CN103305483B - Preparation method of cassava leaf polyphenol oxidase - Google Patents
Preparation method of cassava leaf polyphenol oxidase Download PDFInfo
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- CN103305483B CN103305483B CN201310271582.2A CN201310271582A CN103305483B CN 103305483 B CN103305483 B CN 103305483B CN 201310271582 A CN201310271582 A CN 201310271582A CN 103305483 B CN103305483 B CN 103305483B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 241000658379 Manihot esculenta subsp. esculenta Species 0.000 title 1
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 9
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- 238000007710 freezing Methods 0.000 claims abstract description 7
- 230000008014 freezing Effects 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 44
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 24
- 229940088598 enzyme Drugs 0.000 claims description 24
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 22
- 239000000872 buffer Substances 0.000 claims description 22
- 239000000706 filtrate Substances 0.000 claims description 15
- 108010059892 Cellulase Proteins 0.000 claims description 10
- 229940106157 cellulase Drugs 0.000 claims description 10
- 238000005360 mashing Methods 0.000 claims description 9
- 238000005194 fractionation Methods 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 5
- 235000011837 pasties Nutrition 0.000 claims description 4
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 4
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 4
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 239000012065 filter cake Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 4
- 150000008442 polyphenolic compounds Chemical class 0.000 abstract 3
- 235000013824 polyphenols Nutrition 0.000 abstract 3
- 239000008363 phosphate buffer Substances 0.000 abstract 2
- 229920002678 cellulose Polymers 0.000 abstract 1
- 239000001913 cellulose Substances 0.000 abstract 1
- 238000004140 cleaning Methods 0.000 abstract 1
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- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallyl group Chemical group C1(=C(C(=CC=C1)O)O)O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
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- 244000017020 Ipomoea batatas Species 0.000 description 1
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- 235000004456 Manihot esculenta Nutrition 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- NKQPRNDTEUZYKT-UHFFFAOYSA-N cyclohex-5-ene-1,2,3,4-tetrone Chemical group O=C1C=CC(=O)C(=O)C1=O NKQPRNDTEUZYKT-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a preparation method of cassava leaf polyphenol oxidase. The method comprises the following steps of: cleaning fresh cassava leaves, cutting the cassava leaves into pieces, preparing 4ml of a phosphate buffer with the pH of 6 for each gram of the cassava leaves; adding cellulose of which the mass is 0.8% of the total mass of the cassava leaves to act for 45 minutes at 45 DEG C; triturating the cassava leaves into paste by using a tissue triturating instrument at 5 DEG C, subsequently conducting refrigeration digestion for 5 hours; conducting suction filtration and collecting the filtered liquid; conducting refrigerated centrifugation at 5 DEG C; collecting the centrifugate so as to obtain cassava leaf polyphenol oxidase crude enzyme; purifying the crude enzyme, namely, firstly, conducting grading precipitation by using ammonium sulfate, secondly, dissolving the collected precipitate in phosphate buffer which is pre-cooled at 5 DEG C, and finally purifying by using sephadex G-75 so as to obtain a cassava leaf polyphenol purified liquid; and freezing and drying the cassava leaf polyphenol purified liquid in vacuum so as to obtain cassava leaf polyphenol purified freeze-dried powder. As cassava leaves are low in price, easy to access and high in activity, cassava leaf polyphenol oxidase with high activity is prepared, and the preparation method has practical application values.
Description
Technical field
The present invention relates to a kind of preparation method of cassava leaf polyphenol oxidase.
Background technology
Cassava, also known as cassava, wooden sweet potato (Classification system: Manihot esculenta crantz, English name Cassava), belongs to Euphorbiaceae Euphorbiaceae cassava Manihot, is perennial shrub shape crop.Cassava is extensively planted in torrid areas, the whole world, and China mainly cultivates in area, Guangdong and Guangxi Provinces and Hainan Island.Mainly utilize its tuberous root to process to the utilization of cassava at present and prepare tapioca (flour) and alcohol, fructose, glucose etc., the cassava leaves of One's name is legion is then dropped with cassava stem stalk.Polyphenoloxidase is a kind of proteolytic enzyme of copper ions prothetic group, is extensively present in vegetable cell, and it can be oxidized to adjacent benzene diquinone by catalysis pyrocatechol.Polyphenoloxidase in plant is mainly present in the matrix of the thylakoid of chloroplast(id), thus is unlikely to oxidizing reaction occurs.But when the physiology generation pathology of plant materials or tissue are damaged, the balance that this enzyme-to-substrate region separates is broken, react generation quinone, the result of chain reaction causes browning common in preserving fruit and vegetable utilizing occurs, and directly has influence on the outward appearance of the agricultural-food such as fruits and vegetables, local flavor and nutrition.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation method utilizing cassava leaves to extract cassava leaf polyphenol oxidase, the method can obtain highly active polyphenoloxidase from cassava leaves, important meaning is had, for the comprehensive utilization of cassava provides reference for the zymologic property and application thereof of studying Polyphenol Oxidases In Plants.
