CN102304499B - Purification method of cabbage mustard peroxydase - Google Patents
Purification method of cabbage mustard peroxydase Download PDFInfo
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- CN102304499B CN102304499B CN201110306926.XA CN201110306926A CN102304499B CN 102304499 B CN102304499 B CN 102304499B CN 201110306926 A CN201110306926 A CN 201110306926A CN 102304499 B CN102304499 B CN 102304499B
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- 240000007124 Brassica oleracea Species 0.000 title claims abstract description 17
- 238000000746 purification Methods 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 title abstract description 12
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 32
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 31
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 108010044467 Isoenzymes Proteins 0.000 claims abstract description 14
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims abstract description 8
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 7
- 238000001502 gel electrophoresis Methods 0.000 claims abstract description 7
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 7
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- 239000000287 crude extract Substances 0.000 claims abstract description 5
- 102000004190 Enzymes Human genes 0.000 claims description 20
- 108090000790 Enzymes Proteins 0.000 claims description 20
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 20
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 12
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 11
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 10
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 10
- 239000000499 gel Substances 0.000 claims description 10
- 238000001556 precipitation Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- 238000001962 electrophoresis Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000005342 ion exchange Methods 0.000 claims description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 claims description 4
- 239000003292 glue Substances 0.000 claims description 4
- 239000012160 loading buffer Substances 0.000 claims description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 3
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 230000009182 swimming Effects 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims 1
- 108091006629 SLC13A2 Proteins 0.000 claims 1
- 238000002156 mixing Methods 0.000 claims 1
- 238000010186 staining Methods 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 7
- 230000007547 defect Effects 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 240000001008 Dimocarpus longan Species 0.000 description 3
- 235000000235 Euphoria longan Nutrition 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 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 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- AXAVXPMQTGXXJZ-UHFFFAOYSA-N 2-aminoacetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(O)=O.OCC(N)(CO)CO AXAVXPMQTGXXJZ-UHFFFAOYSA-N 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 240000000950 Hippophae rhamnoides Species 0.000 description 1
- 235000003145 Hippophae rhamnoides Nutrition 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008570 general process Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Abstract
The invention belongs to the technical field of plant biology and discloses a purification method of a cabbage mustard peroxidase isozyme. In the purification method, cabbage mustard protein crude extract liquid is taken as a material, and ammonium sulfate precipitation, hydrophobic chromatography, ion exchange chromatography and gel electrophoresis are sequentially adopted for purifying a cabbage mustard peroxidase. The purification method disclosed by the invention has simple steps, strong pertinence, is convenient to operate and the defect that the specific isozyme is difficult to separate by adopting the other traditional plant peroxidase purification methods can be overcome.
Description
Technical field
The present invention relates to a kind of purification process of cabbage mustard peroxidase, particularly, adopt successively ammonium sulfate precipitation, hydrophobic chromatography, ion-exchange chromatography and gel electrophoresis method, the separated peroxidase that obtains purifying from vegetable-protein crude extract, belongs to plant biotechnology field compactly.
Background technology
Catalyzed Synthesis By Peroxidase H
2o
2or other superoxide are oxidized multiple substrate, as Polyphenols, aminated compounds and some heterogeneous ring compound, the aspects such as, biosensor synthetic in medical science detection, Industrial Wastewater Treatment, organic polymer are widely used.Peroxidase is ubiquity in plant, has multiple isozyme and different physiological functions, as oxygenolysis indolylacetic acid, synthetic xylogen, improve the resistance of plant etc.
The basis of above-mentioned application and physiological function research is to obtain pure peroxidase.At present, from the plants such as horseradish, longan, soybean peel, sea-buckthorn, separation obtains peroxidase, is mainly to utilize the methods such as ammonium sulfate precipitation, ion-exchange, molecular sieve filtration, hydrophobic chromatography.These method general process are complicated, such as Lin Lin etc. when the separated longan peel peroxidase (Lin Lin etc. the separation and purification of longan peel peroxidase. University Of Agriculture and Forestry In Fujian's journal, 2006,35 (2): 157-160) adopt the chromatographic separation such as ammonium sulfate precipitation classification, Streamline Pheneyl, DEAE-Cellose, Phenyl Sepharose High Performance, Superdex200prep grade to go out 4 peroxidase components; In addition the existence of a large amount of isozyme, such as in Fuji apple, have 11 Peroxidase Isoenzymes (Yan Zhongye etc. Apple Fuji Strain Peroxidase Isoenzyme research. Agricultural University Of Shenyang's journal, 2006,37 (4): 578-581) not only increased separated difficulty, and resulting isozyme is not often desirable target protein.
Cabbage mustard is one of distinctive vegetable variety of south China, because of crisp, the clear aquatic foods of its matter, nutritiously liked by consumers in general, and finds a good sale in the countries and regions such as South East Asia.But the research to cabbage mustard peroxidase is limited, not yet there is the report of cabbage mustard peroxidase separation purification method.
