CN115772511A - Method for efficiently extracting full resources from lumbrokinase - Google Patents
Method for efficiently extracting full resources from lumbrokinase Download PDFInfo
- Publication number
- CN115772511A CN115772511A CN202211653322.7A CN202211653322A CN115772511A CN 115772511 A CN115772511 A CN 115772511A CN 202211653322 A CN202211653322 A CN 202211653322A CN 115772511 A CN115772511 A CN 115772511A
- Authority
- CN
- China
- Prior art keywords
- protein
- lumbrokinase
- solution
- buffer solution
- recycling
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010070324 lumbrokinase Proteins 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 56
- 239000007853 buffer solution Substances 0.000 claims abstract description 50
- 238000005185 salting out Methods 0.000 claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 26
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 26
- 241000361919 Metaphire sieboldi Species 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 238000004064 recycling Methods 0.000 claims abstract description 15
- 239000003337 fertilizer Substances 0.000 claims abstract description 12
- 239000012528 membrane Substances 0.000 claims abstract description 11
- 239000000126 substance Substances 0.000 claims abstract description 8
- 239000012460 protein solution Substances 0.000 claims description 27
- 238000002156 mixing Methods 0.000 claims description 21
- 239000000463 material Substances 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 20
- 238000005374 membrane filtration Methods 0.000 claims description 18
- 238000001742 protein purification Methods 0.000 claims description 18
- 238000000855 fermentation Methods 0.000 claims description 15
- 230000004151 fermentation Effects 0.000 claims description 15
- 241001233061 earthworms Species 0.000 claims description 13
- -1 potassium bicarbonate-ammonium acetate Chemical compound 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 11
- 238000002791 soaking Methods 0.000 claims description 11
- 238000004108 freeze drying Methods 0.000 claims description 10
- 238000004821 distillation Methods 0.000 claims description 5
- 239000000243 solution Substances 0.000 abstract description 25
- 238000000605 extraction Methods 0.000 abstract description 17
- 238000000926 separation method Methods 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 8
- 239000002699 waste material Substances 0.000 abstract description 7
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 abstract description 4
- 239000005695 Ammonium acetate Substances 0.000 abstract description 4
- 229940043376 ammonium acetate Drugs 0.000 abstract description 4
- 235000019257 ammonium acetate Nutrition 0.000 abstract description 4
- 229910000028 potassium bicarbonate Inorganic materials 0.000 abstract description 4
- 235000015497 potassium bicarbonate Nutrition 0.000 abstract description 4
- 239000011736 potassium bicarbonate Substances 0.000 abstract description 4
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- 108091000080 Phosphotransferase Proteins 0.000 abstract description 2
- 230000009471 action Effects 0.000 abstract description 2
- 230000036571 hydration Effects 0.000 abstract description 2
- 238000006703 hydration reaction Methods 0.000 abstract description 2
- 102000020233 phosphotransferase Human genes 0.000 abstract description 2
- 235000019441 ethanol Nutrition 0.000 description 12
- 238000000967 suction filtration Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 4
- 241000243684 Lumbricus Species 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- XMWRBQBLMFGWIX-UHFFFAOYSA-N C60 fullerene Chemical compound C12=C3C(C4=C56)=C7C8=C5C5=C9C%10=C6C6=C4C1=C1C4=C6C6=C%10C%10=C9C9=C%11C5=C8C5=C8C7=C3C3=C7C2=C1C1=C2C4=C6C4=C%10C6=C9C9=C%11C5=C5C8=C3C3=C7C1=C1C2=C4C6=C2C9=C5C3=C12 XMWRBQBLMFGWIX-UHFFFAOYSA-N 0.000 description 1
- 206010069729 Collateral circulation Diseases 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000003818 flash chromatography Methods 0.000 description 1
- 229910003472 fullerene Inorganic materials 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 239000003895 organic fertilizer Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Fertilizers (AREA)
Abstract
The invention provides a method for efficiently extracting and recycling lumbrukinase, belonging to the technical field of biological extraction. According to the method, the lumbrokinase protein is extracted by adopting a salting-out method of combining potassium bicarbonate and ammonium acetate with ethanol, and the condition that the pH = 7.5-8.0 provided by a buffer solution of potassium bicarbonate and ammonium acetate can maintain the high activity of the lumbrokinase protein and improve the extraction rate; the ethanol can reduce the electrolytic constant of the solution, so that the attraction of different charges on protein molecules is increased, the solubility is reduced, the hydration membrane of the protein can be damaged under the action of the ethanol and water, the protein is separated out in the ethanol due to the difference of the solubility, and the high-efficiency extraction and separation of lumbrokinase are realized; in addition, the waste liquid obtained after earthworm kinase extraction is converted into liquid fertilizer, residual substances are comprehensively utilized, the problem of resource utilization of the waste liquid is solved, and green environmental protection and full-resource recycling in the earthworm extraction process are realized.