The present invention solves the problems of the technologies described above with following technical scheme: fresh cassava leaves cleaned and shred, by the phosphoric acid buffer 4 milliliters that every gram of cassava leaves preparation pH is 6, at 45 DEG C, add the cellulase effect 45 minutes that quality is cassava leaves total mass 0.8%, be chilled to 5 DEG C; Then smash to pieces to pasty state with high speed tissue mashing instrument, take out freezing lixiviate at being placed in 5 DEG C and carry out suction filtration after 5 hours, discard residue collection filtrate; With 12000r/min frozen centrifugation 25 minutes at 5 DEG C, collect centrifugate and namely obtain cassava leaf polyphenol oxidase crude enzyme liquid.Cassava leaf polyphenol oxidase crude enzyme liquid purifying first adopts ammonium sulfate to carry out fractionation precipitation, collect the precipitation of 40% to 80% saturation ratio, precipitation is dissolved in the phosphoric acid buffer of 5 DEG C, adopt dextrane gel sephadex G-75 purifying again, obtain cassava leaf polyphenol oxidase refined solution, make lyophilized powder through vacuum lyophilization, obtain cassava leaf polyphenol oxidase.
A kind of preparation method of cassava leaf polyphenol oxidase, it is characterized in that fresh cassava leaves to clean to shred, by the phosphoric acid buffer 4 milliliters that every gram of cassava leaves preparation pH is 6, the cellulase effect 45 minutes that quality is cassava leaves total mass 0.8% is added at 45 DEG C, the cross-linked polyvinylpyrrolidone of 0.02 gram is added by every gram of cassava leaves, abundant mixing, smash to pieces with the homogenate of high speed tissue mashing instrument, at 5 DEG C, lixiviate adopted filtered through gauze after 5 hours, filtrate, with 12000r/min5 DEG C of frozen centrifugation 25 minutes, is collected filtrate and is obtained cassava leaf polyphenol oxidase crude enzyme liquid.Cassava leaf polyphenol oxidase crude enzyme liquid purifying first adopts ammonium sulfate to carry out fractionation precipitation, collect the precipitation of 40% to 80% saturation ratio, precipitation is dissolved in the phosphoric acid buffer of 5 DEG C, adopt dextrane gel sephadex G-75 purifying again, obtain cassava leaf polyphenol oxidase refined solution, make lyophilized powder through vacuum lyophilization, obtain cassava leaf polyphenol oxidase.
A kind of preparation method of cassava leaf polyphenol oxidase, it is characterized in that fresh cassava leaves to clean to shred, by the phosphoric acid buffer 4 milliliters that every gram of cassava leaves preparation pH is 6, the cellulase effect 45 minutes that quality is cassava leaves total mass 0.8% is added at 45 DEG C, add the cold acetone of cassava leaves quality 4 times, be pounded homogenate with high-speed tissue mashing machine, suction filtration, filtrate is stand-by; The freezing acetone of filter cake extracts suction filtration again, the white mass obtained, and vacuum-drying obtains cassava leaf polyphenol oxidase acetone powder; Acetone powder is dissolved in the phosphoric acid buffer of pH6.0 and extracts 3h, add above-described stand-by filtrate, with 12000rmp/min centrifugal 25min removing precipitation at 5 DEG C, get its supernatant liquor and be cassava leaf polyphenol oxidase crude enzyme liquid.Cassava leaf polyphenol oxidase crude enzyme liquid purifying first adopts ammonium sulfate to carry out fractionation precipitation, collect the precipitation of 40% to 80% saturation ratio, precipitation is dissolved in the phosphoric acid buffer of 5 DEG C, adopt dextrane gel sephadex G-75 purifying again, obtain cassava leaf polyphenol oxidase refined solution, make lyophilized powder through vacuum lyophilization, obtain cassava leaf polyphenol oxidase.
Present invention employs wide material sources and lacking the cassava leaves utilized is main raw material, cheapness can be obtained, be easy to get and the very high cassava leaf polyphenol oxidase of activity, for the zymologic property research of polyphenoloxidase and application provide highly active enzyme, the comprehensive utilization for cassava provides new way.And have employed microbial enzyme or cross-linked polyvinylpyrrolidone or acetone and assist phosphoric acid buffer extraction method, obtain highly active cassava leaf polyphenol oxidase, there is actual application value.
