CN111394341A - Method for producing serine protease bacillus subtilis and application thereof - Google Patents
Method for producing serine protease bacillus subtilis and application thereof Download PDFInfo
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- 102000012479 Serine Proteases Human genes 0.000 title claims abstract description 43
- 108010022999 Serine Proteases Proteins 0.000 title claims abstract description 43
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 23
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 title abstract description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 34
- 230000004151 fermentation Effects 0.000 claims abstract description 34
- 230000000694 effects Effects 0.000 claims abstract description 14
- 241000243785 Meloidogyne javanica Species 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 12
- 239000001963 growth medium Substances 0.000 claims abstract description 11
- 239000000843 powder Substances 0.000 claims abstract description 8
- 238000005086 pumping Methods 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 6
- 239000000919 ceramic Substances 0.000 claims abstract description 4
- 239000012528 membrane Substances 0.000 claims abstract description 4
- 238000011081 inoculation Methods 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 15
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 235000005822 corn Nutrition 0.000 claims description 7
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 7
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 7
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 7
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 5
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 4
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 4
- 229920002261 Corn starch Polymers 0.000 claims description 3
- 235000019764 Soybean Meal Nutrition 0.000 claims description 3
- 239000008120 corn starch Substances 0.000 claims description 3
- 239000004455 soybean meal Substances 0.000 claims description 3
- 230000002265 prevention Effects 0.000 abstract description 13
- 241000196324 Embryophyta Species 0.000 description 10
- 238000010790 dilution Methods 0.000 description 10
- 239000012895 dilution Substances 0.000 description 10
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 6
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- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 6
- 241000243786 Meloidogyne incognita Species 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000244206 Nematoda Species 0.000 description 3
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- 241000193747 Bacillus firmus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
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- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 244000297179 Syringa vulgaris Species 0.000 description 1
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- 241000223260 Trichoderma harzianum Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 229940005348 bacillus firmus Drugs 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- 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
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21014—Microbial serine proteases (3.4.21.14)
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Abstract
The invention provides a method for producing bacillus subtilis of serine protease, S1: inoculating the bacillus subtilis strain into a culture medium, wherein the culture condition is 24-36 h and the temperature is 37 ℃, and obtaining a fermentation strain; s2: inoculating the fermentation strain in a seed tank, wherein the inoculation amount is 2%, and performing strain expansion culture; s3: s2, inoculating the strain after the enlarged culture into a fermentation tank for fermentation culture, and taking fermentation liquor; s4: pumping the fermentation liquor obtained in the step S3 into a centrifuge, and centrifuging for 4-5 hours at the rotating speed of 2900-3000 rpm; s5: pumping the centrifuged light phase into a ceramic membrane for filtration, wherein the aperture is 500nm, and the filtration time is 4-5 h; s6, filtering, adding a carrier, and drying to obtain powder serine protease. The method for producing the bacillus subtilis of the serine protease, provided by the invention, is simple and strong in operability, and the prepared serine protease is high in activity, can be applied to the prevention and treatment of root-knot nematodes, and is good in prevention and treatment effect.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for producing serine protease bacillus subtilis and application thereof.
Background
Root-knot nematodes are highly obligate parasitic plant pathogenic nematodes, mainly damaging the roots of various plants, and are characterized in that lateral roots and fibrous roots are increased more normally, and form spherical or conical white nodules with different sizes on fibrous roots of young roots, some of which are in the shape of moniliforme, the overground parts of the damaged plants grow short and small, slowly, the leaf color is abnormal, the fruiting rate is low, the yield is low, and even the plants die in advance.
Plant nematode disease is statistically lost to major crops worldwide in the region of $ 1000 billion per year, with more than 50% loss due to root-knot nematodes.
At present, Chinese prevention and control measures for root-knot nematodes mainly comprise agricultural prevention and control, physical prevention and control, chemical prevention and control and biological prevention and control, but mainly chemical prevention and control, chemical nematocides have high toxicity, and can cause a plurality of problems of serious environmental pollution, drug resistance and the like. The cultivation of disease-resistant varieties is the most economic and effective measure for preventing and treating root knot nematode diseases, but the progress of disease-resistant breeding is limited due to pathogenic differentiation and the lack of disease-resistant germplasm resources, and biological prevention is one of the key points and hot points of nematode prevention and treatment at home and abroad at present because the biological prevention has the advantages of cleanness and high efficiency.
At present, biological control related to China only relates to few single biological control strains such as lilac violet spore bacteria, bacillus firmus, trichoderma harzianum and the like which are developed into nematocide, and the prepared biological nematocide has the technical problems of high cost, low efficiency, long time consumption and the like.
