CN103614356A - Method for quickly extracting and separating polyphenol oxidase from plant leaves - Google Patents

Method for quickly extracting and separating polyphenol oxidase from plant leaves Download PDF

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Publication number
CN103614356A
CN103614356A CN201310593709.2A CN201310593709A CN103614356A CN 103614356 A CN103614356 A CN 103614356A CN 201310593709 A CN201310593709 A CN 201310593709A CN 103614356 A CN103614356 A CN 103614356A
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extraction
polyphenoloxidase
crude enzyme
separating plant
polyphenol oxidase
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CN103614356B (en
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杨洋
严东
赵芬芬
叶琴
马万良
胡振兴
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Guangxi University
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Guangxi University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)

Abstract

The invention relates to a method for quickly extracting and separating polyphenol oxidase from plant leaves, and belongs to the field of bioseparation. The method for quickly extracting and separating the polyphenol oxidase from the plant leaves comprises the following steps: cleaning plant leaves; adding a buffer solution, mashing, leaching and carrying out suction filtering; collecting a filtrate, centrifugally collecting crude enzyme; extracting by using reverse micelles after ultra-filtration of the crude enzyme; and concentrating, freezing and drying after extraction is ended, so as to obtain polyphenol oxidase powder. The polyphenol oxidase crude enzyme is extracted by using the reverse micelles, and the obtained polyphenol oxidase is high in purity, fewer in impurities, and high in enzyme activity, and the processing method is simple in technology, large in handling capacity and applicable to industrial production.

