and amino acids by catalyzing peptide bond hydrolysis. Proteases can be classified into acidic proteases (pH 2.5-5.0), neutral proteases (pH 7.0-8.0) and alkaline proteases (pH 9.5-10.5) according to the optimum pH for the protease-catalyzed reaction. Protease occupies an important share in the global enzyme preparation market, and the protease production value in 2019 is about 13.4 hundred million dollars, wherein the protease occupies 28% of the industrial enzyme preparation market, and the protease relates to various industries such as leather processing, meat processing, daily chemical washing, food processing, feed processing, bioactive peptide, raw silk down, dairy product processing and the like. The protease industry in China has developed for 50 years, and has a great progress, but has a certain gap compared with the international advanced enterprises. Such as: the domestic alkaline protease varieties only have the traditional 2709 and 1 gene improved 2709, the foreign alkaline protease varieties have the Norwestine Alcalase, esperase, savinase, duPont Purafect OX, cold water enzyme Properase, P2000/P4000, passion Pro104L and the like, and the neutral protease enzyme-producing strains also have the Bacillus subtilis AS1.398. Therefore, the development of new enzyme-producing strain resources is a problem which needs to be solved in the deep development of protease industry in China at present, and has important strategic value and practical production significance.
Disclosure of Invention
In order to solve the problems, the invention provides bacillus beijerinus (Bacillus velezensis) CYSZG-3. Is deposited with the microorganism strain collection center (address: guangdong province, guangdong, hirschner, 100 th edition, 59 th floor, 5 th China center for Otto, academy of sciences of Guangdong) with deposit number of GDMCC NO:63494, the preservation date is 2023, 05 and 24. The taxonomic name is Bacillus velezensis. Bacillus belicus is used as a bacterial strain for producing neutral protease, and the neutral protease is used in the deep processing industry of protein raw materials such as leather processing, detergents, bioactive peptides, animal processing, feeds and the like. The invention also relates to a screening method, enzyme activity characteristics, a culture medium formula and fermentation production conditions of the strain. The invention provides the following technical scheme:
one of the purposes of the invention is to provide bacillus beijerinus (Bacillus velezensis) CYSZG-3, named after classification: bacillus bailii (Bacillus velezensis).
A second object of the present invention is to provide the use of Bacillus belicus (Bacillus velezensis) CYSZG-3 as described above, wherein the Bacillus belicus (Bacillus velezensis) CYSZG-3 is used for the production of neutral protease, preferably for the further processing industry of protein raw materials such as leather processing, detergents, bioactive peptides, animal processing, feed and the like.
The bacillus belicus (Bacillus velezensis) CYSZG-3 provided by the invention can be fermented to produce enzyme by taking bean pulp powder as a main fermentation medium raw material.
The protease produced by using bacillus belicus (Bacillus velezensis) CYSZG-3 can have higher proteolytic activity under neutral conditions and has a certain tolerance to alkaline conditions.
A neutral proteinase enzyme-producing strain is prepared from bacillus belicus (Bacillus velezensis) CYSZG-3, and can be used for producing neutral proteinase, which can hydrolyze protein under neutral and weak alkaline conditions, and the enzyme-producing strain also comprises a nutrient medium. The nutrient medium is selected from any one of beef extract peptone medium, LB nutrient medium and inorganic salt medium added with carbon and nitrogen source, wherein the carbon and nitrogen source is preferably soybean meal.
The beef extract peptone culture medium comprises the following components in percentage by weight: beef extract 3.0g/L, peptone 10.0g/L, sodium chloride 5.0g/L, pH=7.0-8.0.
The LB nutrient medium comprises the following components in percentage by weight: yeast powder 5.0g/L, sodium chloride 10.0g/L, peptone 10.0g/L, pH=7.0-8.0.
The components and the contents of the fermentation medium are as follows: bean pulp powder 2.0g/L, potassium nitrate 3.8g/L, dipotassium hydrogen phosphate 1.0g/L, ammonium sulfate 1.0g/L, calcium chloride 0.3g/L, magnesium sulfate heptahydrate 0.3g/L, and pH=7.5.