Description
Technical Field
The invention relates to the technical field of biological extraction, in particular to a method for efficiently extracting full resources from lumbrukinase.
Background
Earthworms are one of the largest common groups of soil animals, and play multiple roles in the ecosystem, namely, consumers and creators. It can make contribution to material circulation and energy transfer in the soil process by taking, digesting, excreting (worm cast), secreting (mucus) and digging hole, etc. activities, and can produce important influence on a plurality of processes for determining soil fertility. The earthworm is used as high-protein feed, and the earthworm cast is an organic fertilizer raw material, so that the earthworm cast has good economic value. The earthworm is also a traditional Chinese medicinal material and has great medicinal value, lumbrokinase in the earthworm body has the antithrombotic effect, can track thrombolysis, effectively dissolve micro-thrombus, improve microcirculation, strengthen the collateral circulation of heart and cerebral vessels, openly repair damaged endothelial cells of blood vessels, increase the elasticity of blood vessels, improve the oxygen supply function of blood vessels, reduce the viscosity of blood, reduce the aggregation rate of platelets and inhibit the reformation of thrombus. Repairing peripheral necrotic brain cells after thrombus generation and saving the half dark area.
At present, the existing lumbrokinase extraction method comprises the following steps: NH (NH) 4 Homogenizing Ac-HAc buffer solution, phosphate buffer solution or Tris-HCl buffer solution, salting out with ammonium sulfate, and performing ion exchange chromatography or dialysis to obtain crude enzyme solution. However, the buffer solution and the salting-out solution adopted by the technology cannot be recovered or reused, so that the lumbrokinase extraction cost is increased while risks are brought to the environment.
Disclosure of Invention
The invention aims to provide a method for efficiently extracting and recycling lumbrokinase, which can realize efficient extraction and separation and recycling of lumbrokinase and has low cost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for efficiently extracting full resources from lumbrokinase, which comprises the following steps:
soaking earthworms in a first buffer solution, homogenizing, and performing membrane filtration on the obtained homogenate material to obtain a protein solution;
mixing the protein solution with ethanol, and salting out to obtain supernatant and a salted-out substance;
mixing the salting-out material with a second buffer solution, performing protein purification on the obtained mixed solution, and performing freeze drying to obtain lumbrokinase;
distilling the supernatant under reduced pressure, mixing the obtained residual liquid with the earthworm residue obtained by homogenizing, and fermenting to obtain an ecological fertilizer;
the first buffer solution and the second buffer solution are both potassium bicarbonate-ammonium acetate mixed solutions;
the pH values of the first buffer solution and the second buffer solution are independently 7.5-8.0.
Preferably, the pH of the first buffer solution and the second buffer solution is independently 7.8.
Preferably, the soaking time is 30-60 min.
Preferably, the rotation speed of the homogenate is 20000 to 25000rpm, and the time is 3 to 5min.
Preferably, the molecular weight cut-off of the membrane used for membrane filtration is 50KD, and the molecular weight of the protein in the protein solution is less than or equal to 50KD.
Preferably, the volume ratio of the ethanol to the protein solution is (0.5-3): 1.
Preferably, the mass ratio of the second buffer solution to the salting-out material is (10-100): 1.
Preferably, the temperature of the reduced pressure distillation is 50 to 60 ℃.
Preferably, the fermentation temperature is 30-35 ℃, and the fermentation time is 10-15 days.
Preferably, the fermentation is performed under anaerobic closed conditions.