Description

A kind of method of rapid extraction separating plant leaf polyphenoloxidase
Technical field
A kind of method that the present invention relates to rapid extraction separating plant leaf polyphenoloxidase, belongs to bioseparation field.
Background technology
Polyphenoloxidase (polyphenol oxidase, PPO) be the wide a kind of metalloprotease of occurring in nature distributed pole, be prevalent in the plastid of plant, fungi, insect, the activity of polyphenoloxidase even on the plant residue of soil rot, can be detected.In plant (as apple, lichee, spinach, potato, beans, tealeaves, mulberry leaf, tobacco etc.) tissue, polyphenoloxidase combines with interior utricule film, native state non-activity, but polyphenoloxidase after tissue homogenate or damage is activated, thereby the activity of showing.In fruits and vegetables cell tissue, the position that polyphenoloxidase exists is variant because of the difference of kind, kind and the ripening degree of raw material, and in greenery, polyphenol oxidase activity major part is present in chloroplast(id); Subcellular fraction nearly all in potato tuber all contains polyphenoloxidase, and content is approximately identical with protein portion.It is complicated to the purifying process of polyphenoloxidase that polyphenoloxidase can pass through the purification process such as ammonium sulfate precipitation, dextrane gel sephadex G-75, and equipment requirements is higher, and the purifying time is longer, and impurity is difficult for removing, and enzyme loss alive is larger.
Summary of the invention
A kind of method that the object of this invention is to provide rapid extraction separating plant leaf polyphenoloxidase.Object of the present invention is achieved through the following technical solutions:
A method for rapid extraction separating plant leaf polyphenoloxidase, comprises the following steps:
1) get leaf, add damping fluid, smash to pieces, lixiviate suction filtration, collect filtrate, centrifugal collection crude enzyme liquid;
2) after crude enzyme liquid ultrafiltration, utilize reverse micelle extraction;
3) extraction finishes rear concentrated, lyophilize and obtains polyphenoloxidase powder.
Preferably: the every 100g leaf described in step 1) need add 400ml damping fluid, and described damping fluid is 0.1mol/L phosphoric acid buffer, and PH is 6.
Preferably: the extraction temperature described in step 1) is 4 ℃, extraction time 6h.
The film diameter of the ultra-filtration centrifuge tube that preferably: step 2) described ultrafiltration adopts is 30ku.
Reverse micelle extraction preferably: step 2) comprises front extraction and rear extraction, front aqueous phase extracted is the crude enzyme liquid after ultrafiltration, organic phase is CTAB-isooctane solution, CTAB add-on is 50～100mmol/L, the suitableeest add-on is 75mmol/L, and organic phase and water volume ratio are 1: 1, and front extraction temperature is 28～32 ℃, optimum temperuture is 30 ℃, and front extraction time is 15min; Rear aqueous phase extracted is the NaCl solution of 0.5mol/L, the citrate buffer solution that NaCl solvent is 0.1mol/L, and rear aqueous phase extracted and organic phase volume ratio are 1: 1, and rear extraction temperature is 28～32 ℃, and optimum temperuture is 30 ℃, and rear extraction time is 30min.
Rear extraction stages preferably: step 2) adopts ultrasonic-assisted extraction, and ultrasonic power is 120W, and the time is 30min.
In step 1), every 100ml phosphoric acid buffer contains SODIUM PHOSPHATE, MONOBASIC 1.368g, Sodium phosphate dibasic 0.44g.
Step 2) in, the citrate buffer solution compound method of every 200ml is: get after 0.1mol/L citric acid solution 82ml and 0.1mol/L sodium citrate solution 118ml mix and make.
Beneficial effect of the present invention:
(1) utilize reverse micelle extraction to extract polyphenoloxidase crude enzyme liquid, Reverse Micelles is not high to equipment requirements, and the treatment time, the short polyphenoloxidase purity obtaining was higher, impurity is less, and enzyme is lived higher, and this treatment process technique is simple, can treatment capacity large, be applicable to suitability for industrialized production.
(2) the rear extraction stages in reverse micelle extraction polyphenol oxidase enzymatic process is auxiliary by ultrasonic wave, has improved stripping rate, and effect is better.
Embodiment
Below in conjunction with specific embodiment, the present invention is done to further detailed elaboration, but embodiments of the present invention are not limited to the scope that embodiment represents.These embodiment are only for the present invention is described, but not for limiting the scope of the invention.In addition, after reading content of the present invention, those skilled in the art can do various modifications to the present invention, and these equivalent variations fall within appended claims limited range of the present invention equally.
Embodiment 1
New fresh mulberry leaf is cleaned standby, take 100g mulberry leaf, adding pH is that 6.0 the every 100ml phosphoric acid buffer of 0.1mol/L phosphoric acid buffer 400mL(contains SODIUM PHOSPHATE, MONOBASIC 1.368g, Sodium phosphate dibasic 0.44g), with tissue mashing instrument, smash to pieces to pasty state, after being placed in 4 ℃ of refrigerator leach at low temperature 6h, with 4 layers of gauze, carry out suction filtration, discard filter residue and collect filtrate.Under 4 ℃ of conditions, with 12000r/min frozen centrifugation 30min, collect centrifugate and obtain polyphenoloxidase crude enzyme liquid.
With the ultra-filtration centrifuge tube of film diameter 30ku, with the centrifugal polyphenoloxidase crude enzyme liquid of 6000r/min 20min, concentrate enzyme liquid, enzyme liquid after concentrated extracts polyphenoloxidase by CTAB-isooctane Reversed Micelles system, using 50mmol/LCTAB-isooctane Reversed Micelles solution as organic phase, polyphenoloxidase crude enzyme liquid is as water, organic phase and water are respectively 20ml, on shaking table, with 200r/min concussion, mix 15min, and temperature is controlled at 28 ℃, mixed solution, with the centrifugal 10min of 4000r/min, is obtained to phase-splitting.
