CN103614356B - A kind of method of rapid extraction separating plant leaf polyphenoloxidase - Google Patents
A kind of method of rapid extraction separating plant leaf polyphenoloxidase Download PDFInfo
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- 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)
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Abstract
The present invention relates to a kind of method of rapid extraction separating plant leaf polyphenoloxidase, belong to bioseparation field.A method for rapid extraction separating plant leaf polyphenoloxidase, get leaf clean, add damping fluid, smash to pieces, lixiviate suction filtration, collect filtrate, collected by centrifugation crude enzyme liquid; Reverse micelle extraction is utilized after crude enzyme liquid ultrafiltration; Extraction terminates rear concentrated, lyophilize and obtains polyphenoloxidase powder.The present invention utilizes reverse micelle extraction to extract polyphenoloxidase crude enzyme liquid, and the polyphenoloxidase purity obtained is higher, and 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.
Description
Technical field
The present invention relates to a kind of method of rapid extraction separating plant leaf polyphenoloxidase, belong to bioseparation field.
Background technology
Polyphenoloxidase (polyphenoloxidase, 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 can be detected on the plant residue of soil rot.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, thus show activity.In fruits and vegetables cell tissue, the position that polyphenoloxidase exists is variant because of the difference of the kind of raw material, kind and ripening degree, 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 sephadexG-75, and equipment requirements is higher, and purification time is longer, and impurity not easily removes, and enzyme loss alive is larger.
Summary of the invention
The object of this invention is to provide a kind of method of 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, collected by centrifugation crude enzyme liquid;
2) reverse micelle extraction is utilized after crude enzyme liquid ultrafiltration;
3) extraction terminates 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 DEG C, extraction time 6h.
The film diameter of the ultra-filtration centrifuge tube that the ultrafiltration preferably: step 2) 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 aqueous phase volume ratio are 1: 1, and front extraction temperature is 28 ~ 32 DEG C, optimum temperuture is 30 DEG C, and front extraction time is 15min; Rear aqueous phase extracted is the NaCl solution of 0.5mol/L, and NaCl solvent is the citrate buffer solution of 0.1mol/L, and rear aqueous phase extracted and organic phase volume ratio are 1: 1, and rear extraction temperature is 28 ~ 32 DEG C, and optimum temperuture is 30 DEG C, 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 be: get 0.1mol/L citric acid solution 82ml and 0.1mol/L sodium citrate solution 118ml mix after obtained.
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 obtained 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 ultrasonic assistant in reverse micelle extraction polyphenol oxidase enzymatic process, improve stripping rate, effect is better.
Embodiment
Below in conjunction with specific embodiment, elaboration detailed is further done to the present invention, but embodiments of the present invention are not limited to the scope that embodiment represents.These embodiments only for illustration of the present invention, 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 amendment 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 for subsequent use, take 100g mulberry leaf, add pH be 6.0 the every 100ml phosphoric acid buffer of 0.1mol/L phosphoric acid buffer 400mL(contain SODIUM PHOSPHATE, MONOBASIC 1.368g, Sodium phosphate dibasic 0.44g), smash to pieces to pasty state with tissue mashing instrument, carry out suction filtration with 4 layers of gauze after being placed in 4 DEG C of refrigerator leach at low temperature 6h, discard residue collection filtrate.With 12000r/min frozen centrifugation 30min under 4 DEG C of conditions, collect centrifugate and namely obtain polyphenoloxidase crude enzyme liquid.
Enzyme liquid is concentrated with 6000r/min centrifugal polyphenoloxidase crude enzyme liquid 20min with the ultra-filtration centrifuge tube of film diameter 30ku, enzyme liquid CTAB-isooctane Reversed Micelles system after concentrated extracts polyphenoloxidase, using 50mmol/LCTAB-isooctane Reversed Micelles solution as organic phase, polyphenoloxidase crude enzyme liquid is as aqueous phase, organic phase and aqueous phase are respectively 20ml, and with 200r/min concussion mixing 15min on shaking table, temperature controls at 28 DEG C, by mixed solution with the centrifugal 10min of 4000r/min, obtain phase-splitting.