Furthermore, the literature (Tangna et al. identification of serine protease producing Bacillus and its biocontrol) reports that chitinase and serine protease action can destroy the egg shell of Meloidogyne incognita so that hyphae invade the eggs of Meloidogyne incognita and parasitize in the eggs, and the literature mentions that the filtrate of Bacillus subtilis has a strong insecticidal effect on the second instar larvae of Meloidogyne incognita and a method for identifying serine protease, but does not describe the specific process of producing serine protease by Bacillus subtilis and the biocontrol effect of serine protease on Meloidogyne incognita.
Disclosure of Invention
The invention aims to provide a method for producing serine protease bacillus subtilis and application thereof, which are used for realizing a process for efficiently preparing serine protease and improving the technical effect of enzyme activity.
The invention is realized by the following technical scheme: the method comprises the following steps:
s1: inoculating the bacillus subtilis strain into a culture medium, wherein the culture condition is 24-36 h and the temperature is 37 ℃, and obtaining a fermentation strain;
s2: inoculating the fermentation strain in a seed tank, wherein the inoculation amount is 2%, and performing strain expansion culture;
s3: s2, inoculating the strain after the enlarged culture into a fermentation tank for fermentation culture, and taking fermentation liquor;
s4: pumping the fermentation liquor obtained in the step S3 into a centrifuge, and centrifuging for 4-5 hours at the rotating speed of 2900-3000 rpm;
s5: pumping the centrifuged light phase into a ceramic membrane for filtration, wherein the aperture is 500nm, and the filtration time is 4-5 h;
s6, filtering, adding a carrier, and drying to obtain powder serine protease.
In order to better implement the present invention, the culture medium of the seed tank in the step S2 is selected from 20 g/L of corn, 10 g/L of bean pulp, 10 g/L of bran, Na2CO31g/L，Na2HPO41g/L。
In order to better implement the present invention, further, the culture conditions of step S2 are: the time is 18-20 h, the temperature is 37-38 ℃, the rotating speed is 100rpm, and the air quantity is 40-50 m3/h。
In order to better implement the invention, further, the culture medium in the fermentation tank in the step S3 is selected from 60 g/L of corn, 40 g/L of bean pulp, 20 g/L of bran and Na2CO32g/L，Na2HPO42g/L。
To better implement the invention, furtherThe culture conditions of step S3 are: the culture conditions of the early stage 1-4 h are as follows: the temperature is 37-38 ℃, the rotating speed is 100rpm, and the air quantity is 450-700 m3H; the culture conditions of the medium period of 5-8 h are as follows: the air volume is 450-700 m3The speed is 140rpm, the later period is 9-28 hours, and the air volume is 650-700 m3And the rotation speed is 140rpm, and fermentation liquor is obtained, and the enzyme activity of the fermentation liquor is 21503U/m L.
In order to better implement the present invention, further, the drying conditions in step S6 are: the inlet temperature is 150-200 ℃, and the tower temperature is 70-100 ℃.
In order to better implement the invention, further, the carrier is selected from corn starch.
The serine protease obtained by the above method
The serine protease prepared by the method is applied to root knot nematode.
The method for producing the bacillus subtilis of the serine protease has the advantages of simplicity, strong operability, high activity of the prepared serine protease and 21503U/m L of enzyme activity of fermentation liquid.
The serine protease prepared by the invention can be applied to the prevention and treatment of root-knot nematodes, and has good prevention and treatment effects.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings that are required to be used in the present invention will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and it will be apparent to those skilled in the art that other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 is a graph showing the optimum pH of crude enzyme serine protease according to the present invention;
FIG. 2 is a graph showing the optimum temperature test of the crude enzyme solution serine protease provided by the present invention;
FIG. 3 is a graph showing the optimum reaction time of crude enzyme serine protease according to the present invention.
Detailed Description
The technical solution of the present invention will be described below with reference to the accompanying drawings.
The invention provides a method for producing serine protease bacillus subtilis, which comprises the following steps:
s1, inoculating the bacillus subtilis strain into L B culture medium, wherein the culture condition is 24h and the temperature is 37 ℃, and obtaining the fermentation strain.
