CN110747189A - Method for rapidly extracting matrix metalloproteinase - Google Patents
Method for rapidly extracting matrix metalloproteinase Download PDFInfo
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- CN110747189A CN110747189A CN201910969925.XA CN201910969925A CN110747189A CN 110747189 A CN110747189 A CN 110747189A CN 201910969925 A CN201910969925 A CN 201910969925A CN 110747189 A CN110747189 A CN 110747189A
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 102000002274 Matrix Metalloproteinases Human genes 0.000 title claims abstract description 19
- 108010000684 Matrix Metalloproteinases Proteins 0.000 title claims abstract description 19
- 102000005741 Metalloproteases Human genes 0.000 claims abstract description 38
- 108010006035 Metalloproteases Proteins 0.000 claims abstract description 38
- 239000002002 slurry Substances 0.000 claims abstract description 31
- 238000000605 extraction Methods 0.000 claims abstract description 28
- 241000233866 Fungi Species 0.000 claims abstract description 25
- 239000000243 solution Substances 0.000 claims abstract description 20
- 238000004140 cleaning Methods 0.000 claims abstract description 19
- 239000011324 bead Substances 0.000 claims abstract description 14
- 229920002472 Starch Polymers 0.000 claims abstract description 9
- 239000007864 aqueous solution Substances 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 235000019698 starch Nutrition 0.000 claims abstract description 9
- 239000008107 starch Substances 0.000 claims abstract description 9
- 238000000227 grinding Methods 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 16
- 230000000694 effects Effects 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 12
- 239000012535 impurity Substances 0.000 claims description 12
- 239000012528 membrane Substances 0.000 claims description 8
- 238000000967 suction filtration Methods 0.000 claims description 8
- 238000000108 ultra-filtration Methods 0.000 claims description 8
- 230000007935 neutral effect Effects 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- 210000004102 animal cell Anatomy 0.000 claims description 5
- 230000003204 osmotic effect Effects 0.000 claims description 5
- 102000035195 Peptidases Human genes 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 239000004744 fabric Substances 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 230000002779 inactivation Effects 0.000 claims description 4
- 230000007774 longterm Effects 0.000 claims description 4
- 229920005597 polymer membrane Polymers 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 230000036632 reaction speed Effects 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 4
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 4
- 238000010008 shearing Methods 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 description 17
- 235000009467 Carica papaya Nutrition 0.000 description 7
- 235000006264 Asimina triloba Nutrition 0.000 description 6
- 240000006432 Carica papaya Species 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 3
- 102000010911 Enzyme Precursors Human genes 0.000 description 2
- 108010062466 Enzyme Precursors Proteins 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 241000219173 Carica Species 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000002994 raw material Substances 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)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6489—Metalloendopeptidases (3.4.24)
- C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
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Abstract
The invention discloses a method for quickly extracting matrix metalloproteinase, relates to the technical field of extraction of metalloproteinase, and aims to solve the problem that no complete implementation method is available for extracting metalloproteinase from plants or fungi in the prior art. Step 1: pouring plants or fungi containing the metalloprotease into a crawler-type cleaning machine, and conveying the plants or fungi to a high-speed dicer by the crawler-type cleaning machine to cut the plants or fungi into small pieces so as to facilitate the subsequent cell disruption treatment; step 2: conveying the cut plants or fungi to a bead mill, and grinding to crush cells and release inclusions; and step 3: conveying the ground slurry to an extraction tank, pouring a proper amount of extracting solution into the extraction tank, wherein the extracting solution is an aqueous solution with the pH value of 7-9, and reacting for 10-30 minutes at the temperature of 45-50 ℃ to extract the metalloprotease in the extracting solution; and 4, step 4: after the reaction is finished, the slurry is conveyed to a centrifugal machine, and the starch in the slurry is separated under the action of centrifugal force.
Description
Technical Field
The invention relates to the technical field of extraction of metalloprotease, in particular to a method for quickly extracting matrix metalloprotease.