The invention provides an application of bacillus belicus (Bacillus velezensis) CYSZG-3 in alkaline protease preparation in production, which comprises the following application steps:
a) The culture medium formula of the Bacillus belicus (Bacillus velezensis) CYSZG-3 is configured. The formula of the inorganic salt solution is as follows: 3.8g/L of potassium nitrate, 1.0g/L of dipotassium hydrogen phosphate, 1.0g/L of ammonium sulfate, 0.3g/L of calcium chloride and 0.3g/L of magnesium sulfate heptahydrate. Preferably, the optimal usage amount of the corn protein powder is 4.0g/L, the pH is regulated to 7.5 after the preparation, and the corn protein powder is sterilized at the high temperature of 0.1MPa for 30min at 121 ℃.
b) Bacillus bailii (Bacillus velezensis) CYSZG-3 was inoculated at an inoculum size of 1%, and cultured under shaking culture conditions of 180r/min at 37℃as a preferred example. Preferably, the optimal incubation time is 40h. The enzyme yield under this culture condition was highest.
The invention has the beneficial effects that the Bacillus bailii (Bacillus velezensis) CYSZG-3 can produce neutral protease under the preferable culture condition.
Drawings
FIG. 1 is a colony morphology of Bacillus belicus (Bacillus velezensis) CYSZG-3 in example 2 of the invention;
FIG. 2 shows the enzyme activities of the protease produced by Bacillus bailii (Bacillus velezensis) CYSZG-3 in example 3 under different pH conditions;
FIG. 3 shows the enzyme activities of the protease produced by Bacillus bailii (Bacillus velezensis) CYSZG-3 in example 4 under different temperature conditions;
FIG. 4 shows the enzyme activity of the protease produced by Bacillus bailii (Bacillus velezensis) CYSZG-3 in example 5 under different NaCl salt concentrations;
FIG. 5 shows the enzyme productivity of Bacillus bailii (Bacillus velezensis) CYSZG-3 in different carbon source media according to example 6 of the invention;
FIG. 6 is a growth curve of Bacillus belicus (Bacillus velezensis) CYSZG-3 of example 7 of the invention when produced in optimized medium using a fermenter.
FIG. 7 shows the enzyme production profile of Bacillus belicus (Bacillus velezensis) CYSZG-3 of example 8 of the invention in optimized medium using fermenter production.
Detailed Description
For the purpose of promoting an understanding of the principles and advantages of the invention, reference will now be made in detail to the drawings and specific examples.
The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below, unless otherwise specified, are all conventional biochemical reagents and are commercially available.
Example 1
Screening to obtain Bacillus belicus (Bacillus velezensis) CYSZG-3 for producing neutral protease, wherein the screening process is as follows:
step 1), taking an activated sludge sample from an aerobic treatment tank of casing wastewater;
step 2), 10mL of activated sludge is inoculated into 100mL of skimmed milk powder enrichment culture medium (10% skimmed milk powder, 1.0g/L of dipotassium hydrogen phosphate, 5.0g/L of sodium chloride and 1mL/L of microelement solution, and pH is adjusted to 7.0), and enrichment culture is carried out for 5 days at 37 ℃ and 180r/min, so that enrichment culture solution is obtained.
And 3) absorbing the enrichment culture solution in the step 2), adopting a dilution plate method, separating and primary screening and culturing a screening and separating culture medium (10 g/L of casein, 1.0g/L of dipotassium hydrogen phosphate, 1.0g/L of magnesium sulfate, 1.0g/L of calcium chloride, 0.1g/L of ferric chloride, 3.0g/L of sodium chloride and 10g/L of agar, adjusting pH7.2, and sterilizing at the high temperature and the high pressure of 121 ℃), observing once every 12 hours, picking out the first 10 bacterial colonies with the largest degradation circle, inoculating in an LB culture medium, purifying and naming.
Step 4), inoculating the strains named in the step 3) into a rescreening culture medium (10 g/L of casein, 1.0g/L of dipotassium hydrogen phosphate, 1.0g/L of magnesium sulfate, 1.0g/L of calcium chloride, 0.1g/L of ferric chloride and 3.0g/L of sodium chloride, adjusting pH7.2, and sterilizing at high temperature and high pressure at 121 ℃) for shake flask fermentation culture, wherein the culture conditions are 37 ℃ and 180r/min, sampling once every 24 hours, measuring the protease activity of the fermentation broth by referring to a standard protease activity measuring method (GB/T28715-2012), and selecting bacteria with the highest protease activity as protease-producing strains of the final rescreening.