Organic phase and each 20ml of strip aqueous of getting load enzyme are placed in 100ml triangular flask, first use ultrasonication 30min, and ultrasonic power is 120W, mix 30min afterwards on shaking table with 200r/min concussion, and it is 28 ℃ that temperature is controlled.Strip aqueous is 0.5mol/L NaCl solution, the citrate buffer solution that solvent is 0.1mol/LpH6.0 (every 200ml citrate buffer solution makes after being mixed by 0.1mol/L citric acid solution 82ml and 0.1mol/L sodium citrate solution 118ml).Mixed solution, with the centrifugal 10min of 4000r/min, is obtained to water.Then utilize polyethylene glycol 6000 embedding concentrated, obtain polyphenoloxidase refined solution, through vacuum lyophilization, make lyophilized powder, obtain polyphenoloxidase powder, unit enzyme is lived as 180U/mg after measured.
Embodiment 2
Fresh tea leaf is cleaned standby, take 100g leaf of tea tree, adding pH is that 6.0 the every 100ml phosphoric acid buffer of 0.1mol/L phosphoric acid buffer 400mL(contains SODIUM PHOSPHATE, MONOBASIC 1.368g, Sodium phosphate dibasic 0.44g), with tissue mashing instrument, smash to pieces to pasty state, be placed in 4 ℃ of refrigerator freezing lixiviates and with 4 layers of gauze, carry out suction filtration after 6 hours, discard filter residue and collect filtrate.Under 4 ℃ of conditions, with 12000r/min frozen centrifugation 30 minutes, collect centrifugate and obtain polyphenoloxidase crude enzyme liquid.
Utilize organic solvent-acetone to carry out protein precipitation to crude enzyme liquid, the acetone that adds-20 ℃ of precoolings to crude enzyme liquid, crude enzyme liquid and acetone volume ratio are 1: 3, be placed in 4 ℃ of standing 3h of refrigerator, the centrifugal rear phosphoric acid buffer dissolution precipitation with 50ml pH6.0 of 6000r/min, with the ultra-filtration centrifuge tube of film diameter 30ku with the centrifugal 20min of 6000r/min, by CTAB-isooctane Reversed Micelles system, the polyphenoloxidase enzyme liquid of collecting is extracted again, using 75mmol/LCTAB-isooctane Reversed Micelles solution as organic phase, polyphenoloxidase crude enzyme liquid is as water, organic phase and water are respectively 20ml, on shaking table, with 200r/min concussion, mix 15min, temperature is controlled at 30 ℃, by mixed solution with the centrifugal 10min of 4000r/min, obtain phase-splitting.
Organic phase and each 20ml of strip aqueous of getting load enzyme are placed in 100ml triangular flask, first use ultrasonication 30min, and ultrasonic power is 120W, mix 30min afterwards on shaking table with 200r/min concussion, and it is 30 ℃ that temperature is controlled.Strip aqueous is 0.5mol/L NaCl solution, the citrate buffer solution that solvent is 0.1mol/L (every 200ml citrate buffer solution makes after being mixed by 0.1mol/L citric acid solution 82ml and 0.1mol/L sodium citrate solution 118ml).Afterwards by mixed solution with the centrifugal 10min of 4000r/min, obtain water.Then utilize polyethylene glycol 6000 embedding concentrated, obtain polyphenoloxidase refined solution, through vacuum lyophilization, make lyophilized powder, obtain polyphenoloxidase powder.Unit enzyme is lived as 260U/mg after measured.
Embodiment 3
Fresh tea leaf is cleaned standby, take 100g leaf of tea tree, adding pH is that 6.0 the every 100ml phosphoric acid buffer of 0.1mol/L phosphoric acid buffer 400mL(contains SODIUM PHOSPHATE, MONOBASIC 1.368g, Sodium phosphate dibasic 0.44g), with tissue mashing instrument, smash to pieces to pasty state, be placed in 4 ℃ of refrigerator freezing lixiviates and with 4 layers of gauze, carry out suction filtration after 6 hours, discard filter residue and collect filtrate.Under 4 ℃ of conditions, with 12000r/min frozen centrifugation 30 minutes, collect centrifugate and obtain polyphenoloxidase crude enzyme liquid.
Utilize organic solvent-acetone to carry out protein precipitation to crude enzyme liquid, the acetone that adds-20 ℃ of precoolings to crude enzyme liquid, crude enzyme liquid and acetone volume ratio are 1: 3, be placed in 4 ℃ of standing 3h of refrigerator, the centrifugal rear phosphoric acid buffer dissolution precipitation with 50ml pH6.0 of 6000r/min, with the ultra-filtration centrifuge tube of film diameter 30ku with the centrifugal 20min of 6000r/min, by CTAB-isooctane Reversed Micelles system, the polyphenoloxidase enzyme liquid of collecting is extracted again, using 100mmol/L CTAB-isooctane Reversed Micelles solution as organic phase, polyphenoloxidase crude enzyme liquid is as water, organic phase and water are respectively 20ml, on shaking table, with 200r/min concussion, mix 15min, temperature is controlled at 32 ℃, by mixed solution with the centrifugal 10min of 4000r/min, obtain phase-splitting.
Organic phase and each 20ml of strip aqueous of getting load enzyme are placed in 100ml triangular flask, first use ultrasonication 30min, and ultrasonic power is 120W, mix 30min afterwards on shaking table with 200r/min concussion, and it is 32 ℃ that temperature is controlled.Strip aqueous is 0.5mol/L NaCl solution, the citrate buffer solution that solvent is 0.1mol/L (every 200ml citrate buffer solution makes after being mixed by 0.1mol/L citric acid solution 82ml and 0.1mol/L sodium citrate solution 118ml).Afterwards by mixed solution with the centrifugal 10min of 4000r/min, obtain water.Then utilize polyethylene glycol 6000 embedding concentrated, obtain polyphenoloxidase refined solution, through vacuum lyophilization, make lyophilized powder, obtain polyphenoloxidase powder.Unit enzyme is lived as 240U/mg after measured.