S2, inoculating 2% of fermentation strain in a seed tank for strain expansion culture, wherein the culture medium in the seed tank comprises 20 g/L of corn, 10 g/L of soybean meal, 10 g/L of bran and Na2CO31g/L，Na2HPO41 g/L, the culture conditions are that the time is 18h, the temperature is 37-38 ℃, the rotating speed is 100rpm, and the air quantity is 40-50 m3/h。
S3, inoculating the strain after the expanded culture in the step S2 into a fermentation tank for fermentation culture, wherein the culture medium in the fermentation tank comprises 60 g/L of corn, 40 g/L of bean pulp, 20 g/L of bran and Na2CO32g/L，Na2HPO42 g/L, wherein the culture conditions of the first period of 1-4 h are that the temperature is 37-38 ℃, the rotating speed is 100rpm, and the air volume is 450-700 m3H; the culture conditions of the medium period of 5-8 h are as follows: the air volume is 450-700 m3The speed is 140rpm, the later period is 9-28 hours, and the air volume is 650-700 m3And the rotation speed is 140rpm, and fermentation liquor is obtained, and the enzyme activity of the fermentation liquor is 21503U/m L.
S4: pumping the fermentation liquor obtained in the step S3 into a centrifugal machine, rotating at the speed of 2950rpm, and centrifuging for 4 hours;
s5: pumping the centrifuged light phase into a ceramic membrane for filtration, wherein the aperture is 500nm, and the filtration time is 4-5 h;
s6: after filtration, 60% of a carrier selected from corn starch is added to dry the fermentation broth according to the volume of the fermentation broth, and the drying conditions are as follows: the inlet temperature is 150-200 ℃, the tower temperature is 70-100 ℃, and powder serine protease is obtained, wherein the enzyme activity is 82534U/g.
The detection method comprises the following steps:
according to the enzyme-producing culture medium 20 g/L corn, 10 g/L soybean meal, 10 g/L bran and Na2CO31g/L，Na2HPO41 g/L9.0.0, sterilizing at 121 deg.C for 30min, subpackaging, adding Bacillus subtilis strain under aseptic condition, culturing on shaker at 37 deg.C for 36 hr to obtain crude enzyme solution of serine protease.
The determination method is carried out according to national standard 23527;
at pH 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10.0; 10.5 boric acid buffer measurement of OD of the crude serine protease680As shown in FIG. 1, the optimum pH of serine protease was determined to be 9.0.
Measuring the crude serine protease OD at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, and 60 deg.C680As shown in FIG. 2, the optimum temperature for serine protease was determined to be 40 ℃.
The reaction time is 5min, 10min, 15min, 20min, 25min, 30min, and 35min, respectively, and the crude enzyme liquid serine protease OD is measured680As shown in FIG. 3, the optimal reaction time for serine protease was determined to be 30 min.
The application experiment of the powder serine protease prepared by the invention in root-knot nematodes is as follows:
selecting a test point:
the altitude of a test site is 1850 m, the annual rainfall is 1050mm or so, the annual average temperature is 11.8 ℃, the test soil is rice soil, the texture is medium soil, the fertility is medium, the test plant is Chinese cabbage, and 600 plants are selected for the test.
Grouping into 6 groups, wherein each group comprises 100 Chinese cabbages, and the 6 groups are respectively: a control group; treating the original powder; 50-fold dilution treatment group; 100-fold dilution treatment group; 200-fold dilution treatment group; treatment group was diluted 300 times.
Arranging a seedbed in a greenhouse, sowing on the same day, starting to grow seedlings after a period of time, applying no fertilizer to the seedbed field, preparing the field after a period of time, applying base fertilizer once in each test area according to requirements, transplanting the Chinese cabbage seedlings to the field on the same day, cultivating the Chinese cabbage seedlings in the open field, harvesting all the Chinese cabbage seedlings once after a period of time, wherein the field growth time of the Chinese cabbage crop is 47 days, the whole growth period is 67 days, and no pesticide for preventing and controlling pests is sprayed during the period.
When harvesting, slowly taking out each test plant, cleaning the root system in water, and observing the root knot condition of each plant.
| Treatment of | CK (control group) | ) |
50 times dilution | 100 times dilution | 200 times dilution | 300 times dilution |
| Root and knot rate | 65.43％ | 6.54％ | 9.63％ | 10.12％ | 29.54％ | 52.38％ |
From the results, the serine protease produced by the bacillus subtilis has a good effect of preventing and treating the root-knot nematode, wherein the effect of preventing and treating the root-knot nematode by raw powder treatment is the best, the difference between 50-fold dilution and 100-fold dilution is not obvious, and the 100-fold dilution is recommended to be used for preventing and treating the root-knot nematode of plants in consideration of the cost.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. A method of producing a serine protease-producing bacillus subtilis, comprising the steps of:
s1: inoculating the bacillus subtilis strain into a culture medium, wherein the culture condition is 24-36 h and the temperature is 37 ℃, and obtaining a fermentation strain;
s2: inoculating the fermentation strain in a seed tank, wherein the inoculation amount is 2%, and performing strain expansion culture;
s3: s2, inoculating the strain after the enlarged culture into a fermentation tank for fermentation culture, and taking fermentation liquor;
s4: pumping the fermentation liquor obtained in the step S3 into a centrifuge, and centrifuging for 4-5 hours at the rotating speed of 2900-3000 rpm;
s5: pumping the centrifuged light phase into a ceramic membrane for filtration, wherein the aperture is 500nm, and the filtration time is 4-5 h;
s6, filtering, adding a carrier, and drying to obtain powder serine protease.