Step 5), absorbing the re-screened strain in the step 4), and obtaining single colonies with the same colony morphology in an LB culture medium by adopting a dilution coating method;
and 6), picking the single colony in the step 5), placing the single colony into a centrifuge tube, mixing the single colony with glycerin (volume fraction is 30%) in a volume ratio of 1:1 to prepare bacterial liquid, and storing the bacterial liquid in an ultralow temperature refrigerator at-80 ℃.
Example 2
The strains screened in example 1 were identified.
In example 1 of the present invention, 1 strain of the most preferred neutral protease producing strain was selected and designated CYSZG-3 in the present invention. Wherein, CYSZG-3 is identified as bacillus bailii (Bacillus velezensis).
Bacillus belicus (Bacillus velezensis) grows well on LB agar plate culture medium, and as shown in figure 1, the colony is in a regular shape, round and convex, transparent, uniform in size, 2-4 mm in diameter, dry in surface, flat in edge, and has mobility and facultative aerobic growth. The growth temperature of the strain is 25-40 ℃, the pH is 6-9, and the optimal growth conditions are as follows: the growth temperature was 37℃and the pH 7.5.
DNA was extracted from the single colony obtained in step 5) of example 1, and the strain was subjected to DNA sequencing, the 16S rRNA gene sequence of which is shown in SEQ ID No. 1. The strain is also called Bacillus bailii (Bacillus velezensis) CYSZG-3 in the invention.
Example 3
The neutral protease producing culture conditions of Bacillus belicus (Bacillus velezensis) CYSZG-3 obtained in example 2 were optimized. The method comprises the following specific steps:
step 1), respectively selecting different types of carbon sources as main fermentation medium materials of bacillus bailii (Bacillus velezensis) CYSZG-3 to prepare culture mediums for optimal screening, wherein the culture mediums comprise peptone, corn meal, soybean meal, peanut meal, soybean meal, fish meal and the like.
Step 2) Bacillus belicus (Bacillus velezensis) CYSZG-3 obtained in example 1 was inoculated into the above culture media of different carbon and nitrogen sources, and fermented with shake flask, and the neutral protease activity of the fermentation broth was measured on a 24-hour sample.
Step 3) taking the fermentation broth in step 2) and measuring the protease activity of the fermentation broth according to a standard protease activity measuring method (GB/T28715-2012).
The results are detailed in FIG. 2. As can be seen from FIG. 2, the carbon source with the number 8508 has the highest enzyme yield.
Example 4
The enzyme solution produced by Bacillus belicus (Bacillus velezensis) CYSZG-3 obtained in example 3 was subjected to performance evaluation, and the change of enzyme activity of the bacterium producing enzyme at different temperatures, pH and salt concentrations was evaluated. The method comprises the following specific steps:
step 1) Bacillus bailii (Bacillus velezensis) CYSZG-3 obtained in example 1 was inoculated into a medium and cultured at 37℃for 180 r/min.
Step 2) taking the fermentation broth in the step 1), and filtering and sterilizing by using a microporous filter membrane with the diameter of 0.22 mu m to obtain a crude enzyme liquid CYSZG-3.
Step 3), protease activity of the crude enzyme liquid CYSZG-3 at different temperatures is determined by adopting a standard protease activity determination method (GB/T28715-2012). The reaction temperatures at the time of enzyme activity measurement were set at 30℃at 40℃at 50℃at 60℃at 70℃at 80℃at 90 ℃.
The results are detailed in FIG. 3. The results show that: the results show that the protease produced by Bacillus belicus (Bacillus velezensis) CYSZG-3 has the highest enzyme activity at 60 ℃.
Step 4), protease activity of the crude enzyme liquid CYSZG-3 in buffers with different pH values is determined by adopting a standard protease activity determination method (GB/T28715-2012). Measuring the activity of protease produced by Bacillus bailii (Bacillus velezensis) CYSZG-3 under neutral condition when the pH of the buffer for enzyme activity measurement is set to 7.0; measuring the activity of protease produced by Bacillus bailii (Bacillus velezensis) CYSZG-3 under alkaline condition when the pH of the buffer for enzyme activity measurement is set to 10.5; the activity of the protease produced by Bacillus bailii (Bacillus velezensis) CYSZG-3 under acidic conditions was measured at a buffer pH of 4.0 for the enzyme activity assay.