Claims (6)

1. a method for rapid extraction separating plant leaf polyphenoloxidase, comprises the following steps:
1) get leaf, add damping fluid, smash to pieces, lixiviate suction filtration, collect filtrate, centrifugal collection crude enzyme liquid;
2) after crude enzyme liquid ultrafiltration, utilize reverse micelle extraction;
3) extraction finishes rear concentrated, lyophilize and obtains polyphenoloxidase powder.
2. the method for rapid extraction separating plant leaf polyphenoloxidase according to claim 1, is characterized in that: the every 100g leaf described in step 1) need add 400ml damping fluid, and described damping fluid is 0.1mol/L phosphoric acid buffer, and PH is 6.
3. the method for rapid extraction separating plant leaf polyphenoloxidase according to claim 1, is characterized in that: the extraction temperature described in step 1) is 4 ℃, extraction time 6h.
4. the method for rapid extraction separating plant leaf polyphenoloxidase according to claim 1, is characterized in that: step 2) the film diameter of the ultra-filtration centrifuge tube that adopts of described ultrafiltration is 30ku.
5. the method for rapid extraction separating plant leaf polyphenoloxidase according to claim 1, it is characterized in that: step 2) in reverse micelle extraction comprise front extraction and rear extraction, front aqueous phase extracted is the crude enzyme liquid after ultrafiltration, organic phase is CTAB-isooctane solution, and CTAB add-on is 50～100mmol/L, and the suitableeest add-on is 75mmol/L, organic phase and water volume ratio are 1: 1, front extraction temperature is 28～32 ℃, and optimum temperuture is 30 ℃, and front extraction time is 15min; Rear aqueous phase extracted is the NaCl solution of 0.5mol/L, the citrate buffer solution that NaCl solvent is 0.1mol/L, and rear aqueous phase extracted and organic phase volume ratio are 1: 1, and rear extraction temperature is 28～32 ℃, and optimum temperuture is 30 ℃, and rear extraction time is 30min.
6. the method for rapid extraction separating plant leaf polyphenoloxidase according to claim 5, is characterized in that: step 2) rear extraction stages adopt ultrasonic-assisted extraction, ultrasonic power is 120W, the time is 30min.
CN201310593709.2A 2013-11-22 2013-11-22 A kind of method of rapid extraction separating plant leaf polyphenoloxidase Active CN103614356B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108503683A (en) * 2018-04-03 2018-09-07 重庆汇达柠檬科技集团有限公司 A kind of method that HPMC precipitations assist limonin in reverse micelle extraction lemon seed
CN109198075A (en) * 2018-11-22 2019-01-15 成都农业科技职业学院 A kind of technique preparing black tea using polyphenol oxidase in potato

Citations (3)

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Publication number Priority date Publication date Assignee Title
CN101760453A (en) * 2008-12-11 2010-06-30 湖州来色生物基因工程有限公司 Apple polyphenol oxidase extracting and measuring method
CN103013937A (en) * 2012-11-27 2013-04-03 南昌大学 Method for extracting polyphenol oxidase from lotus seedpod
CN103305483A (en) * 2013-07-01 2013-09-18 广西大学 Preparation method of cassava leaf polyphenol oxidase

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101760453A (en) * 2008-12-11 2010-06-30 湖州来色生物基因工程有限公司 Apple polyphenol oxidase extracting and measuring method
CN103013937A (en) * 2012-11-27 2013-04-03 南昌大学 Method for extracting polyphenol oxidase from lotus seedpod
CN103305483A (en) * 2013-07-01 2013-09-18 广西大学 Preparation method of cassava leaf polyphenol oxidase

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108503683A (en) * 2018-04-03 2018-09-07 重庆汇达柠檬科技集团有限公司 A kind of method that HPMC precipitations assist limonin in reverse micelle extraction lemon seed
CN108503683B (en) * 2018-04-03 2020-11-10 重庆汇达生物科技股份有限公司 Method for extracting limonin from lemon seeds by HPMC precipitation-assisted reverse micelle
CN109198075A (en) * 2018-11-22 2019-01-15 成都农业科技职业学院 A kind of technique preparing black tea using polyphenol oxidase in potato

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