The invention provides a method for efficiently extracting full resources from lumbrukinase, which comprises the following steps: soaking earthworms in a first buffer solution, homogenizing, and performing membrane filtration on the obtained homogenate material to obtain a protein solution; mixing the protein solution with ethanol, and salting out to obtain supernatant and a salted-out substance; mixing the salting-out material with a second buffer solution, performing protein purification on the obtained mixed solution, and performing freeze drying to obtain lumbrukinase; distilling the supernatant under reduced pressure, mixing the obtained residual liquid with the earthworm residue obtained by homogenizing, and fermenting to obtain an ecological fertilizer; the first buffer solution and the second buffer solution are both potassium bicarbonate-ammonium acetate mixed solutions, and the pH values of the first buffer solution and the second buffer solution are independently 7.5-8.0. According to the method, the lumbrokinase protein is extracted by adopting a salting-out method of combining potassium bicarbonate and ammonium acetate with ethanol, and the condition that the pH = 7.5-8.0 provided by a buffer solution of potassium bicarbonate and ammonium acetate can maintain the high activity of the lumbrokinase protein and improve the extraction rate; the ethanol can reduce the electrolytic constant of the solution, so that the attraction of different charges on protein molecules is increased, the solubility is reduced, the hydration membrane of the protein can be damaged under the action of the ethanol and water, the protein is separated out in the ethanol due to the difference of the solubility, and the high-efficiency extraction and separation of lumbrokinase are realized; in addition, the waste liquid obtained after earthworm kinase extraction is converted into liquid fertilizer, residual substances are comprehensively utilized, the problem of resource utilization of the waste liquid is solved, and green environmental protection and full-resource recycling in the earthworm extraction process are realized.
The method adopts the steps of buffer solution dissolution, membrane filtration, salting out and protein purification to realize the extraction of the high-quality lumbrokinase; waste residues and waste solution generated in the extraction process can be processed into ecological fertilizer through advanced treatment, so that zero emission in the whole earthworm extraction process is realized, and a green new path is provided for the production of lumbrokinase. Compared with the prior art, the method can realize the recovery and resource utilization of the extraction waste liquid, has the advantages of simple method, easy operation, low cost and the like, and has remarkable economic and environmental benefits.
Detailed Description
The invention provides a method for efficiently extracting full resources from lumbrukinase, which comprises the following steps:
soaking earthworms in a first buffer solution, homogenizing, and performing membrane filtration on the obtained homogenate material to obtain a protein solution;
mixing the protein solution with ethanol, and salting out to obtain supernatant and a salted-out substance;
mixing the salting-out material with a second buffer solution, performing protein purification on the obtained mixed solution, and performing freeze drying to obtain lumbrokinase;
distilling the supernatant under reduced pressure, mixing the obtained residual liquid with the earthworm residue obtained by homogenizing, and fermenting to obtain an ecological fertilizer;
the first buffer solution and the second buffer solution are both potassium bicarbonate-ammonium acetate mixed solutions;
the pH values of the first buffer solution and the second buffer solution are independently 7.5-8.0.
In the present invention, unless otherwise specified, all the materials or reagents required are commercially available products well known to those skilled in the art.
According to the invention, earthworms are soaked in a first buffer solution, homogenate is carried out, and membrane filtration is carried out on the homogenate material to obtain a protein solution.
The source of the earthworms is not particularly limited in the present invention, and the earthworms can be obtained in a manner well known in the art.
In the invention, the first buffer solution is a potassium bicarbonate-ammonium acetate mixed solution, and the pH value of the first buffer solution is 7.5-8.0, preferably 7.8. The dosage of the first buffer solution is not particularly limited, and the corresponding earthworms can be completely soaked.
In the present invention, the soaking time is preferably 30 to 60min, and more preferably 40 to 50min.
After the soaking is finished, the obtained earthworms are homogenized by a homogenizer; the rotation speed of the homogenate is preferably 20000 to 25000rpm, more preferably 23000rpm; the time is preferably 3 to 5min, more preferably 4min. The refiner is not particularly limited in the present invention and may be any of those known in the art.