Background
Matrix metalloproteinases are a large family named for their requirement of metal ions such as Ca2+, Zn2+, etc. as cofactors. The family members have similar structures and generally consist of 5 functionally distinct domains (1) a hydrophobic signal peptide sequence; (2) the main function of the propeptide region is to stabilize the proenzyme. When the region is cut off by exogenous enzyme, the MMPs zymogen is activated; (3) the catalytic active region has a zinc ion binding site and is crucial to the exertion of the catalytic action of the enzyme; (4) a proline-rich hinge region; (5) the carboxy-terminal region, which is related to the substrate specificity of the enzyme. Wherein the enzyme catalytic active region and the propeptide region are highly conserved, and the MMPs members have the characteristics based on the structure. Various MMPs have some substrate specificity among themselves, but not absolutely. The same MMP degrades various extracellular matrix components, and one extracellular matrix component can be degraded by various MMPs, but the degradation efficiency of different enzymes can be different.
However, no complete set of implementation methods for extracting the metalloprotease from the plants or the fungi in the prior art is available; thus, the existing needs are not met, and a method for rapidly extracting matrix metalloproteinase is provided for the method.
Disclosure of Invention
The invention aims to provide a method for quickly extracting matrix metalloproteinase, which aims to solve the problem that no complete implementation method is available for extracting the metalloproteinase from plants or fungi in the prior art.
In order to achieve the purpose, the invention provides the following technical scheme: a method for rapidly extracting matrix metalloproteinase comprises the following steps:
step 1: pouring plants or fungi containing the metalloprotease into a crawler-type cleaning machine, and conveying the plants or fungi to a high-speed dicer by the crawler-type cleaning machine to cut the plants or fungi into small pieces so as to facilitate the subsequent cell disruption treatment;
step 2: conveying the cut plants or fungi to a bead mill, and grinding to crush cells and release inclusions;
and step 3: conveying the ground slurry to an extraction tank, pouring a proper amount of extracting solution into the extraction tank, wherein the extracting solution is an aqueous solution with the pH value of 7-9, and reacting for 10-30 minutes at the temperature of 45-50 ℃ to extract the metalloprotease in the extracting solution;
and 4, step 4: after the reaction is finished, conveying the slurry to a centrifugal machine, and separating starch in the slurry under the action of centrifugal force;
and 5: the slurry with the starch removed is conveyed to an ultrafilter in a suction filtration mode, and in the suction filtration process, solid matters such as yeast, mould, plant cells and the like in the slurry can be filtered out through filter cloth, so that the rough filtration effect is achieved;
step 6: sending the slurry after rough filtration to an ultrafilter, filtering out residual biomacromolecule impurities by an ultrafiltration membrane, and concentrating and purifying;
and 7: the treated metalloprotease concentrate is sent to a vacuum freeze drying oven for long-term storage.
Preferably, in the step 1, the plant or fungus is selected as an extraction carrier, and the sensitivity of the plant or fungus to sound waves, machinery, osmotic pressure and temperature is smaller in the extraction process compared with that of animal cells, so that the extraction process of the metalloprotease can be more stable.
Preferably, the crawler-type cleaning machine in step 2 is provided with a plurality of spray heads, so that the crawler-type cleaning machine can clean plants or fungi while conveying the plants or the fungi to remove surface impurities.
Preferably, the glass beads are arranged inside the bead mill in the step 2, and collide with plants or fungi during stirring, shearing is carried out, cells are crushed, contents are released, and during crushing, the temperature inside the machine is controlled below 55 ℃ in a jacket cooling mode, so that a large amount of inactivation of the metalloprotease is avoided.
Preferably, the aqueous solution with a pH value of 7-9 is used as the extracting solution in the step 3, because most of the metalloproteases are neutral proteins, the optimal pH value is 7-9, proteases outside the pH range can be inactivated after the reaction is finished, the effect of extracting the metalloproteases is achieved, and the reaction speed can be effectively improved by controlling the temperature to be 45-50 ℃.
Preferably, the ultrafiltration membrane in the step 6 is a polymer membrane, and the size of the intercepted particles is within the range
The particle size is-0.2 um, so that biomacromolecule impurities in the slurry can be effectively filtered out, and the purification effect is achieved.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the raw material containing the metalloprotease is preprocessed by arranging the automatic equipment such as the crawler-type cleaning machine, the high-speed dicer, the bead mill and the like, and the equipment can continuously work, so that a complete metalloprotease extraction production line is formed, and the extraction efficiency of the matrix metalloprotease is effectively improved.
2. Compared with animal cells, the extraction carrier of the plant or fungus has smaller sensitivity to sound wave, machinery, osmotic pressure and temperature during extraction, so that the extraction process of the metalloprotease is more stable.