Therefore, the purification process of exploitation cabbage mustard peroxidase, simplifies the purification step of existing plant peroxidases, from and a plurality of Peroxidase Isoenzymes of depositing isolate specific kind, there is certain value.
Summary of the invention
The object of the present invention is to provide a kind of purification process of cabbage mustard peroxidase, simplify step, improve the specific aim of isozyme separation.
The technical scheme that realizes above-mentioned purpose is as follows:
(1) ammonium sulfate precipitation
Cabbage mustard protein crude extract slowly adds while stirring ammonium sulfate at 4 ℃, and when ammonium sulfate mass volume ratio concentration reaches 31%, 10000-12000rpm is centrifugal, discards precipitation; Continue to add ammonium sulfate, to mass volume ratio concentration be 62%; 10000-12000rpm centrifugation subsequently, abandoning supernatant adds phosphoric acid buffer (20mmol/L in every 1g precipitation, pH7.5) 10-15mL slowly stirs 1h at 4 ℃, and 10000-12000rpm is centrifugal subsequently, discard precipitation, supernatant liquor is enzyme liquid I.
(2) hydrophobic chromatography
The enzyme liquid I that step (1) is obtained is splined on Phenyl Sefinose6Fast Flow, then with containing the phosphoric acid buffer (20mmol/L that mass volume ratio concentration is 26-0% ammonium sulfate, pH7.5) gradient elution, collects the component with peroxidase activity, is enzyme liquid II.
(3) ion-exchange chromatography
The enzyme liquid II that step (2) is obtained is splined on DEAE Sefinose Fast Flow or Mono Q ion exchange column, then with containing the phosphoric acid buffer (20mmol/L that mass volume ratio concentration is 0-12%NaCl, pH7.5) gradient elution, collection has the component of peroxidase activity, add in ultrafiltration and concentration centrifuge tube, 3000rpm, 4 ℃ are centrifugal, and the concentrated solution obtaining is enzyme liquid III.
(4) gel electrophoresis
By the enzyme liquid III obtaining in step (3) and the sample-loading buffer (Tris-HCl of 20mmol/L, pH6.8; Mass volume ratio concentration is 10% glycine; Mass volume ratio concentration is the sodium laurylsulfonate of 0.1-1%) mix on the separation gel that concentrated glue that application of sample is 5-6% in acrylamide mass concentration and acrylamide mass concentration are 10-12% electrophoresis.The gel of getting a swimming lane after electrophoresis carries out enzymic activity dyeing with p-diaminodiphenyl, extracts all the other gels at corresponding colour generation pillar location place, puts into deionized water and soaks 12h, and supernatant liquor freeze-drying is the Peroxidase Isoenzyme of purifying.
It is 12% acrylamide that the purity of above-mentioned enzyme can adopt mass concentration, mass volume ratio concentration is 1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis detection, electrophoretic buffer is 25mmol/L Tris-glycine (pH8.3), with coomassie brilliant blue R_250 dyeing, methyl alcohol/Glacial acetic acid/distilled water (1: 1: 8) decolouring.
Compared with prior art, the present invention has following beneficial effect:
(1) the present invention adopts ammonium sulfate precipitation, hydrophobic chromatography, ion-exchange chromatography, gel electrophoresis method purifying cabbage mustard superoxide isozyme successively, and purification step is few.(2) adopt the gel electrophoresis method can the specific superoxide isozyme of purifying, with strong points.The enzyme liquid of purifying of the present invention is detected by described detection method, and result is single band, shows that product purity is high.
Embodiment
Embodiment 1
Utilize method purifying cabbage mustard peroxidase of the present invention, be specially:
(1) ammonium sulfate precipitation
Cabbage mustard protein crude extract slowly adds while stirring ammonium sulfate at 4 ℃, and when ammonium sulfate mass volume ratio concentration reaches 31%, 10000rpm is centrifugal, discards precipitation; Continue to add ammonium sulfate, to mass volume ratio concentration be 62%; 10000rpm centrifugation subsequently, abandoning supernatant adds phosphoric acid buffer (20mmol/L, pH7.5) 12mL in every 1g precipitation, slowly stirs 1h at 4 ℃, and 10000rpm is centrifugal subsequently, discards precipitation, and supernatant liquor is enzyme liquid I.
(2) hydrophobic chromatography
The enzyme liquid I that step (1) is obtained is splined on Phenyl Sefinose6Fast Flow, then with containing the phosphoric acid buffer (20mmol/L that mass volume ratio concentration is 26-0% ammonium sulfate, pH7.5) gradient elution, collects the component with peroxidase activity, is enzyme liquid II.
(3) ion exchange chromatography
The enzyme liquid II that step (2) is obtained is splined on DEAE Sefinose Fast Flow ion exchange column, then with containing the phosphoric acid buffer (20mmol/L that mass volume ratio concentration is 0-12%NaCl, pH7.5) gradient elution, collection has the component of peroxidase activity, add in ultrafiltration and concentration centrifuge tube, 3000rpm, 4 ℃ are centrifugal, and the concentrated solution obtaining is enzyme liquid II.
