CN113980872A - Bacillus mojavensis strain for producing lipopeptide biosurfactant - Google Patents
Bacillus mojavensis strain for producing lipopeptide biosurfactant Download PDFInfo
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- 241001249117 Bacillus mojavensis Species 0.000 title claims abstract description 17
- 239000003876 biosurfactant Substances 0.000 title claims abstract description 15
- 108010028921 Lipopeptides Proteins 0.000 title description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 20
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 16
- 239000004094 surface-active agent Substances 0.000 claims abstract description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 10
- 239000001888 Peptone Substances 0.000 claims abstract description 7
- 108010080698 Peptones Proteins 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 235000019319 peptone Nutrition 0.000 claims abstract description 7
- 229940057995 liquid paraffin Drugs 0.000 claims abstract description 6
- 239000003921 oil Substances 0.000 claims description 25
- 235000019198 oils Nutrition 0.000 claims description 21
- 238000000855 fermentation Methods 0.000 claims description 20
- 230000004151 fermentation Effects 0.000 claims description 20
- 235000019484 Rapeseed oil Nutrition 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 6
- 238000003916 acid precipitation Methods 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000010779 crude oil Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 230000035755 proliferation Effects 0.000 claims description 2
- 241000193830 Bacillus <bacterium> Species 0.000 claims 2
- 230000003321 amplification Effects 0.000 claims 1
- 238000003199 nucleic acid amplification method Methods 0.000 claims 1
- 239000001963 growth medium Substances 0.000 abstract description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 abstract description 6
- 239000000843 powder Substances 0.000 abstract description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 abstract description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 abstract description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 abstract description 4
- 235000011130 ammonium sulphate Nutrition 0.000 abstract description 4
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- 229910052943 magnesium sulfate Inorganic materials 0.000 abstract description 3
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- 241000894006 Bacteria Species 0.000 description 2
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
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- 239000012138 yeast extract Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241000194103 Bacillus pumilus Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- AFWTZXXDGQBIKW-UHFFFAOYSA-N C14 surfactin Natural products CCCCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 AFWTZXXDGQBIKW-UHFFFAOYSA-N 0.000 description 1
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- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
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- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 description 1
- 229960005484 daptomycin Drugs 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- UDKIYKILOKEDQK-UHFFFAOYSA-N esperin Chemical compound OC(=O)C(C)NC(=O)C(CC(C)C)NC(=O)C1CC(=O)OC(C)CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)N1 UDKIYKILOKEDQK-UHFFFAOYSA-N 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 108010002015 fengycin Proteins 0.000 description 1
- CUOJDWBMJMRDHN-VIHUIGFUSA-N fengycin Chemical compound C([C@@H]1C(=O)N[C@H](C(=O)OC2=CC=C(C=C2)C[C@@H](C(N[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CCC(N)=O)C(=O)N1)[C@@H](C)O)=O)NC(=O)[C@@H](CCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)C[C@H](O)CCCCCCCCCCCCC)[C@@H](C)CC)C1=CC=C(O)C=C1 CUOJDWBMJMRDHN-VIHUIGFUSA-N 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 108010018095 pumilacidin Proteins 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
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- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- NJGWOFRZMQRKHT-UHFFFAOYSA-N surfactin Natural products CC(C)CCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-UHFFFAOYSA-N 0.000 description 1
- NJGWOFRZMQRKHT-WGVNQGGSSA-N surfactin C Chemical compound CC(C)CCCCCCCCC[C@@H]1CC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-WGVNQGGSSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- Life Sciences & Earth Sciences (AREA)
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
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- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a bacillus mojavensis strain for producing a biosurfactant, and the GenBank accession number is OK 605547. The strain is separated from the Clarity oil field soil, and the strain is determined to be the bacillus mojavensis (through morphological feature observation and 16S rDNA sequencing analysis of the strain: (a)Bacillus mojavensis) The strain is added with 2mL of colza oil, 0.4g of ammonium sulfate and KH per 100mL2PO40.1 g，Na2HPO4·12H2O 0.1 g，MgSO4·7H2And (3) shaking and culturing the mixture in a culture medium containing 0.05g of O and 0.2g of yeast powder at 35 ℃ and 160 rpm for more than 2 days to obtain a surfactant product with better quality. Performing single condition optimization experiment to obtain the surfactant with 2% peptone as nitrogen source, 2% liquid paraffin as carbon source and cultured at 35 deg.cThe quality is optimal. The surfactant belongs to glycolipid biological surfactants, and has wide application prospect and development possibility in fields of petrochemical industry, medicine, washing and nursing, environmental protection and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus mojavensis strain for high-yield lipopeptide biosurfactant, wherein the GenBank entry number of the strain is OK 605547.