The organic phase and each 20ml of strip aqueous that get load enzyme are placed in 100ml triangular flask, and first use ultrasonication 30min, ultrasonic power is 120W, and on shaking table, mix 30min with 200r/min concussion afterwards, it is 28 DEG C that temperature controls.Strip aqueous is 0.5mol/LNaCl solution, and solvent is the citrate buffer solution (every 200ml citrate buffer solution is obtained after being mixed by 0.1mol/L citric acid solution 82ml and 0.1mol/L sodium citrate solution 118ml) of 0.1mol/LpH6.0.By mixed solution with the centrifugal 10min of 4000r/min, obtain aqueous phase.Then utilize polyethylene glycol 6000 to embed concentrated, obtain polyphenoloxidase refined solution, make lyophilized powder through vacuum lyophilization, obtain polyphenoloxidase powder, unit enzyme is lived as 180U/mg after measured.
Embodiment 2
Fresh tea leaf is cleaned for subsequent use, take 100g leaf of tea tree, add pH be 6.0 the every 100ml phosphoric acid buffer of 0.1mol/L phosphoric acid buffer 400mL(contain SODIUM PHOSPHATE, MONOBASIC 1.368g, Sodium phosphate dibasic 0.44g), smash to pieces to pasty state with tissue mashing instrument, be placed in 4 DEG C of refrigerator freezing lixiviates and carry out suction filtration with 4 layers of gauze after 6 hours, discard residue collection filtrate.With 12000r/min frozen centrifugation 30 minutes under 4 DEG C of conditions, collect centrifugate and namely obtain polyphenoloxidase crude enzyme liquid.
Organic solvent-acetone is utilized to carry out protein precipitation to crude enzyme liquid, the acetone of-20 DEG C of precoolings is added to crude enzyme liquid, crude enzyme liquid and acetone volume ratio are 1: 3, be placed in 4 DEG C of refrigerators and leave standstill 3h, the phosphoric acid buffer dissolution precipitation of the centrifugal rear 50mlpH6.0 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 collected is extracted again, using 75mmol/LCTAB-isooctane Reversed Micelles solution as organic phase, polyphenoloxidase crude enzyme liquid is as aqueous phase, organic phase and aqueous phase are respectively 20ml, with 200r/min concussion mixing 15min on shaking table, temperature controls at 30 DEG C, by mixed solution with the centrifugal 10min of 4000r/min, obtain phase-splitting.
The organic phase and each 20ml of strip aqueous that get load enzyme are placed in 100ml triangular flask, and first use ultrasonication 30min, ultrasonic power is 120W, and on shaking table, mix 30min with 200r/min concussion afterwards, it is 30 DEG C that temperature controls.Strip aqueous is 0.5mol/LNaCl solution, and solvent is the citrate buffer solution (every 200ml citrate buffer solution is obtained after being mixed by 0.1mol/L citric acid solution 82ml and 0.1mol/L sodium citrate solution 118ml) of 0.1mol/L.Afterwards by mixed solution with the centrifugal 10min of 4000r/min, obtain aqueous phase.Then utilize polyethylene glycol 6000 to embed concentrated, obtain polyphenoloxidase refined solution, make lyophilized powder through vacuum lyophilization, obtain polyphenoloxidase powder.Unit enzyme is lived as 260U/mg after measured.
Embodiment 3
Fresh tea leaf is cleaned for subsequent use, take 100g leaf of tea tree, add pH be 6.0 the every 100ml phosphoric acid buffer of 0.1mol/L phosphoric acid buffer 400mL(contain SODIUM PHOSPHATE, MONOBASIC 1.368g, Sodium phosphate dibasic 0.44g), smash to pieces to pasty state with tissue mashing instrument, be placed in 4 DEG C of refrigerator freezing lixiviates and carry out suction filtration with 4 layers of gauze after 6 hours, discard residue collection filtrate.With 12000r/min frozen centrifugation 30 minutes under 4 DEG C of conditions, collect centrifugate and namely obtain polyphenoloxidase crude enzyme liquid.