Embodiment
Polyphenoloxidase all extensively exists in each tissue of plant, because it has multiple physiological significance to plant itself, plays an important role especially in plant disease-resistant.
The present invention utilizes biological and chemical extracting method to organically combine and extracts cassava leaf polyphenol oxidase.
The substrate that cassava leaf polyphenol oxidase binding ability is the strongest is pyrogallol, and reaction optimum pH is 7.0, and when temperature is 40 DEG C, the Km value of enzymatic oxidn reaction is 0.8mol/L, Vmax=0.1510 (U/min).Total enzyme 26000U, total protein 269.4mg, Rate activity 96.51U/mg alive.
Clean to cassava leaves the size shredded not to be strict with, the chances are about 1 ~ 3cm just because also will smash to pieces below!
Embodiment 1
Gather fresh cassava leaves to clean and shred about 1cm, adding 4 milliliters of pH according to every gram of cassava leaves is the phosphoric acid buffer of 6, adds the cellulase effect 45 minutes that quality is cassava leaves total mass 0.8% at 45 DEG C.Then be chilled to 5 DEG C, smash to pieces to pasty state with high speed tissue mashing instrument, take out and be placed in 5 DEG C of refrigerator freezing lixiviates 5 hours.Carry out suction filtration with 2 layers of gauze again, discard residue collection filtrate, with 12000r/min frozen centrifugation 25 minutes under 5 DEG C of conditions, collect centrifugate and namely obtain cassava leaf polyphenol oxidase crude enzyme liquid.Cassava leaf polyphenol oxidase crude enzyme liquid purifying first adopts ammonium sulfate to carry out fractionation precipitation, collect the precipitation of 40% to 80% saturation ratio, precipitation is dissolved in the phosphoric acid buffer of 5 DEG C, adopt dextrane gel sephadex G-75 purifying again, obtain cassava leaf polyphenol oxidase refined solution, make lyophilized powder through vacuum lyophilization, obtain cassava leaf polyphenol oxidase.Unit enzyme is lived as 260U/mL after measured.
Embodiment 2
Gather fresh cassava leaves clean shred as 1cm; adding 4 milliliters of pH according to every gram of cassava leaves is the phosphoric acid buffer of 6; the cellulase effect 45 minutes that quality is cassava leaves total mass 0.8% is added at 45 DEG C; the cross-linked polyvinylpyrrolidone of 0.02 gram is added by every gram of cassava leaves; abundant mixing; smash 1 minute to pieces with the homogenate of high speed tissue mashing instrument, shut down 1 minute and then smash 1 minute to pieces.Gained homogenate adopts 2 layers of filtered through gauze 5 DEG C of lixiviates after 5 hours, and filtrate, with 12000r/min5 DEG C of frozen centrifugation 25 minutes, is collected filtrate and obtained cassava leaf polyphenol oxidase crude enzyme liquid.Cassava leaf polyphenol oxidase crude enzyme liquid purifying first adopts ammonium sulfate to carry out fractionation precipitation, collect the precipitation of 40% to 80% saturation ratio, precipitation is dissolved in the phosphoric acid buffer of 5 DEG C, adopt dextrane gel sephadex G-75 purifying again, obtain cassava leaf polyphenol oxidase refined solution, make lyophilized powder through vacuum lyophilization, obtain cassava leaf polyphenol oxidase.Unit enzyme is lived as 310U/mL after measured.
Embodiment 3
Gather fresh cassava leaves clean shred as 2cm, adding 4 milliliters of pH according to every gram of cassava leaves is the phosphoric acid buffer of 6, the cellulase effect 45 minutes that quality is cassava leaves total mass 0.8% is added at 45 DEG C, add-20 DEG C of acetone of every gram of cassava leaves 4mL again, in tissue mashing machine, the cassava leaves after enzymolysis is broken into homogenate, 2 metafiltration paper suction filtrations, filtrate is stand-by; The acetone of filter residue precooling washes into white powder, vacuum-drying, obtains cassava leaf polyphenol oxidase acetone powder; Add the phosphoric acid buffer of 5 DEG C, and add above-described stand-by filtrate, be placed in 5 DEG C of refrigerator magnet suspension stirrers and stir 5 hours, 12000r/min5 DEG C of centrifugal 25min gets supernatant, obtains cassava leaf polyphenol oxidase crude enzyme liquid.Cassava leaf polyphenol oxidase crude enzyme liquid purifying first adopts ammonium sulfate to carry out fractionation precipitation, collect the precipitation of 40% to 80% saturation ratio, precipitation is dissolved in the phosphoric acid buffer of 5 DEG C, adopt dextrane gel sephadex G-75 purifying again, obtain cassava leaf polyphenol oxidase refined solution, make lyophilized powder through vacuum lyophilization, obtain cassava leaf polyphenol oxidase.At this moment total enzyme is lived as 28170U, total protein 189mg, Rate activity 149U/mg, and unit enzyme is lived as 414U/mL.