2. The method of producing a serine protease-producing bacillus subtilis according to claim 1, wherein:
the culture medium of the seed tank in step S2 is selected from corn 20 g/L, soybean meal 10 g/L, bran 10 g/L, Na2CO31g/L，Na2HPO41g/L。
3. The method of producing a serine protease-producing bacillus subtilis according to claim 2, wherein: the culture conditions of the step S2 are as follows: the time is 18-20 h, the temperature is 37-38 ℃, the rotating speed is 100rpm, and the air quantity is 40-50 m3/h。
4. The method of Bacillus subtilis for producing serine protease according to claim 1, wherein the culture medium in the fermentor in step S3 is selected from corn 60 g/L, bean pulp 40 g/L, bran 20 g/L, Na2CO32g/L，Na2HPO42g/L。
5. The method of producing a serine protease-producing Bacillus subtilis according to claim 3, wherein the culture conditions of step S3 are: the culture conditions of the early stage 1-4 h are as follows: the temperature is 37-38 ℃, the rotating speed is 100rpm, and the air quantity is 450-700 m3H; the culture conditions of the medium period of 5-8 h are as follows: the air volume is 450-700 m3The speed is 140rpm, the later period is 9-28 hours, and the air volume is 650-700 m3And the rotation speed is 140rpm, and fermentation liquor is obtained, and the enzyme activity of the fermentation liquor is 21503U/m L.
6. The method of producing a serine protease-producing bacillus subtilis according to claim 1, wherein the drying conditions in step S6 are: the inlet temperature is 150-200 ℃, and the tower temperature is 70-100 ℃.
7. The method of producing a serine protease-producing bacillus subtilis according to claim 1, wherein the vector is selected from the group consisting of corn starch.
8. The serine protease produced by the method according to any one of claims 1 to 7.
9. Use of the serine protease of claim 8 for root knot nematode disease.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151065A (en) * | 2021-03-30 | 2021-07-23 | 山东蔚蓝生物科技有限公司 | Bacillus subtilis for preventing and treating nematode diseases and application thereof |
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| CN1456668A (en) * | 2003-05-21 | 2003-11-19 | 云南大学 | Alkaline fungus serine proteinase and its preapring method and application |
| CN108823191A (en) * | 2018-06-13 | 2018-11-16 | 天津科技大学 | A kind of fermentation process for improving bacillus and producing protease |
| CN109439601A (en) * | 2018-12-25 | 2019-03-08 | 河南省科学院生物研究所有限责任公司 | One plant of method for producing the bacterial strain of protease and its preparing alkali protease |
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Patent Citations (4)
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|---|---|---|---|---|
| US5585253A (en) * | 1989-11-27 | 1996-12-17 | The Regents Of The University Of California | Extracellular serine protease and a Bacillus subtilis alkaline neutral an serine protease mutant strain |
| CN1456668A (en) * | 2003-05-21 | 2003-11-19 | 云南大学 | Alkaline fungus serine proteinase and its preapring method and application |
| CN108823191A (en) * | 2018-06-13 | 2018-11-16 | 天津科技大学 | A kind of fermentation process for improving bacillus and producing protease |
| CN109439601A (en) * | 2018-12-25 | 2019-03-08 | 河南省科学院生物研究所有限责任公司 | One plant of method for producing the bacterial strain of protease and its preparing alkali protease |
Non-Patent Citations (1)
| Title |
|---|
| 唐娜等: "产丝氨酸蛋白酶芽胞杆菌的鉴定及其生防作用" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151065A (en) * | 2021-03-30 | 2021-07-23 | 山东蔚蓝生物科技有限公司 | Bacillus subtilis for preventing and treating nematode diseases and application thereof |
| CN113151065B (en) * | 2021-03-30 | 2022-08-05 | 山东蔚蓝生物科技有限公司 | Bacillus subtilis for preventing and treating nematode diseases and application thereof |
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Application publication date: 20200710 |