Background
Biosurfactants are natural compounds with surface activity that are metabolically synthesized by microorganisms under certain conditions. The water-oil composite material has both hydrophilic group and hydrophobic group, can be directionally arranged on a water-oil two-phase interface to form a molecular layer, and has various functions of emulsification, bacteriostasis, solubilization, interface tension reduction and the like. In addition, the biosurfactant is used as a secondary metabolite, the molecular structure is more complex, the biosurfactant occupies larger space, and the stability is generally not influenced by environmental conditions such as temperature, salinity, pH value and the like.
Compared with a chemical synthesis surfactant, the biosurfactant can obviously reduce the interfacial tension, is used as an excellent-performance and environment-friendly surfactant, accords with the trend of 'green development' in China, and has wide application prospect and development possibility in various fields such as petrochemical industry, medicines, washing and nursing, environmental protection and the like.
Lipopeptide biosurfactants are the most effective biosurfactants, can greatly reduce surface and interface tension and have a plurality of outstanding excellent characteristics. Although the microbial resources capable of producing lipopeptides are very abundant, it is not much that the production level is really achieved, and Surfactin, Iturin and Fengycin produced by bacillus subtilis are mainly used; daptomycin produced by streptomyces roseosporus; lichenyishin produced by bacillus licheniformis; bacillus pumilus produces Pumilacidin and Esperin.
Due to the restriction of factors such as high separation and purification difficulty, high production cost and the like, most lipopeptides are still in the laboratory research stage, and the main research at present focuses on the identification of chemical structures. Therefore, the novel efficient lipopeptide substance is excavated, so that the diversified functions of the lipopeptide substance are fully exerted, and the lipopeptide substance has important scientific significance and practical application value.
Disclosure of Invention
In order to solve the problems that the existing biosurfactant has high cost and is difficult to realize large-scale industrialization, the invention aims to provide a high-yield strain for producing the biosurfactant and a production method thereof.
The strain is sampled in the oil field soil of the Clarity and is obtained by screening after enrichment culture.
The strain is determined to be bacillus mojavensis (through morphological feature observation, 16S rDNA sequencing, comparison and evolutionary tree construction analysis of the strainBacillus mojavensis) The accession number of the gene is OK605547 in GenBank.
The strain grows well on the nutrient agar plate, the bacterial colony is yellow and opaque, and the edge is irregular; the cells are rod-shaped, spore-growing and oval.
The invention provides a method for producing a biosurfactant by using bacillus mojavensis, which comprises the following steps:
(1) performing seed culture on the strain;
(2) performing enrichment culture on seeds obtained by seed culture by taking crude oil or rapeseed oil as a carbon source, wherein the enrichment culture time is determined according to the change condition of an oil discharge ring of a culture system, and obtaining fermentation liquor after the culture is finished;
(3) and carrying out acid precipitation, centrifugation, extraction, concentration and drying on the fermentation liquor to obtain the surfactant.
(4) And optimizing the carbon source, the nitrogen source and the temperature of the fermentation culture solution through a single factor experiment.
Preferably, the seeds are cultured at 28-32 ℃ under shaking till logarithmic phase, and the bacillus mojavensis seeds are obtained.