Claims (3)
1. a preparation method for cassava leaf polyphenol oxidase, cleans fresh cassava leaves and shreds, smash to pieces to pasty state after adding phosphoric acid buffer and cellulase effect, more freezing lixiviate, suction filtration, filtrate centrifugation obtain cassava leaf polyphenol oxidase crude enzyme liquid; Crude enzyme liquid adopts ammonium sulfate to carry out fractionation precipitation, and precipitation is dissolved in phosphoric acid buffer, then adopts dextrane gel sephadex G-75 purifying, and gained cassava leaf polyphenol oxidase refined solution makes cassava leaf polyphenol oxidase through vacuum lyophilization; It is characterized in that:
(1) every gram of cassava leaves preparation pH is the phosphoric acid buffer 4 milliliters of 6, adds the cellulase effect 45 minutes of cassava leaves total mass 0.8% at 45 DEG C;
(2) to smash to pasty state freezing lixiviate to pieces 5 hours with tissue mashing instrument at 5 DEG C, use gauze suction filtration, discard residue collection filtrate;
At 5 DEG C with 12000r/min frozen centrifugation 25 minutes, collect centrifugate and obtain cassava leaf polyphenol oxidase crude enzyme liquid;
(4) cassava leaf polyphenol oxidase crude enzyme liquid purifying first adopts ammonium sulfate to carry out fractionation precipitation, collects the precipitation of 40% to 80% saturation ratio, and precipitation is dissolved in the phosphoric acid buffer of 5 DEG C, makes cassava leaf polyphenol oxidase after purifying through vacuum lyophilization.
2. the preparation method of cassava leaf polyphenol oxidase as claimed in claim 1, it is characterized in that after cassava leaves adds phosphoric acid buffer and cellulase effect, the cross-linked polyvinylpyrrolidone of 0.02 gram is added by every gram of cassava leaves, abundant mixing, smash to pieces with the homogenate of high speed tissue mashing instrument, at 5 DEG C, lixiviate adopted filtered through gauze after 5 hours, discarded residue collection filtrate.
3. the preparation method of cassava leaf polyphenol oxidase as claimed in claim 1, is characterized in that, after cassava leaves adds phosphoric acid buffer and cellulase effect, add the cold acetone of cassava leaves quality 4 times, be pounded homogenate with high-speed tissue mashing machine, suction filtration, filtrate is stand-by; The freezing acetone of filter cake extracts suction filtration again, the white mass obtained, and vacuum-drying obtains cassava leaf polyphenol oxidase acetone powder; Be dissolved in by acetone powder in the phosphoric acid buffer of pH6.0 and extract 3h, add above-described stand-by filtrate, with the centrifugal 25min of 12000rmp/min at 5 DEG C, removing precipitation, gets its supernatant liquor and is cassava leaf polyphenol oxidase crude enzyme liquid.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103614356B (en) * | 2013-11-22 | 2015-12-02 | 广西大学 | A kind of method of rapid extraction separating plant leaf polyphenoloxidase |
| CN113951346B (en) * | 2021-11-08 | 2024-02-13 | 彭加林 | Tea beverage and preparation method thereof |
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Non-Patent Citations (5)
| Title |
|---|
| Plant Sciences》.2011,第9卷(第3期),第1169-1179页. * |
| POLYPHENOL OXIDASES FROM CASSAVA (MANIHOT ESCULENTA C.)ROOT: EXTRACTION, PURIFICATION AND CHARACTERIZATION;Véronique J. Barthet;《eScholarship_McGill》;19971231;摘要，第56-61页 * |
| Seu Jonathan GOGBEU等.Study of some characteristics of soluble polyphenol oxidases from six cultivars callus of cassava (Manihot esculenta Crantz)..《Journal of Animal & * |
| 吴炫柯.不同木薯品种生长发育及一些光合衰老生理特性的研究.《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》.2005,(第5期),D047-178. * |
| 甘薯叶多酚氧化酶的分离纯化及部分性质与功能基团研究;梁建荣;《中国优秀硕士学位论文全文数据库农业科技辑》;20110915(第9期);D047-53 * |
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