Organic solvent-acetone is utilized to carry out protein precipitation to crude enzyme liquid, the acetone of-20 DEG C of precoolings is added to crude enzyme liquid, crude enzyme liquid and acetone volume ratio are 1: 3, be placed in 4 DEG C of refrigerators and leave standstill 3h, the phosphoric acid buffer dissolution precipitation of the centrifugal rear 50mlpH6.0 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 collected is extracted again, using 100mmol/LCTAB-isooctane Reversed Micelles solution as organic phase, polyphenoloxidase crude enzyme liquid is as aqueous phase, organic phase and aqueous phase are respectively 20ml, with 200r/min concussion mixing 15min on shaking table, temperature controls at 32 DEG C, by mixed solution with the centrifugal 10min of 4000r/min, obtain phase-splitting.
The organic phase and each 20ml of strip aqueous that get load enzyme are placed in 100ml triangular flask, and first use ultrasonication 30min, ultrasonic power is 120W, and on shaking table, mix 30min with 200r/min concussion afterwards, it is 32 DEG C that temperature controls.Strip aqueous is 0.5mol/LNaCl solution, and solvent is the citrate buffer solution (every 200ml citrate buffer solution is obtained after being mixed by 0.1mol/L citric acid solution 82ml and 0.1mol/L sodium citrate solution 118ml) of 0.1mol/L.Afterwards by mixed solution with the centrifugal 10min of 4000r/min, obtain aqueous phase.Then utilize polyethylene glycol 6000 to embed concentrated, obtain polyphenoloxidase refined solution, make lyophilized powder through vacuum lyophilization, obtain polyphenoloxidase powder.Unit enzyme is lived as 240U/mg after measured.
Claims (4)
1. rapid extraction is separated a method for mulberry leaf or leaf of tea tree polyphenoloxidase, comprises the following steps:
1) get leaf, add damping fluid, smash to pieces, lixiviate suction filtration, collect filtrate, collected by centrifugation crude enzyme liquid; 2) reverse micelle extraction is utilized after crude enzyme liquid ultrafiltration;
3) extraction terminates rear concentrated, lyophilize and obtains polyphenoloxidase powder;
Step 1) described in every 100g leaf need add 400ml damping fluid, described damping fluid is 0.1mol/L phosphoric acid buffer, and PH is 6;
Step 1) described in extraction temperature be 4 DEG C, extraction time 6h;
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, CTAB add-on is 50 ~ 100mmol/L, organic phase and aqueous phase volume ratio are 1:1, front extraction temperature is 28 ~ 32 DEG C, and front extraction time is 15min; Rear aqueous phase extracted is the NaCl solution of 0.5mol/L, and solvent is the citrate buffer solution of 0.1mol/L, and rear aqueous phase extracted and organic phase volume ratio are 1:1, and rear extraction temperature is 28 ~ 32 DEG C, and rear extraction time is 30min.
2. rapid extraction according to claim 1 is separated the method for mulberry leaf or leaf of tea tree polyphenoloxidase, it is characterized in that: step 2) described in CTAB add-on be 75mmol/L, described front extraction temperature is 30 DEG C, and described rear extraction temperature is 30 DEG C.
3. rapid extraction according to claim 1 is separated the method for mulberry leaf or leaf of tea tree polyphenoloxidase, it is characterized in that: step 2) described in the film diameter of ultra-filtration centrifuge tube that adopts of ultrafiltration be 30ku.
4. rapid extraction according to claim 1 is separated the method for mulberry leaf or leaf of tea tree polyphenoloxidase, it is characterized in that: step 2) rear extraction stages adopt ultrasonic-assisted extraction, ultrasonic power is 120W, and the time is 30min.
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| 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)
| 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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Patent Citations (3)
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| Convenient partial purification of polyphenol oxidase from apple skin by cationic reverse micellar extraction;Jee-Young Imm et al.;《Food Chemistry》;20091231;第113卷;摘要，第303页左边第1-5段 * |
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