(4) gel electrophoresis
By the enzyme liquid II in step (3) and the sample-loading buffer (Tris-HCl of 20mmol/L, pH6.8; Mass volume ratio concentration is 10% glycine; Mass volume ratio concentration is 0.5% sodium laurylsulfonate) mix on the separation gel that concentrated glue that application of sample is 5% in acrylamide mass concentration and acrylamide mass concentration are 12% electrophoresis.After electrophoresis, get the gel of a swimming lane, with p-diaminodiphenyl, carry out enzymic activity dyeing, extract all the other gels at corresponding colour generation pillar location place, put into deionized water and soak 12h, supernatant liquor freeze-drying is the Peroxidase Isoenzyme of purifying.The acrylamide that the peroxidase of purifying is 12% through mass concentration, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis that mass volume ratio concentration is 1% detects as single protein band.
Embodiment 2
Substantially the same manner as Example 1, its difference is
What in step (3), use is Mono Q ion exchange column, with containing phosphoric acid buffer (20mmol/L, the pH7.5) wash-out that mass volume ratio concentration is 0-8.8%NaCl.
Embodiment 3
Substantially the same manner as Example 2, its difference is
In step (4) sample-loading buffer, the mass volume ratio concentration of sodium laurylsulfonate is 0.1%; The acrylamide mass concentration of concentrated glue is 6%, and the acrylamide mass concentration of separation gel is 10%.
Claims (1)
1. a purification process for cabbage mustard peroxidase, is characterized in that, concrete operation step is as follows:
(1) ammonium sulfate precipitation: cabbage mustard protein crude extract, at 4 ℃, slowly add while stirring ammonium sulfate, when ammonium sulfate mass volume ratio concentration reaches 31%, 10000-12000rpm is centrifugal, discards precipitation; Continue to add ammonium sulfate, to mass volume ratio concentration be 62%; 10000-12000rpm centrifugation subsequently, abandoning supernatant, the phosphoric acid buffer 10-15mL to adding 20mmol/L, pH7.5 in every 1g precipitation slowly stirs 1h at 4 ℃, and 10000-12000rpm is centrifugal subsequently, discards precipitation, and supernatant liquor is enzyme liquid I;
(2) hydrophobic chromatography: the enzyme liquid I that step (1) is obtained is splined on Phenyl Sefinose6Fast Flow, then with containing the 20mmol/L that mass volume ratio concentration is 26-0% ammonium sulfate, the phosphoric acid buffer gradient elution of pH7.5, collection has the component of peroxidase activity, is enzyme liquid II;
(3) ion-exchange chromatography: the enzyme liquid II that step (2) is obtained is splined on DEAE Sefinose Fast Flow or Mono Q ion exchange column, then with containing the 20mmol/L that mass volume ratio concentration is 0-12%NaC1, the phosphoric acid buffer gradient elution of pH7.5, collection has the component of peroxidase activity, add in ultrafiltration and concentration centrifuge tube, 3000rpm, 4 ℃ centrifugal, and the concentrated solution obtaining is enzyme liquid III;
(4) gel electrophoresis: on the separation gel that the concentrated glue that the sample-loading buffer mixing application of sample of the glycine that is 10% by the enzyme liquid III obtaining in step (3) and the Tris-HC1 that contains 20mmol/L, pH6.8, mass volume ratio concentration, the sodium laurylsulfonate that mass volume ratio concentration is 0.1-1% is 5-6% in acrylamide mass concentration and acrylamide mass concentration are 10-12%, electrophoresis; After electrophoresis, get the gel benzidine staining of a swimming lane, extract all the other gels at corresponding colour generation pillar location place, put into deionized water and soak 12h, supernatant liquor freeze-drying is the superoxide isozyme of purifying.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201110306926.XA CN102304499B (en) | 2011-10-12 | 2011-10-12 | Purification method of cabbage mustard peroxydase |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201110306926.XA CN102304499B (en) | 2011-10-12 | 2011-10-12 | Purification method of cabbage mustard peroxydase |
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| CN102304499A CN102304499A (en) | 2012-01-04 |
| CN102304499B true CN102304499B (en) | 2014-01-22 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2011119706A1 (en) * | 2010-03-26 | 2011-09-29 | E.I. Dupont De Nemours And Company | Perhydrolase providing improved specific activity |
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Non-Patent Citations (4)
| Title |
|---|
| 付伟丽.甘薯叶过氧化物酶的分离纯化及其部分性质研究.《食品科学》.2010,第31卷(第7期),第223-227页. |
| 甘蓝型、白菜型油菜和白花芥蓝的同工酶比较研究;程必芳;《中国油料》;19910702(第2期);摘要 * |
| 甘薯叶过氧化物酶的分离纯化及其部分性质研究;付伟丽;《食品科学》;20100401;第31卷(第7期);摘要 * |
| 程必芳.甘蓝型、白菜型油菜和白花芥蓝的同工酶比较研究.《中国油料》.1991,(第2期),第24-27页. |
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