After the homogenization is finished, the obtained material is preferably centrifuged, supernatant is taken and transferred to a filter flask for suction filtration, and the obtained earthworm residue (insoluble earthworm residue generated by centrifugal separation) is retained; the solution obtained after suction filtration (homogeneous slurry) was subjected to membrane filtration. In the invention, the molecular weight cut-off of the membrane used for membrane filtration is preferably 50KD, and the molecular weight of the protein in the protein solution is preferably less than or equal to 50KD. The membrane used by the membrane filtration membrane is preferably a flat membrane, and the protein separation equipment used by the membrane filtration is from Fulemm corporation of Qingdao; the invention intercepts protein with molecular weight more than 50KD and flows out with molecular weight less than 50KD through membrane filtration, and the protein solution is formed through separation. The flat membrane is not specially limited, and the interception effect can be achieved.
After obtaining the protein solution, the invention mixes the protein solution with ethanol, and carries out salting out to obtain supernatant and a salting-out substance.
In the present invention, the ethanol is preferably anhydrous ethanol; the volume ratio of the ethanol to the protein solution is preferably (0.5-3): 1, and more preferably (1.5-2): 1. The protein in the protein solution is separated out through salting out, the obtained salting-out matter comprises lumbrukinase and other proteins, and the lumbrukinase is extracted through subsequent protein purification.
After the salting-out material is obtained, the salting-out material is mixed with a second buffer solution, and the obtained mixed solution is subjected to protein purification and freeze drying to obtain the lumbrokinase.
In the invention, the second buffer solution is a potassium bicarbonate-ammonium acetate mixed solution, and the pH value of the second buffer solution is 7.5-8.0, preferably 7.8; the mass ratio of the second buffer solution to the salting-out product is preferably (10 to 100): 1, and more preferably (10 to 50): 1.
In the present invention, the protein purification is preferably carried out in a protein purification apparatus, which is not particularly limited in the present invention and may be any of those known in the art; in the examples of the present invention, the protein purification apparatus used was a protein purification apparatus manufactured by Shanghai flash chromatography. The protein with the molecular weight of more than 20KD is separated by a protein purification device, the lumbrokinase is a compound protein product, the molecular weight range is 20-45 KD, and the protein with the molecular weight of more than 20KD extracted from the earthworm body is the lumbrokinase. The present invention does not specifically limit the specific conditions for purifying the protein, and the purification can be performed according to the procedures well known in the art. The present invention preferably uses a separation column, preferably SephadexG-100 or SephadexG-50, to obtain a protein of the desired molecular weight.
After the protein purification is completed, the lumbrukinase solution is subjected to freeze drying to obtain lumbrukinase; the freeze-drying is not particularly limited in the present invention, and may be performed according to a procedure well known in the art.
After the supernatant is obtained, the supernatant is distilled under reduced pressure, and the obtained residual liquid and the earthworm residue obtained by homogenizing are mixed and fermented to obtain the ecological fertilizer. In the present invention, the supernatant liquid comprises water, ethanol and a non-salting-out protein.
In the present invention, the temperature of the reduced pressure distillation is preferably 50 to 60 ℃, more preferably 55 to 57 ℃. The invention recovers the absolute ethyl alcohol while carrying out reduced pressure distillation.
After the reduced pressure distillation is finished, mixing the obtained residual liquid (protein solution with molecular weight larger than 50KD intercepted by membrane filtration) with the earthworm residue obtained by homogenate, and feeding the mixture into a closed container for sealing; and maintaining the normal pressure state of the closed container, releasing pressure through a pressure reducing valve, and fermenting to obtain the ecological fertilizer.
In the present invention, the closed vessel is preferably a fermenter; the fermentation temperature is preferably 30-35 ℃, and more preferably 33 ℃; the time is preferably 10 to 15 days, more preferably 12 days; the fermentation is preferably carried out under anaerobic closed conditions.
In the fermentation process, under the anoxic condition, the earthworm residue and the protein solution in the residual liquid are fermented to form enzyme.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
In the following examples, the molecular weight cut-off of the flat membrane used for membrane filtration was 50KD, and the protein separation equipment used was from the company fullerene, qingdao;
the protein purification device is a protein purification device manufactured by Shanghai flash spectrum, and the separation column is SephadexG-50.