Preferably, the enrichment fermentation culture adopts an inorganic salt culture medium added with rapeseed oil; the conditions of proliferation culture are as follows: performing shaking culture at 28-32 deg.C and 150-170 rpm for more than 2 d.
Preferably, the formula of the inorganic salt culture medium is as follows: 2mL of rapeseed oil and 0.4g of ammonium sulfate and KH are added into each 100mL of rapeseed oil2PO40.1g，Na2HPO4·12H2O 0.1g，MgSO4·7H20.05g of O and 0.2g of yeast powder.
Preferably, in the single-factor experiment, 2% of peptone is used as a nitrogen source, 2% of liquid paraffin is used as a carbon source, the diameter of an oil discharge ring of the fermentation liquid obtained by culturing at 35 ℃ is the largest, and the quality of the obtained surfactant is optimal.
Drawings
FIG. 1 is a tree of 16S rDNA of strain PGF 23.
FIG. 2 is a graph showing the diameter change of an oil drain ring of the strain PGF23 in a culture medium containing different carbon sources.
FIG. 3 is a graph showing the change of the diameter of the oil drain ring of the strain PGF23 in the culture media with different contents of liquid paraffin.
FIG. 4 is a graph showing the change in the diameter of the oil drain ring of the strain PGF23 in the medium containing different nitrogen sources.
FIG. 5 is a graph showing the change in the diameter of the oil drain of strain PGF23 in media with different peptone contents.
FIG. 6 is a graph showing the change in the diameter of the oil drain ring of the strain PGF23 cultured at different temperatures.
Detailed Description
The invention is explained in more detail below with reference to the figures and examples
Formula of culture medium
1. Enrichment of a fermentation medium: 2mL of rapeseed oil and 0.4g of ammonium sulfate and KH are added into each 100mL of rapeseed oil2PO4 0.1g，Na2HPO4·12H2O 0.1g，MgSO4·7H20.05g of O and 0.2g of yeast powder, and sterilizing for 20min at 121 ℃ for later use.
2. Seed medium (fermentation medium under optimized conditions): yeast Extract (1 g), Peptone (2 g) and glucose (2 g) are added into each 100ml, and sterilized at 121 ℃ for 20min for later use.
3. Solid medium: every 100ml is added with Yeast Extract 1g, Peptone 2g, glucose 2g, agar powder 2g, sterilized at 121 deg.C for 20min for use.
(II) Bacillus mojavensis (II)Bacillus mojavensis) And (4) screening strains.
1. Collecting and enriching culture of sample
Adding 10 g of soil sample stored at low temperature into 100mL of sterile water, culturing for 30 min in a shaking table at 30 ℃ and 160 rpm, taking out, standing for 1 h, inoculating 10 mL of solution into 100mL of enrichment fermentation medium using rapeseed oil as a carbon source, placing into the shaking table at 30 ℃ and culturing for 2-3 days at 160 rpm (a control group is additionally set, and the steps are the same as above except that the soil sample is not added).
2. Preliminary screening and separation purification of bacterial strain
The oil drainage method is used for primarily screening the strains generating the surfactant. The specific method comprises the following steps: and adding 40 mL of deionized water into the culture dish, dropwise adding 3 mL of rapeseed oil on the water surface, slowly dropwise adding a proper amount of bacterial liquid in the previous step into the center of the oil film, and roughly measuring the diameter of the oil discharge ring. The bacteria solution without the sample is used as a control group, and the bacteria solution with the oil discharge ring may contain a strain which generates a surfactant. And (3) carrying out streak separation on the bacterial liquid generating the oil discharge ring on a solid culture medium, and culturing for 3-5 days in a 30 ℃ thermostat to obtain a single pure strain.
3. Bacterial strain rescreening
Inoculating the pure strain obtained by primary screening into 100mL enrichment fermentation medium with the inoculation amount of 5%, culturing at 30 ℃ and 160 rpm for 2-3 days, measuring the oil discharge activity of the fermentation liquor, and screening out the strain with the oil discharge diameter larger than 3 cm.
The invention obtains a bacterial strain PGF23 with stronger surfactant producing ability from 8 screened single bacterial strains.
And (III) optimizing the fermentation conditions of the strains.
1. Influence of the carbon Source
The carbon source is an important influence factor of microbial metabolism, and glucose, liquid paraffin, rapeseed oil and sucrose are respectively selected as the carbon sources for comparison. Inoculating the strain into sterilized 100mL fermentation medium, and performing constant temperature shaking culture at 30 deg.C and 160r/min for 3 d. And (4) measuring the diameter of the oil discharge ring of the fermentation liquor, and selecting a proper carbon source. The content of the most suitable carbon source in the culture medium is then determined.
2. Influence of Nitrogen Source
Respectively taking yeast powder, ammonium sulfate, ammonium chloride and peptone as nitrogen sources of the fermentation culture solution, taking the carbon sources as carbon sources determined in a single-factor experiment, keeping other conditions unchanged, carrying out shake culture in a shaking table for 3d under the conditions of 30 ℃ and 160r/min, measuring the diameter of an oil discharge ring of the fermentation culture solution, and determining the optimal nitrogen source. The content of the most suitable nitrogen source in the medium is then determined.
3. Effect of culture temperature
The carbon source and the nitrogen source determined by the single-factor test are adopted, other components are unchanged, the culture is carried out for 3d at different temperatures at the oscillation rate of 160r/min, the diameter of an oil discharge ring of the fermentation culture solution is measured, and the optimal culture temperature is determined.
(IV) identification of Individual strains
The separated strain is determined by comparing 16S rDNA sequence, and sent to Nanjing Ongjingkidaceae Biotech limited company for PCR amplification and Sanger sequencing
The upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCA-3'
A downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3'
The resulting sequences were BLAST (NCBI) followed by the construction of a phylogenetic tree using MEGA software (FIG. 1). Finally, the strain PGF23 was determined to belong to the genus Bacillus mojavensis (B.) (Bacillus mojavensis) Is named asBacillus mojavensis PGF23。
Purification of surfactant from strain PGF23
The PGF23 strain with larger oil discharge circle of the fermentation liquor is inoculated into an enrichment medium for culture for three days, and the pH value of the medium is adjusted to about 7.0 by NaOH. Centrifuging at 10000 rpm and 4 deg.C for 10 min, separately pouring out supernatant, adding hydrochloric acid into the supernatant, adjusting pH to 2.0, standing at 4 deg.C for 24 hr, and centrifuging under the same conditions. And (3) mixing the supernatant with dichloromethane according to a volume ratio of 1: 1, performing rotary evaporation on the extract liquid at 40 ℃ to obtain a solid, and combining the solid with the second centrifugal precipitate to obtain a crude extract.
Claims (3)
1. A high-yield strain of biosurfactant is Bacillus mojavensis PGF23 (B.mojavensis)Bacillus mojavensisPGF 23), accession number OK605547 in GenBank.
2. A method for preparing a biosurfactant is characterized by comprising the following steps: the method comprises the following steps:
1) carrying out seed expansion culture on the bacillus mojavensis PGF 23;
2) carrying out proliferation culture on the seeds obtained by the amplification culture by taking crude oil or rapeseed oil or liquid paraffin as a carbon source for 2-3 days to obtain fermentation liquor;
3) and treating the fermentation liquor by adopting methods such as acid precipitation, centrifugation, extraction, concentration, drying and the like to obtain the surfactant.
3. The method for preparing a surfactant according to claim 2, wherein: 2 percent of peptone is taken as a nitrogen source, 2 percent of liquid paraffin is taken as a carbon source, the diameter of an oil discharge ring of fermentation liquor obtained by culturing at 35 ℃ reaches about 1.85cm, the oil discharge performance is optimal, and the bacillus mojavensis PGF23 (PGF 23)Bacillus mojavensisPGF 23) produces surfactants with optimum performance.
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Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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