Drawings
FIG. 1 is a schematic view of the overall structure of the present invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Referring to fig. 1, an embodiment of the present invention: a method for rapidly extracting matrix metalloproteinase comprises the following embodiments:
example 1:
step 1: pouring the pawpaw into a crawler-type cleaning machine, and conveying the pawpaw to a high-speed dicer by the crawler-type cleaning machine to be diced so as to facilitate subsequent cell disruption treatment;
step 2: conveying the cut pawpaw to a bead mill, and grinding to break cells and release inclusion;
and step 3: conveying the ground slurry to an extraction tank, pouring a proper amount of extracting solution into the extraction tank, wherein the extracting solution is a neutral aqueous solution with a pH value of 7, and reacting for 30 minutes at the temperature of 45 ℃ to extract the metalloprotease in the extracting solution;
and 4, step 4: after the reaction is finished, conveying the slurry to a centrifugal machine, and separating starch in the slurry under the action of centrifugal force;
and 5: the slurry with the starch removed is conveyed to an ultrafilter in a suction filtration mode, and in the suction filtration process, solid matters such as yeast, mould, plant cells and the like in the slurry can be filtered out through filter cloth, so that the rough filtration effect is achieved;
step 6: sending the slurry after rough filtration to an ultrafilter, filtering out residual biomacromolecule impurities by an ultrafiltration membrane, and concentrating and purifying;
and 7: the treated metalloprotease concentrate is sent to a vacuum freeze drying oven for long-term storage.
Furthermore, the pawpaw is selected as an extraction carrier in the step 1, compared with animal cells, the sensitivity of plants to sound waves, machinery, osmotic pressure and temperature is low during extraction, and the extraction process of the metalloprotease can be more stable.
Further, install a plurality of shower nozzles on the crawler-type cleaning machine in step 2, can carry out cleaning work to it when carrying papaya, get rid of surface impurity.
Furthermore, a plurality of glass beads are arranged in the bead mill in the step 2, and collide with pawpaw during stirring and cut to break cells, so that the content is released, and in the breaking process, the temperature in the machine is controlled below 55 ℃ in a jacket cooling mode, so that a large amount of inactivation of metalloproteinase is avoided.
Further, the aqueous solution with the pH value of 7 is adopted as the extracting solution in the step 3, because most of the metalloproteases in the pawpaw are neutral proteins, the optimal pH value is 7, the proteases outside the pH value can be inactivated after the reaction is finished, the effect of extracting the metalloproteases is achieved, and the reaction speed can be effectively improved by controlling the temperature to be 45 ℃.
Further, the ultrafiltration membrane in the step 6 is a polymer membrane, and the size of the intercepted particles is within the range
Between-0.2 um, thereforeCan effectively filter biomacromolecule impurities in the slurry, and achieve the purification effect.
Example 2:
step 1: pouring the figs into a crawler-type cleaning machine, and conveying the figs into a high-speed dicer by the crawler-type cleaning machine to be diced so as to facilitate subsequent cell disruption treatment;
step 2: conveying the figs to a bead mill, and grinding to break cells and release contents;
and step 3: conveying the ground slurry to an extraction tank, pouring a proper amount of extracting solution into the extraction tank, wherein the extracting solution is a neutral aqueous solution with a pH value of 7, and reacting for 10 minutes at 50 ℃ to extract the metalloprotease in the extracting solution;
and 4, step 4: after the reaction is finished, conveying the slurry to a centrifugal machine, and separating starch in the slurry under the action of centrifugal force;
and 5: the slurry with the starch removed is conveyed to an ultrafilter in a suction filtration mode, and in the suction filtration process, solid matters such as yeast, mould, plant cells and the like in the slurry can be filtered out through filter cloth, so that the rough filtration effect is achieved;
step 6: sending the slurry after rough filtration to an ultrafilter, filtering out residual biomacromolecule impurities by an ultrafiltration membrane, and concentrating and purifying;
and 7: the treated metalloprotease concentrate is sent to a vacuum freeze drying oven for long-term storage.
Furthermore, fig is selected as an extraction carrier in the step 1, compared with animal cells, the sensitivity of plants to sound waves, machinery, osmotic pressure and temperature is lower during extraction, and the extraction process of the metalloprotease can be more stable.