Example 1
Soaking earthworms in a potassium bicarbonate-ammonium acetate buffer solution with the pH =7.8 for 40min, and homogenizing for 4min at 25000rpm by using a homogenizer;
centrifuging the homogenized solution, transferring the supernatant into a filter flask for suction filtration, and reserving the obtained earthworm residue;
performing membrane filtration on the solution after suction filtration to obtain a protein solution with the protein molecular weight of below 50 KD;
mixing the protein solution with absolute ethyl alcohol according to a volume ratio of 1;
dissolving the obtained salting-out material and a potassium bicarbonate-ammonium acetate buffer solution with the pH =7.8 according to a mass ratio of 1; separating the dissolved solution into protein with the molecular weight more than 20KD by a protein purification device, and freeze-drying the obtained lumbrokinase solution to obtain lumbrokinase powder;
distilling the supernatant obtained after salting out at 55 deg.C under reduced pressure, recovering anhydrous ethanol, mixing the obtained residue with the above filtered Lumbricus residue, and fermenting in a fermentation tank under oxygen-free sealed condition; maintaining the normal pressure state of the fermentation tank, releasing pressure through a pressure reducing valve, and fermenting at 35 ℃ for 10 days to obtain the ecological fertilizer.
Example 2
Soaking earthworms in a potassium bicarbonate-ammonium acetate buffer solution with the pH =7.8 for 50min, and then homogenizing for 5min at 20000rpm by a homogenizer;
centrifuging the homogenized solution, transferring the supernatant into a filter flask for suction filtration, and reserving the obtained earthworm residue;
performing membrane filtration on the solution after suction filtration to obtain a protein solution with the protein molecular weight of below 50 KD;
mixing the protein solution with absolute ethyl alcohol according to a volume ratio of 1.5, and salting out to obtain a salting-out substance and a supernatant;
dissolving the obtained salting-out material with a potassium bicarbonate-ammonium acetate buffer solution with the pH =7.7 according to a mass ratio of 1; separating the dissolved solution into protein with the molecular weight more than 20KD by a protein purification device, and freeze-drying the obtained lumbrokinase solution to obtain lumbrokinase powder;
distilling the supernatant at 60 deg.C under reduced pressure, recovering anhydrous ethanol, mixing the residue with the above Lumbricus residue, and fermenting in a fermentation tank under oxygen-free sealed condition; maintaining the normal pressure state of the fermentation tank, releasing pressure through a pressure reducing valve, and fermenting for 15 days at 30 ℃ to obtain the ecological fertilizer.
Example 3
Soaking earthworms in a potassium bicarbonate-ammonium acetate buffer solution with the pH =7.8 for 30min, and homogenizing for 3min at 23000rpm by using a homogenizer;
centrifuging the homogenized solution, transferring the supernatant into a filter flask for suction filtration, and reserving the obtained earthworm residue;
performing membrane filtration on the solution after suction filtration to obtain a protein solution with the protein molecular weight of below 50 KD;
mixing the protein solution with absolute ethyl alcohol according to a volume ratio of 1;
dissolving the obtained salting-out material with a potassium bicarbonate-ammonium acetate buffer solution with the pH =7.9 according to a mass ratio of 1; separating the dissolved solution into protein with the molecular weight more than 20KD by a protein purification device, and freeze-drying the obtained lumbrokinase solution to obtain lumbrokinase powder;
distilling the supernatant at 57 deg.C under reduced pressure, recovering anhydrous ethanol, mixing the residue with the above Lumbricus residue, and fermenting in a fermentation tank under oxygen-free sealed condition; maintaining the normal pressure state of the fermentation tank, releasing pressure through a pressure reducing valve, and fermenting at 33 ℃ for 12 days to obtain the ecological fertilizer.