The results are shown in detail in FIG. 4. The results show that the protease produced by Bacillus bailii (Bacillus velezensis) CYSZG-3 has activity close to 0 under the acidic (4.0) condition, the highest enzyme activity under the neutral (7.0) condition and the second enzyme activity under the alkaline (10.5) condition.
Step 5), protease activity of the crude enzyme liquid CYSZG-3 in different salt solutions is determined by adopting a standard protease enzyme activity determination method (GB/T28715-2012). NaCl is added into the reaction system during enzyme activity measurement to ensure that the salt concentration is 5, 10, 20 and 50g/L.
The results are detailed in FIG. 5. The results showed that the activity of the protease produced by Bacillus belicus (Bacillus velezensis) CYSZG-3 decreased with the increase of the salt concentration in the reaction system.
Example 5
The optimized medium obtained for Bacillus belicus (Bacillus velezensis) CYSZG-3 obtained in example 3 was subjected to fermentation culture. The method comprises the following specific steps:
step 1), optimal medium formulation for Bacillus bailii (Bacillus velezensis) CYSZG-3 obtained in example 1.
Step 2), adjusting the pH value of the culture medium formula to 7, and carrying out enzyme production culture on bacillus bailii (Bacillus velezensis) CYSZG-3 by using a 20L fermentation tank under the conditions of 37 ℃ and ventilation of 1.0 vvm.
Step 3) taking the fermentation liquor in the step 2) at intervals, and measuring the number of viable bacteria in the fermentation process by colony counting through a dilution plate method. The growth curve of Bacillus belicus (Bacillus velezensis) CYSZG-3 during fermentation was determined. The pH value of the culture medium formula is 5.5, 7.0 and 8.5, and bacillus belicus (Bacillus velezensis) CYSZG-3 is subjected to enzyme production culture at 37 ℃ to respectively determine the enzyme activities at different times.
The results are shown in detail in FIG. 6. The results showed that the number of viable bacteria was highest at 8-10h, when the activity of the bacteria was highest.
Step 4) taking the fermentation broth in the step 2) at intervals, filtering and sterilizing by using a filter membrane with the diameter of 0.22 mu m, and carrying out enzyme activity test on the sterilized filtrate by using a standard protease enzyme activity measuring method (GB/T28715-2012).
The results are shown in detail in FIG. 7. The results showed that the enzyme yield of the protease increased sharply and then increased slowly and remained stable for 0-8h, and the optimal fermentation time under the fermentation conditions of step 1) was 22h. The highest enzyme activity of the fermentation broth is 270U/mL.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. Bacillus bailii @ and its preparationBacillus velezensis) The application of CYSZG-3 is characterized in that the bacillus bailii is used for preparing the strainBacillus velezensis) CYSZG-3 is used for producing neutral protease.
2. The bacillus belicus according to claim 1Bacillus velezensis) The application of CYSZG-3 is characterized in that the bacillus bailii is used for preparing the strainBacillus velezensis) The CYSZG-3 is used for producing neutral protease and is applied to the decomposition of proteins and polypeptides under neutral and alkalescent conditions.
3. Bacillus belicus according to any one of claims 1 or 2Bacillus velezensis) The application of CYSZG-3 is characterized in that the reaction condition for decomposing protein and polypeptide substances is neutral or weak alkaline condition.
4. Bacillus belicus according to any one of claims 1 or 2Bacillus velezensis) The application of CYSZG-3 is characterized by applying bacillus bailiiBacillus velezensis) Production of neutral protease by CYSZG-3 is carried out under aerobic conditions.
5. Bacillus belicus according to any one of claims 1 or 2Bacillus velezensis) The application of CYSZG-3 is characterized by using bacillus bailiiBacillus velezensis) Production of neutral protease by CYSZG-3 was carried out using a special medium (soybean meal).
6. Bacillus belicus according to any one of claims 1 or 2Bacillus velezensis) The application of CYSZG-3 is characterized in that the bacillus bailii is used for preparing the strainBacillus velezensis) The protease produced by CYSZG-3 was 270U/mL.
7. A neutral protease preparation, characterized in that the neutral protease preparation adopts Bacillus belicus according to claim 1Bacillus velezensis) The CYSZG-3 is prepared.
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