Further, install a plurality of shower nozzles on the crawler-type cleaning machine in step 2, can carry the fig and carry out cleaning work to it when, get rid of surface impurity.
Furthermore, a plurality of glass beads are arranged inside the bead mill in the step 2, and collide with fig during stirring, shearing is carried out, cells are crushed, contents are released, and in the crushing process, the temperature inside the machine is controlled below 55 ℃ in a jacket cooling mode, so that a large amount of inactivation of metalloproteinase is avoided.
Furthermore, the aqueous solution with pH 7 is adopted as the extracting solution in the step 3, because most of the metalloproteases in the figs are neutral proteins, the optimal pH value is 7, proteases outside the pH can be inactivated after the reaction is finished, the effect of extracting the metalloproteases is achieved, and the reaction speed can be effectively improved by controlling the temperature at 50 ℃.
Further, the ultrafiltration membrane in the step 6 is a polymer membrane, and the size of the intercepted particles is within the range
The particle size is-0.2 um, so that biomacromolecule impurities in the slurry can be effectively filtered out, and the purification effect is achieved.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (6)
1. A method for rapidly extracting matrix metalloproteinase is characterized by comprising the following steps:
step 1: pouring plants or fungi containing the metalloprotease into a crawler-type cleaning machine, and conveying the plants or fungi to a high-speed dicer by the crawler-type cleaning machine to cut the plants or fungi into small pieces so as to facilitate the subsequent cell disruption treatment;
step 2: conveying the cut plants or fungi to a bead mill, and grinding to crush cells and release inclusions;
and step 3: conveying the ground slurry to an extraction tank, pouring a proper amount of extracting solution into the extraction tank, wherein the extracting solution is an aqueous solution with the pH value of 7-9, and reacting for 10-30 minutes at the temperature of 45-50 ℃ to extract the metalloprotease in the extracting solution;
and 4, step 4: after the reaction is finished, conveying the slurry to a centrifugal machine, and separating starch in the slurry under the action of centrifugal force;
and 5: the slurry with the starch removed is conveyed to an ultrafilter in a suction filtration mode, and in the suction filtration process, solid matters such as yeast, mould, plant cells and the like in the slurry can be filtered out through filter cloth, so that the rough filtration effect is achieved;
step 6: sending the slurry after rough filtration to an ultrafilter, filtering out residual biomacromolecule impurities by an ultrafiltration membrane, and concentrating and purifying;
and 7: the treated metalloprotease concentrate is sent to a vacuum freeze drying oven for long-term storage.
2. The method for rapidly extracting matrix metalloproteinase according to claim 1, wherein the method comprises the following steps: compared with animal cells, the plant or fungus is less sensitive to sound waves, machinery, osmotic pressure and temperature during extraction, so that the extraction process of the metalloprotease is more stable.
3. The method for rapidly extracting matrix metalloproteinase according to claim 1, wherein the method comprises the following steps: the crawler-type cleaning machine in the step 2 is provided with a plurality of spray heads, so that the crawler-type cleaning machine can clean plants or fungi while conveying the plants or the fungi to remove surface impurities.
4. The method for rapidly extracting matrix metalloproteinase according to claim 1, wherein the method comprises the following steps: the glass beads are arranged in the bead mill in the step 2, and collide with plants or fungi during stirring, shearing is carried out, cells are crushed, contents are released, and in the crushing process, the temperature in the machine is controlled to be below 55 ℃ in a jacket cooling mode, so that a large amount of inactivation of metalloproteinase is avoided.
5. The method for rapidly extracting matrix metalloproteinase according to claim 1, wherein the method comprises the following steps: the aqueous solution with the pH value of 7-9 is used as the extracting solution in the step 3, because the metalloprotease is mostly neutral protein, the optimum pH value is 7-9, the protease outside the pH range can be inactivated after the reaction is finished, the effect of extracting the metalloprotease is achieved, and the reaction speed can be effectively improved by controlling the temperature to be 45-50 ℃.
6. The method for rapidly extracting matrix metalloproteinase according to claim 1, wherein the method comprises the following steps: the ultrafiltration membrane in the step 6 is a polymer membrane, and the size of the intercepted particles is within the rangeMeanwhile, the biomacromolecule impurities in the slurry can be effectively filtered, and the purification effect is achieved.
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