Lumbrukinase activity assay
Adding 1mL of the lumbrukinase solution obtained in the embodiments 1-3 into a clean glass plate, uniformly mixing the fibrinogen solution and the melted agarose solution which are kept at 40 ℃, quickly pouring the mixture into the plate added with the lumbrukinase, shaking and uniformly mixing the mixture, and standing the mixture at room temperature for 1h to obtain the lumbrukinase-containing composite membrane, wherein 10 muL of each of the lumbrukinase standard solution and the sample solution with different concentrations are arranged according to a conventional method, respectively spotting the lumbrukinase standard solution and the sample solution into the same plate, covering the plate, and placing the plate in a 37 ℃ incubator for heat preservation for 18h. Taking out the lumbrukinase standard product, measuring two perpendicular diameters of the solvent ring by using a vernier caliper, drawing a standard curve by taking the unit number of the lumbrukinase standard product as a horizontal coordinate and the product of the two perpendicular diameters as a vertical coordinate, and calculating a regression equation. Substituting the product of the two vertical diameters of the lumbrokinase solution into a regression equation to calculate the titer unit number of the sample.
The results show that the lumbrokinase activity obtained in example 1 is 30000IU/mg, the lumbrokinase activity obtained in example 2 is 24000IU/mg, and the lumbrokinase activity obtained in example 3 is 27000IU/mg.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (10)
1. A method for efficiently extracting full resources from lumbrukinase is characterized by comprising the following steps:
soaking earthworms in a first buffer solution, homogenizing, and performing membrane filtration on the obtained homogenate material to obtain a protein solution;
mixing the protein solution with ethanol, and salting out to obtain supernatant and a salted-out substance;
mixing the salting-out material with a second buffer solution, performing protein purification on the obtained mixed solution, and performing freeze drying to obtain lumbrokinase;
distilling the supernatant under reduced pressure, mixing the obtained residual liquid with the earthworm residue obtained by homogenizing, and fermenting to obtain an ecological fertilizer;
the first buffer solution and the second buffer solution are both potassium bicarbonate-ammonium acetate mixed solutions;
the pH values of the first buffer solution and the second buffer solution are independently 7.5-8.0.
2. The method for efficiently extracting and recycling lumbrokinase according to claim 1, wherein the pH values of the first buffer solution and the second buffer solution are 7.8.
3. The method for efficiently extracting and recycling lumbrokinase according to claim 1, wherein the soaking time is 30-60 min.
4. The method for efficiently extracting and recycling lumbrukinase as claimed in claim 1, wherein the rotation speed of the homogenate is 20000-25000 rpm for 3-5 min.
5. The method for efficiently extracting and recycling lumbrokinase according to claim 1, wherein the molecular weight cut-off of the membrane used for membrane filtration is 50KD, and the molecular weight of the protein in the protein solution is less than or equal to 50KD.
6. The method for efficiently extracting and fully recycling lumbrukinase as claimed in claim 1 or 5, wherein the volume ratio of ethanol to protein solution is (0.5-3): 1.
7. The method for efficiently extracting and recycling lumbrukinase as claimed in claim 1, wherein the mass ratio of the second buffer solution to the salting-out material is (10-100): 1.
8. The method for efficiently extracting and recycling lumbrokinase according to claim 1, wherein the temperature of the reduced pressure distillation is 50-60 ℃.
9. The method for efficiently extracting and recycling lumbrokinase according to claim 1, wherein the fermentation temperature is 30-35 ℃ and the fermentation time is 10-15 days.
10. The method for efficiently extracting and recycling lumbrokinase according to claim 9, wherein the fermentation is performed under anaerobic closed conditions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211653322.7A CN115772511A (en) | 2022-12-22 | 2022-12-22 | Method for efficiently extracting full resources from lumbrokinase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211653322.7A CN115772511A (en) | 2022-12-22 | 2022-12-22 | Method for efficiently extracting full resources from lumbrokinase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115772511A true CN115772511A (en) | 2023-03-10 |
Family
ID=85392712
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211653322.7A Pending CN115772511A (en) | 2022-12-22 | 2022-12-22 | Method for efficiently extracting full resources from lumbrokinase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115772511A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907738A (en) * | 2016-07-06 | 2016-08-31 | 奥为（天津）环保科技有限公司 | Method for efficiently extracting lumbrokinase from earthworms |
| CN108048434A (en) * | 2017-12-28 | 2018-05-18 | 天津百利食品有限公司 | The extracting method of earthworm protein and Lumbrokinase in a kind of earthworm |
| CN111763127A (en) * | 2020-01-07 | 2020-10-13 | 宁夏大学 | Preparation method for producing fresh earthworm full-nutrient fermented liquid fertilizer in large scale |
-
2022
- 2022-12-22 CN CN202211653322.7A patent/CN115772511A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907738A (en) * | 2016-07-06 | 2016-08-31 | 奥为（天津）环保科技有限公司 | Method for efficiently extracting lumbrokinase from earthworms |
| CN108048434A (en) * | 2017-12-28 | 2018-05-18 | 天津百利食品有限公司 | The extracting method of earthworm protein and Lumbrokinase in a kind of earthworm |
| CN111763127A (en) * | 2020-01-07 | 2020-10-13 | 宁夏大学 | Preparation method for producing fresh earthworm full-nutrient fermented liquid fertilizer in large scale |
Non-Patent Citations (3)
| Title |
|---|
| 国家食品药品监督管理局: "国家食品药品监督管理局国家药品标准修订件", 中国药品标准, vol. 12, no. 5, 31 December 2011 (2011-12-31), pages 395 - 396 * |
| 涂清波 等: "蚓激酶的提取工艺改进研究", 天津中医药, vol. 35, no. 1, 31 January 2018 (2018-01-31), pages 2 - 2 * |
| 郑慧: "蚓激酶提取分离工艺的研究", 中国优秀硕士学位论文全文数据库医药卫生科技辑(月刊), no. 12, 15 December 2014 (2014-12-15), pages 47 - 48 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101863822B (en) | Production method for extracting tryptophan from fermentation liquor by one-step refining | |
| CN108752487A (en) | A method of detaching fungi exocellular polysaccharide using three-phase system | |
| CN108299278A (en) | A method of extraction separation L-Trp | |
| CN103073652A (en) | Method for extracting polysaccharide of spirulina platensis | |
| FI66905B (en) | FRAMEWORK FOR CONTAINING AN ETHANOL GENOM CONTAINER CONTAINER | |
| CN113088542A (en) | Biomass pretreatment process and method for recovering solvent used in treatment process | |
| CN103554247B (en) | A kind of method utilizing ionic liquid mixed solvent to prepare collagen microfibril | |
| CN110791532A (en) | Method for preparing ethanol from cellulose and comprehensively utilizing biomass | |
| CN106631852A (en) | Method for extracting L-ornithine hydrochloride from L-ornithine fermentation broth | |
| CN113480580B (en) | Method for selectively and efficiently extracting flavonoids from licorice residues and co-producing biological methane | |
| CN103215335B (en) | The method of mold oxide is prepared in mold fermentation biosynthesizing | |
| CN115772511A (en) | Method for efficiently extracting full resources from lumbrokinase | |
| CN106893686A (en) | A kind of Enrichment culture method of compound caproic acid bacteria solution | |
| CN109485559B (en) | Method for extracting shikimic acid from star anise | |
| CN107050217A (en) | A kind of preparation method of aloe gel | |
| CN105400754B (en) | High-valued comprehensive utilization method of traditional Chinese medicine residues rich in cellulose substances | |
| CN104610282B (en) | A kind of method of purification of cefazolin | |
| CN106349402A (en) | Method for removing protein in polysaccharide extraction liquid by dialdehyde cellulose | |
| BR112017001669B1 (en) | PROCESSES FOR THE PRODUCTION OF AN ENZYME COCKTAIL FROM A CELLULOLYTIC MICROORGANISM PRODUCING CELLULASES AND/OR HEMICELLULASES, WHERE THE MICROORGANISM IS A FILAMENTOUS FUNGUS, AND FOR THE ENZYME HYDROLYSIS OF A FOOD | |
| CN104762359A (en) | Recycling process of amoxicillin in enzymatically synthesized amoxicillin mother liquor | |
| CN107099555A (en) | A kind of method for carrying out biogas production as raw material with Chinese medicine dreg | |
| CN203402929U (en) | Production system for producing vegetative fulvic acid, potassium sulfate concentrated solution and vegetative organic bacterial manure by alcohol biological wastewater | |
| KR100405316B1 (en) | In situ extraction of hydrocarbon from the culture of microalgae | |
| CN104307368B (en) | Method for screening active ingredients in plant extracting liquid and special electrophoretic separation device | |
| CN113122606B (en) | Fermentation method of riboflavin and fermentation culture medium used in fermentation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |