CN111269898B - Extraction and purification method of lentinus edodes polyphenol oxidase - Google Patents
Extraction and purification method of lentinus edodes polyphenol oxidase Download PDFInfo
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- 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
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- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03001—Catechol oxidase (1.10.3.1), i.e. tyrosinase
Abstract
The invention belongs to the technical field of enzyme separation and purification methods, and particularly relates to a method for extracting and purifying lentinan polyphenol oxidase. The invention provides a method for extracting and purifying polyphenol oxidase from shiitake mushrooms, which comprises the following steps: extracting crude enzyme liquid, carrying out ammonium sulfate fractional precipitation on the crude enzyme liquid, dialyzing enzyme extract, purifying the enzyme extract by ion exchange chromatography and purifying by gel chromatography, wherein 0.1-0.5 mol.L is used in the ion exchange chromatography ‑1 Carrying out gradient elution on the NaCl solution from low concentration to high concentration, wherein the whole process is carried out at 0-4 ℃; the polyphenol oxidase extracted and purified by the method has extremely high purity.
Description
Technical Field
The invention belongs to the technical field of enzyme separation and purification methods, and particularly relates to a method for extracting and purifying lentinan oxidase.
Background
Polyphenol oxidase, a nuclear-encoded metalloenzyme containing copper, is widely distributed in the natural world and roughly classified into three major classes, monophenols, bisphenols and urushiol. It is commonly found in insects, fungi, plants and other organisms, and in most cases, it is found in microorganisms, like laccase, monophenol oxidase. Bisphenol oxidase, also known as catechol oxidase, is mostly distributed in plants. The functions of polyphenol oxidase in organisms are many. Under the condition of oxygen, polyphenol oxidase can catalyze and oxidize phenolic substances to form quinone, and further browning phenomena are caused. Since the distribution of phenolics and polyphenol oxidase in normal tissue cells exhibits a certain regionality, most of the polyphenol oxidase activities are latent in plants.
Related researches find that the resistance of plants is closely related to phenolic substances. After the cucumber scab is inoculated to a plant body, the polyphenol oxidase activity in leaf tissues is found to be rapidly improved in a newly formed disease-resistant variety, and is obviously higher than that of a control variety and a variety infected with germs. When grape is in the severe stage of the anthracnose attack, the polyphenol oxidase activity in the intact leaf is obviously higher than that in the diseased leaf, and the polyphenol oxidase activity in the leaf with stronger disease resistance is also obviously increased. Polyphenol oxidase has not only the above-mentioned action function but also many additional functions such as demethylation to function as lignin degradation, and is closely linked to biosynthesis of β -anthocyanin.
As more uses of polyphenol oxidase have been developed, research on polyphenol oxidase attracts more and more attention of scholars, and polyphenol oxidase from different sources has a greatly different structure and a greatly different extraction and purification method.
The mushroom is an edible mushroom which is wide in source, low in price and easy to obtain, polyphenol oxidase is extracted from the mushroom, and the extraction cost of the polyphenol oxidase can be reduced to a certain extent.
Disclosure of Invention
The first purpose of the invention is to provide a method for extracting and purifying polyphenol oxidase from lentinus edodes, and in order to realize the purpose, the invention adopts the following technical scheme:
the extraction and purification method of the lentinus edodes polyphenol oxidase comprises the following steps:
(1) extraction of mushroom polyphenol oxidase crude enzyme liquid
Homogenizing fresh mushrooms and a phosphate mixed buffer solution added with PVP (polyvinyl pyrrolidone), leaching for 5-25 min, and centrifuging to obtain a supernatant, namely a mushroom polyphenol oxidase crude enzyme solution A, wherein the whole process is carried out at 0-4 ℃;
feed-to-liquid ratio (g.mL) of fresh shiitake mushrooms to phosphate mixed buffer solution added with PVP -1 )1:1 to 1:5, the pH of the phosphate mixed buffer solution with PVP is 6 to 8.5, and the addition amount (g/mL) of PVP in the phosphate mixed buffer solution with PVP -1 )10 to 18 percent;
(2) ammonium sulfate fractional precipitation of crude enzyme solution
Firstly, adding ammonium sulfate into a crude enzyme solution A, stirring until the ammonium sulfate is completely dissolved, so that the saturation degree of the ammonium sulfate in the crude enzyme solution A is 20-50%, standing and centrifuging, wherein the whole process is carried out at 0-4 ℃, and the centrifuged supernatant is a lentinus edodes polyphenol oxidase extracting solution B;
secondly, adding ammonium sulfate into the extracting solution B, stirring until the ammonium sulfate is completely dissolved, keeping the saturation degree of the ammonium sulfate in the extracting solution B at 60-90%, standing, centrifuging, removing supernate, collecting precipitated precipitate and oily floating substances, performing the whole process at 0-4 ℃, and dissolving the collected precipitate and oily floating substances by using a phosphate buffer solution with the pH of 6-8.5 to obtain a lentinus edodes polyphenol oxidase extracting solution C;
(3) dialysis treatment of enzyme extract
Adding the extracting solution C into a pretreated dialysis bag, sealing the dialysis bag, completely immersing the sealed dialysis bag into phosphate buffer solution with the pH value of 6-8.5, standing, and replacing the buffer solution in due time until BaCl is used 2 Detecting that the dialyzed external liquid has no precipitate by using the solution, transferring the liquid out of the dialysis bag to obtain a lentinus edodes polyphenol oxidase extracting solution D, and carrying out the whole process at 0-4 ℃;
(4) chromatographic purification of enzyme extract
Ion exchange chromatography: adding the enzyme extracting solution D into an ion exchange resin column balanced by phosphate buffer solution, standing, and respectively using 0.1-0.5 mol.L -1 Carrying out gradient elution on the NaCl solution from low concentration to high concentration, wherein the whole process is carried out at 0-4 ℃; collecting enzyme solution adjacent to the activity peak to obtain lentinus edodes polyphenol oxidase extracting solution E;
gel chromatography: and adding the extracting solution E into a gel chromatography column balanced by a phosphate buffer solution, eluting by using the buffer solution, carrying out the whole process at 0-4 ℃, and collecting enzyme solution near an active peak to obtain the lentinus edodes polyphenol oxidase extracting solution F.
Since the activity of lentinus edodes polyphenol oxidase (lentinus edodes PPO) is easily affected by temperature, the lentinus edodes PPO is easily inactivated as the temperature rises. The invention proves that the temperature is strictly controlled to be carried out at low temperature in the crude extraction process of the mushroom PPO, and the method can fully inhibit the catalytic reaction and the decomposition process of the mushroom PPO.
In order to protect the activity of polyphenol oxidase, PVP is added in the extraction process of the crude enzyme liquid of the lentinus edodes polyphenol oxidase, and the PVP is used as a phenol complexing agent and can strongly associate with phenolic substances, so that the activity of the enzyme is protected.
In the ammonium sulfate fractional precipitation of the crude enzyme solution, attention should be paid not to adding ammonium sulfate too quickly to avoid precipitation caused by insoluble ammonium sulfate, and the stirring rate should not be too fast to avoid bubbles in the crude enzyme extract.
The purpose of the first step of ammonium sulfate fractional precipitation is to remove most of the impure proteins in the crude enzyme solution A and retain the desired protein, namely the lentinus edodes PPO. The purpose of the second step of ammonium sulfate fractional precipitation is to precipitate the desired protein (lentinus edodes PPO) from the enzyme extract B from which most of the impure proteins have been removed, for separation.
The ion exchange resin column of the present invention is preferably DEAE cellulose DE-52, and other ion exchange resins of the same type may be tried by those skilled in the art.
The gel column of the present invention is preferably Sephadex G-100, but those skilled in the art may try other gel columns having similar screening effects.
During the chromatographic purification of the enzyme extract, the OD was measured by UV spectrophotometer as the active peak 280 And (4) determining.
In the extraction process of the crude enzyme liquid of the lentinus edodes polyphenol oxidase, the feed-liquid ratio (g.mL) of fresh lentinus edodes and phosphate mixed buffer solution added with PVP -1 ) Preferably 1:1 to 1:3, the pH of the phosphate mixed buffer solution with PVP is preferably 6.5 to 7.5, and the addition amount (g/mL) of PVP in the phosphate mixed buffer solution with PVP is preferably -1 ) Preferably 12-16%, and the leaching time is preferably 10-15 min.
Further optimization of the extraction process of the crude enzyme solution of lentinus edodes polyphenol oxidase, fresh lentinus edodes and phosphate mixed buffer added with PVPLiquid-to-liquid ratio (g.mL) -1 ) Preferably 1:2 to 1:3, the pH of the phosphate mixed buffer solution with PVP is preferably 7 to 7.5, and the addition amount (g/mL) of PVP in the phosphate mixed buffer solution with PVP -1 ) Preferably 12-14%, and the leaching time is preferably 10-15 min.
PVP is a surfactant, and when the lentinus edodes polyphenol oxidase is extracted, a proper amount of PVP is added to promote the extraction effect, so that the addition of the PVP increases the polyphenol oxidase activity of the crude enzyme liquid within a certain range; however, if the amount of PVP added is excessive, the polyphenol oxidase activity is inhibited.
The optimization of the crude enzyme liquid ammonium sulfate fractional precipitation process is carried out, the first step of the crude enzyme liquid ammonium sulfate fractional precipitation is carried out at 1-4 ℃, and the saturation degree of ammonium sulfate in the crude enzyme liquid A is 30-40%; the second step of the fractional precipitation of the crude enzyme liquid by ammonium sulfate is carried out at 1-4 ℃, and the saturation degree of the ammonium sulfate in the extracting solution B is 60-70%.
Preferably, the chromatographic purification of the enzyme extract is performed by ion exchange chromatography: adding the enzyme extracting solution D into an ion exchange resin column balanced by phosphate buffer solution, standing, and respectively using 0.1-0.5 mol.L -1 The NaCl solution is subjected to gradient elution from low concentration to high concentration, and the whole process is carried out at 1-4 ℃; collecting enzyme liquid adjacent to an active peak, namely lentinus edodes polyphenol oxidase extracting solution E; gel chromatography: and adding the extracting solution E into a gel chromatography column balanced by a phosphate buffer solution, eluting by using the buffer solution, carrying out the whole process at 1-4 ℃, and collecting enzyme solution near an active peak to obtain the lentinus edodes polyphenol oxidase extracting solution F.
The second purpose of the invention is to provide the lentinus edodes polyphenol oxidase obtained by the extraction and purification method of the lentinus edodes polyphenol oxidase.
The third purpose of the invention is to provide the application of the lentinus edodes polyphenol oxidase obtained by the extraction and purification method of the lentinus edodes polyphenol oxidase as a polyphenol oxidase standard product.
The invention has the beneficial effects that:
the invention provides a method for extracting and purifying polyphenol oxidase from shiitake mushrooms in a complete system, and provides a new method for extracting and purifying polyphenol oxidase (mushroom);
the polyphenol oxidase obtained by the extraction and purification method of the lentinus edodes polyphenol oxidase provided by the invention has extremely high purity, can be completely used as a polyphenol oxidase standard product, and has uniform protein bands on electrophoresis gel bands detected by SDS-PAGE electrophoresis test.
Drawings
FIG. 1 is a graph showing the effect of PVP addition on lentinan oxidase extraction;
FIG. 2 is a graph showing the effect of extraction time on the extraction of polyphenol oxidase from Lentinus edodes;
FIG. 3 is a graph showing the effect of pH of the leach solution on the extraction of lentinan oxidase;
FIG. 4 is the effect of feed liquid ratio on lentinan oxidase extraction;
FIG. 5 is DEAE cellulose DE-52 elution curve of lentinan oxidase of the present invention;
FIG. 6 is Sephadex G-100 elution curve of lentinus edodes polyphenol oxidase of the present invention;
FIG. 7 is a SDS-PAGE picture of the purified lentinan oxidase of the present invention; marker is standard protein, A1 and A2 are two parallel samples of enzyme extract F.
Detailed Description
Method for measuring activity of mushroom polyphenol oxidase (by extinction value method)
Setting the wavelength at 410nm, adding 2.0mL of acetic acid buffer solution with the pH value of 4.75 and 0.1 mol.L into the cuvette in sequence -1 Adding 0.5mL of enzyme extract into 1.0mL of catechol solution as substrate, quickly shaking and measuring, timing when adding the enzyme extract, recording light absorption value A410 every 30s, and continuously recording for 3 min. One polyphenol oxidase activity unit (U) is defined as: the amount of enzyme required to cause a change in absorbance of 0.01 per minute per ml of sample under the conditions of the assay was measured in U.min -1 ·g -1 And (4) expressing the fresh mushroom.
Method for calculating activity of polyphenol oxidase
One unit of enzyme activity (U) PPO (Δ a410 × V) as a change (increase) in OD per minute of 0.01 t )/(W×V s ×0.01×t)U·min -1 ·g -1
Δ A410: change in absorbance over reaction time;
w: fresh mass of shiitake mushrooms, g;
t: reaction time, min;
V t : the total volume of the crude enzyme extract is mL;
V s : the volume of enzyme solution, mL, was used for the assay.
Pretreatment of the dialysis bag:
cutting the dialysis bag into 10cm pieces, completely soaking in distilled water, and decocting in boiling water bath for 30 min. Then clamping the dialysis bag out by using forceps, and cooling the dialysis bag in distilled water at normal temperature for later use; the size of the dialysis bag is selected to be moderate, if the dialysis bag is too large, the enzyme extracting solution cannot fill the dialysis bag, and the dialysis efficiency is greatly reduced; in the process of treating the dialysis bag, attention is paid to the operation of wearing gloves in the whole process, and the dialysis bag cannot be directly contacted by hands, is not contacted with protein, and is not damaged.
Pretreatment of DEAE cellulose DE-52 particles:
soaking DE-52 granule in deionized water for 3 hr, and soaking with 0.5 mol.L -1 Soaking in HCl solution for 2 hr, washing with distilled water to neutrality, detecting pH with pH paper, and detecting with 0.5 mol.L -1 Soaking in NaOH solution for 2h, drying, and taking the granules for later use; cleaning and fixing a chromatographic column on a chromatographic frame, adding a proper amount of deionized water, then opening a lower liquid outlet and keeping water flow smooth, pouring pretreated and standby DE-52 particles into the column, slowly immersing the particles into the bottom of the column, filling about one third of the column volume, stopping filling the column, opening the lower liquid outlet again to allow distilled water to flow out, and then closing the liquid outlet.
Pretreatment of Sephadex G-100 of cross-linked glucan:
soaking the dry powder in 50% ethanol for 3 hr, stirring to swell, washing with distilled water to remove residual ethanol until neutral, and filtering to dry. Then swelling in distilled water for 24 hours, stirring intermittently to ensure complete swelling of the gel, then using 0.2mol · L -1 Soaking in hydrochloric acid for 12 hr, stirring intermittently, mixingThe gel was drained and rinsed with distilled water until neutral. And (3) carrying out ultrasonic treatment on the swollen gel particles for 10min to play a role in degassing, pouring the required particles into the column according to the column packing requirement, keeping the column packed in a wet state, and avoiding bubbles or faults in the column.
SDS-PAGE electrophoresis test method:
the sample loading amount of each hole is 5 mu L, the voltage of an electrophoresis apparatus is initially 80V, when the bromophenol blue marker line is electrophoresed to the junction of the concentrated gel and the separation gel and becomes a thin blue line, the voltage is adjusted to 120V until the bromophenol blue marker line reaches the front edge of the gel, and the electrophoresis is finished. Taking out the gel sheet, soaking in 45% ethanol 10% glacial acetic acid staining solution, and slowly shaking on a constant temperature oscillator for 1 h; then pouring out the staining solution, washing the film for 3 times by using distilled water, then placing the film in 10% glacial acetic acid-10% ethanol decolorizing solution, placing the film on a constant temperature oscillator for continuous oscillation for decolorizing until most of the film is colorless and transparent, and changing the decolorizing solution every 0.5h, wherein the decolorizing solution can be absorbed by sponge to accelerate the decolorizing speed.
Example 1
Extraction of mushroom polyphenol oxidase crude enzyme liquid
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 ) At a ratio of 1:4, phosphate buffer mixture (PBS: pH7, 12% (g.mL) was taken -1 ) PVP)200mL is put into a precooled homogenizer for homogenate, leached for 15min and transferred into a centrifugal tube for low-temperature centrifugation (10000 r.min) -1 ) And (3) 10min, wherein the obtained supernatant is the enzyme extracting solution A, and the whole process is carried out at the temperature of 0-4 ℃.
Example 2
The amounts of PVP added in example 1 were replaced with 8%, 10%, 14%, 16%, and 18%, respectively.
Example 3
The leaching time in example 1 was replaced by 5min, 10min, 20min, 25min and 30min, respectively.
Example 4
The pH of the phosphate mixed buffer in example 1 was replaced with 6, 6.5, 7.5, 8, 8.5, respectively.
Example 5
The feed-liquid ratios in example 1 were replaced by 1:1, 1:2, 1:3, 1:5, 1:6, respectively.
Example 6
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 ) At a ratio of 1:3, a phosphate buffer mixture (PBS: pH7.5, containing 14% (g. mL) -1 ) PVP)150mL, placing in a precooling homogenizer for homogenizing, leaching for 15min, transferring into a centrifugal tube for low-temperature centrifugation (8000r min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 7
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:2, phosphate buffer mixture (PBS: pH7, 12% (g.mL) -1 ) PVP)100mL is put into a precooled homogenizer for homogenate, leached for 10min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 8
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:4, taking a phosphate mixed buffer (PBS: pH7, containing 12% (g.mL) -1 ) PVP)200mL is put into a precooled homogenizer for homogenate, leached for 20min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 9
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:3, phosphate buffer mixture (PBS: pH7.5, containing 14% (g.mL) -1 ) PVP)150mL, placing in a precooling homogenizer for homogenizing, leaching for 15min, transferring into a centrifugal tube for low-temperature centrifugation (8000r min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 10
Taking fresh Lentinus Edodes 5 without physical damage, disease and pest damage0g, based on the ratio of material to liquid (g.mL) -1 )1:3, phosphate buffer mixture (PBS: pH7.5, containing 14% (g.mL) -1 ) PVP)150mL, placing in a precooled homogenizer for homogenizing, leaching for 5min, and transferring into a centrifugal tube for low-temperature centrifugation (8000r min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 11
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:4, phosphate buffer mixture (PBS: pH7, 16% (g.mL) was taken -1 ) PVP)200mL is put into a precooled homogenizer for homogenate, leached for 10min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 12
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:4, phosphate buffer mixture (PBS: pH8, 12% (g.mL) -1 ) PVP)200mL is put into a precooled homogenizer for homogenate, leached for 10min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 13
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:3, phosphate buffer mixture (PBS: pH7.5, containing 10% (g.mL) -1 ) PVP)150mL, placing in a precooling homogenizer for homogenizing, leaching for 15min, transferring into a centrifugal tube for low-temperature centrifugation (8000r min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 14
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:4, phosphate buffer mixture (PBS: pH8, 16% (g.mL) was taken -1 ) PVP)200mL is put into a precooled homogenizer for homogenate, leached for 10min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, and collecting supernatant as enzyme extractive solutionAnd A, the whole process is carried out at (4 +/-1) DEG C.
Example 15
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:2, phosphate buffer mixture (PBS: pH8, 16% (g.mL) was taken -1 ) PVP)100mL is put into a precooled homogenizer for homogenate, leached for 20min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 16
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:3, phosphate mixed buffer (PBS: pH7.5, containing 14% (g. mL) -1 ) PVP)150mL, placing in a precooling homogenizer for homogenizing, leaching for 25min, transferring into a centrifugal tube for low-temperature centrifugation (8000r min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 17
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:2, phosphate buffer mixture (PBS: pH7, 16% (g.mL) was taken -1 ) PVP)100mL is put into a precooled homogenizer for homogenate, leached for 20min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 18
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:2, phosphate buffer mixture (PBS: pH7, 12% (g.mL) -1 ) PVP)100mL into a precooled homogenizer for homogenization, leaching for 20min, and transferring into a centrifuge tube for low-temperature centrifugation (8000 r-min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 19
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:4, taking phosphate mixed buffer solution(PBS: pH8, containing 16% (g.mL) -1 ) PVP)200mL is put into a precooled homogenizer for homogenate, leached for 20min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 20
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:4, phosphate buffer mixture (PBS: pH8, 12% (g.mL) -1 ) PVP)200mL is put into a precooled homogenizer for homogenate, leached for 20min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 21
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:1, phosphate mixed buffer (PBS: pH7.5, containing 14% (g. mL) -1 ) PVP)50mL is put into a precooled homogenizer for homogenate, leached for 15min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 22
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:4, phosphate buffer mixture (PBS: pH7, 12% (g.mL) -1 ) PVP)200mL is put into a precooled homogenizer for homogenate, leached for 10min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 23
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:5, phosphate buffer mixture (PBS: pH7.5, containing 14% (g.mL) -1 ) PVP)250mL is put into a precooled refiner for homogenate, leached for 15min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 24
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:3, phosphate buffer mixture (PBS: pH7.5, containing 18% (g.mL) -1 ) PVP)150mL, placing in a precooling homogenizer for homogenizing, leaching for 15min, transferring into a centrifugal tube for low-temperature centrifugation (8000r min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 25
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:4, phosphate mixed buffer (PBS: pH7, 16% in g.mL) -1 ) PVP)200mL is put into a precooled homogenizer for homogenate, leached for 20min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 26
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:3, phosphate buffer mixture (PBS: pH8.5, containing 14% (g.mL) -1 ) PVP)150mL, placing in a precooling homogenizer for homogenizing, leaching for 15min, transferring into a centrifugal tube for low-temperature centrifugation (8000r min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 27
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:2, phosphate mixed buffer (PBS: pH8, 16% in g.mL) -1 ) PVP)100mL is put into a precooled homogenizer for homogenate, leached for 10min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 28
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:2, phosphate buffer mixture (PBS: pH8, 12% (g.mL) -1 ) PVP)100mL into the pre-cooled homogenateHomogenizing in machine, leaching for 10min, and centrifuging at low temperature (8000r min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 29
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:2, phosphate buffer mixture (PBS: pH8, 12% (g.mL) -1 ) PVP)100mL is put into a precooled homogenizer for homogenate, leached for 20min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 30
Taking 50g of fresh shiitake mushroom with no physical damage, no diseases and no insect pests on the fresh surface according to the material-liquid ratio (g.mL) -1 )1:2, phosphate buffer mixture (PBS: pH7, 16% (g.mL) was taken -1 ) PVP)100mL is put into a precooled homogenizer for homogenate, leached for 10min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
Example 31
Taking 50g of fresh shiitake mushroom with fresh surface without physical damage, diseases and insect pests according to the material-liquid ratio (g.mL) -1 )1:3, phosphate mixed buffer (PBS: pH6.5, containing 14% (g. mL) -1 ) PVP)150mL is put into a precooling homogenizer for homogenate, leached for 15min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
The activity of lentinus edodes polyphenol oxidase (lentinus edodes PPO) is easily influenced by temperature, the lentinus edodes PPO is easily inactivated along with the temperature rise, the activity of the lentinus edodes PPO is influenced when the temperature for extraction and purification is higher than 4 ℃, and the purified lentinus edodes PPO is extracted at a low temperature of 0-4 ℃ which is easy to control through a large number of experimental optimizations.
TABLE 1 scheme and results of crude enzyme extraction of lentinan polyphenol oxidase
Example 32
1) Extraction of mushroom polyphenol oxidase crude enzyme liquid
Taking 50g of fresh shiitake mushroom, and mixing according to the material-liquid ratio (g.mL) -1 )1:2, phosphate buffer mixture (PBS: pH7, 12% (g.mL) -1 ) PVP)100mL into a precooled homogenizer for homogenization, leaching for 15min, and transferring into a centrifuge tube for low-temperature centrifugation (8000 r-min) -1 )15min, the obtained supernatant is the enzyme extract A, and the whole process is carried out at 0 ℃.
2) Fractional precipitation of ammonium sulfate in crude enzyme liquid
Firstly, adding 8.2g of solid ammonium sulfate into 50mL of crude enzyme extracting solution A stirred by a digital display constant temperature magnetic stirrer at the temperature of (0 +/-1) DEG C, namely the saturation degree of the ammonium sulfate in the crude enzyme extracting solution A is 30%, and after the addition is finished within 20min, continuously stirring for 10 min. Standing the crude enzyme extractive solution for 10min, and keeping the temperature at (0 + -1) deg.C under 10000 r.min -1 Centrifuging for 20min, and collecting supernatant to obtain enzyme extractive solution B.
And secondly, adding solid ammonium sulfate into the collected enzyme extracting solution B under the condition of slowly stirring by a digital display constant temperature magnetic stirrer at the temperature of (0 +/-1) DEG C to ensure that the saturation degree of the ammonium sulfate in the enzyme extracting solution B is 60 percent, finishing the adding within 30min, and then continuously stirring for 10 min. Standing at (0 + -1) deg.C for 12 hr, and standing at (0 + -1) deg.C under 10000r min -1 Centrifuging for 30min, discarding supernatant, collecting precipitate and oily floating substance, and dissolving the precipitate and oily floating substance at saturation with phosphate buffer solution of pH7 to obtain enzyme extract C.
3) Dialysis treatment of enzyme extract
Adding the enzyme extract C into the pretreated dialysis bag, clamping with dialysis clamp, and completely immersing in 500mL of 0.05 mol.L -1 In phosphate buffer of (4), standing at 0 ℃ and replacing the buffer every 4 hours until BaCl is used 2 Detecting whether the dialyzed external solution has no precipitate by using the solution, and transferring the liquid from the dialysis bag to obtain enzyme extract D.
4) Chromatographic purification of enzyme extract
(ii) DEAE cellulose anion exchange chromatography
Equilibration, phosphate buffer at pH7 was poured into the pretreated column until the pH of the effluent from the lower port of the column was consistent with the pH of the buffer.
Adding enzyme extractive solution D into the balanced chromatographic column for several times, adding 5mL once, adding 25mL totally, closing the outlet at the lower end of the chromatographic column after adding enzyme extractive solution D each time, standing for 10min, opening the outlet at the lower end of the chromatographic column, collecting the effluent with 1.5mL small centrifuge tube at flow rate of 0.5 mL/min -1 One tube was taken 1.5mL, i.e., one tube was taken 3 min.
Respectively using prepared 0.1, 0.2, 0.3, 0.4 and 0.5 mol.L -1 The NaCl solution is subjected to gradient elution from low concentration to high concentration, 15mL of the NaCl solution is eluted at each concentration, and the flow rate is controlled to be 0.5 mL/min -1 One tube is used for 1.5mL, namely one tube is used for 3min, and the whole process is finished at 0 ℃.
Detecting the protein collection effect of each tube by using an ultraviolet spectrophotometer to determine OD 280 Several tubes of enzyme solutions adjacent to the active peak were collected as enzyme extract E.
② Sephadex G-100 chromatography of Sephadex
After the gel chromatography column is pretreated, phosphate buffer solution with pH7 is used for balancing until the pH of effluent liquid at the lower end of the chromatography column is consistent with the pH of the buffer solution. Then, the sample loading was started, and the collected enzyme extract E was added to the column in its entirety, and 0.5mL of the enzyme extract E was taken every minute, and one tube was 1.5mL, i.e., one tube was connected for three minutes.
Eluting with buffer solution at flow rate of 0.5 mL/min -1 The whole process is carried out at 0 ℃.
Detecting the OD of each tube of eluent by using an ultraviolet spectrophotometer 280 And collecting a plurality of tubes of enzyme liquid near the activity peak to obtain an enzyme extracting solution F, and measuring the PPO activity of the shiitake mushrooms.
Example 33
1) Extraction of mushroom polyphenol oxidase crude enzyme liquid
Taking 50g of fresh shiitake mushroom, and mixing according to the material-liquid ratio (g.mL) -1 )1:1, phosphate buffer mixture (PBS: pH6.5, containing 14% (g.mL) -1 ) PVP)100mL is put into a precooled refiner for homogenate, leached for 25min and transferred into a centrifugal tubeWarm centrifugation (8000r min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (1 + -1) deg.C.
2) Fractional precipitation of ammonium sulfate in crude enzyme liquid
Firstly, adding 11.3g of solid ammonium sulfate into 50mL of crude enzyme extracting solution A stirred by a digital display constant temperature magnetic stirrer at the temperature of (1 +/-1) DEG C, namely the saturation degree of the ammonium sulfate in the crude enzyme extracting solution is 40%, and after the addition is finished within 20min, continuously stirring for 10 min. Standing the crude enzyme extractive solution for 10min, and keeping the temperature at (1 + -1) deg.C under 10000 r.min -1 Centrifuging for 20min, and collecting supernatant to obtain enzyme extractive solution B.
And secondly, adding solid ammonium sulfate into the collected enzyme extracting solution B under the condition of slowly stirring by a digital display constant temperature magnetic stirrer at the temperature of (1 +/-1) DEG C to ensure that the saturation degree of the ammonium sulfate in the enzyme extracting solution B is 70 percent, finishing the adding within 30min, and then continuously stirring for 10 min. Standing at (1 + -1) deg.C for 12 hr, and standing at (1 + -1) deg.C below 10000r min -1 Centrifuging for 30min, discarding supernatant, collecting precipitate and oily floating substance, and dissolving precipitate and oily floating substance at saturation with phosphate buffer solution to obtain enzyme extract C.
3) Dialysis treatment of enzyme extract
Adding the enzyme extract C into the pretreated dialysis bag, clamping with dialysis clamp, and completely immersing in 500mL of 0.05 mol.L -1 The buffer solution was allowed to stand at (1. + -. 1) ℃ and the buffer solution was changed every 4 hours until BaCl was used 2 Detecting whether the dialyzed external solution has no precipitate by using the solution, and transferring the liquid from the dialysis bag to obtain enzyme extract D.
4) Chromatographic purification of enzyme extract
(ii) DEAE cellulose anion exchange chromatography
Equilibration, phosphate buffer at pH6.5 was poured into the pretreated column until the pH of the effluent from the lower port of the column was consistent with the pH of the buffer.
Adding enzyme extractive solution D into the balanced chromatographic column for several times, adding 5mL once, adding 25mL totally, closing the outlet at the lower end of the chromatographic column after adding enzyme extractive solution D each time, standing for 10min, opening the lower end of the chromatographic columnThe outlet of the reactor was used to receive the effluent from a 1.5mL small centrifuge tube, and the flow rate was controlled to 0.5 mL/min -1 One tube was taken 1.5mL, i.e., one tube was taken 3 min.
Gradient elution is carried out from low concentration to high concentration by prepared NaCl solution of 0.1, 0.2, 0.3, 0.4 and 0.5 mol.L-1 respectively, each concentration is eluted by 15mL, and the flow rate is controlled to be 0.5 mL.min -1 One tube is connected with 1.5mL, namely one tube is connected in 3min, and the whole process is finished at the temperature of (1 +/-1) ° C.
Detecting the protein collection effect of each tube by using an ultraviolet spectrophotometer to determine OD 280 Several tubes of enzyme solutions adjacent to the active peak were collected as enzyme extract E.
② Sephadex G-100 chromatography of Sephadex
After the gel chromatography column is pretreated, the gel chromatography column is balanced by phosphate buffer solution until the pH of effluent liquid at the lower end of the chromatography column is consistent with the pH of the buffer solution. Then, the sample loading was started, and the collected enzyme extract E was added to the column in its entirety, and 0.5mL of the enzyme extract E was taken every minute, and one tube was 1.5mL, i.e., one tube was connected for three minutes.
Eluting with buffer solution at flow rate of 0.5 mL/min -1 The whole process is carried out at (1 +/-1) DEG C. Detecting the OD of each tube of eluent by using an ultraviolet spectrophotometer 280 And collecting a plurality of tubes of enzyme liquid near the activity peak to obtain an enzyme extracting solution F, and measuring the PPO activity of the shiitake mushrooms.
Example 38
1) Extraction of mushroom polyphenol oxidase crude enzyme liquid
Taking 50g of fresh shiitake mushroom, and mixing according to the material-liquid ratio (g.mL) -1 )1:3, phosphate buffer mixture (PBS: pH7.5, containing 16% (g.mL) -1 ) PVP)100mL is put into a precooled homogenizer for homogenate, leached for 5min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (2 +/-1) deg.C.
2) Fractional precipitation of ammonium sulfate in crude enzyme liquid
Firstly, adding 14.55g of solid ammonium sulfate into 50mL of crude enzyme extracting solution A stirred by a digital display constant temperature magnetic stirrer at the temperature of (2 +/-1) DEG C, namely the saturation degree of the ammonium sulfate in the crude enzyme extracting solution A is 50%, and continuing stirring for 10min within 20 min. Subjecting the crude enzyme to a treatmentStanding the extractive solution for 10min, and keeping at (2 + -1) deg.C under 10000 r.min -1 Centrifuging for 20min, and collecting supernatant to obtain enzyme extractive solution B.
And secondly, adding solid ammonium sulfate into the collected enzyme extracting solution B under the condition of slowly stirring by a digital display constant temperature magnetic stirrer at the temperature of (2 +/-1) DEG C to ensure that the saturation degree of the ammonium sulfate in the enzyme extracting solution B is 80 percent, finishing the adding within 30min, and then continuously stirring for 10 min. Standing at (2 + -1) deg.C for 12 hr, and standing at (2 + -1) deg.C under 10000r min -1 Centrifuging for 30min, discarding supernatant, collecting precipitate and oily floating substance, and dissolving precipitate and oily floating substance at saturation with phosphate buffer solution of pH7.5 to obtain enzyme extract C.
3) Dialysis treatment of enzyme extract
Adding the enzyme extract C into the pretreated dialysis bag, clamping with dialysis clamp, and completely immersing in 500mL of 0.05 mol.L -1 The resulting mixture was allowed to stand at (2. + -. 1) ℃ and the buffer was replaced every 4 hours until BaCl was added 2 Detecting whether the dialyzed external solution has no precipitate by using the solution, and transferring the liquid from the dialysis bag to obtain enzyme extract D.
4) Chromatographic purification of enzyme extract
(ii) DEAE cellulose anion exchange chromatography
Equilibration, phosphate buffer at pH7.5 was poured into the pretreated column until the pH of the effluent from the lower port of the column was consistent with the pH of the buffer.
Adding enzyme extractive solution D into the balanced chromatographic column for several times, adding 5mL once, adding 25mL totally, closing the outlet at the lower end of the chromatographic column after adding enzyme extractive solution D each time, standing for 10min, opening the outlet at the lower end of the chromatographic column, collecting the effluent with 1.5mL small centrifuge tube at flow rate of 0.5 mL/min -1 One tube was taken 1.5mL, i.e., one tube was taken 3 min.
Respectively using prepared 0.1, 0.2, 0.3, 0.4 and 0.5 mol.L -1 The NaCl solution is subjected to gradient elution from low concentration to high concentration, 15mL of the NaCl solution is eluted at each concentration, and the flow rate is controlled to be 0.5 mL/min -1 One tube is connected with 1.5mL, namely one tube is connected in 3min, and the whole process is finished at the temperature of (2 +/-1) ° C.
Detecting the protein collection effect of each tube by using an ultraviolet spectrophotometer to determine OD 280 Several tubes of enzyme solution with adjacent active peaks are collected to obtain enzyme extract E.
② Sephadex G-100 chromatography of Sephadex
After the gel chromatography column is pretreated, the gel chromatography column is balanced by phosphate buffer solution until the pH of effluent liquid at the lower end of the chromatography column is consistent with the pH of the buffer solution. Then, the sample loading was started, and the collected enzyme extract E was added to the column in its entirety, and 0.5mL of the enzyme extract E was taken every minute, and one tube was 1.5mL, i.e., one tube was connected for three minutes.
Eluting with buffer solution at flow rate of 0.5 mL/min -1 The whole process is carried out at (2 +/-1) DEG C.
Detecting the OD of each tube of eluent by using an ultraviolet spectrophotometer 280 And collecting a plurality of tubes of enzyme liquid near the activity peak to obtain an enzyme extracting solution F, and measuring the PPO activity of the shiitake mushrooms.
Example 39
1) Extraction of mushroom polyphenol oxidase crude enzyme liquid
Taking 50g of fresh shiitake mushroom, and mixing according to the material-liquid ratio (g.mL) -1 )1:5, phosphate buffer mixture (PBS: pH8.5, containing 18% (g.mL) -1 ) PVP)250mL is put into a precooled refiner for homogenate, leached for 25min and transferred into a centrifugal tube for low-temperature centrifugation (10000 r.min) -1 )5min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
2) Fractional precipitation of ammonium sulfate in crude enzyme liquid
Firstly, 50mL of crude enzyme extract A stirred by a digital display constant temperature magnetic stirrer is added with solid ammonium sulfate under the environment of (4 +/-1) DEG C to ensure that the saturation degree of the ammonium sulfate in the crude enzyme extract is 20 percent, the addition is finished within 20min, and then the stirring is continued for 10 min. Standing the crude enzyme extractive solution for 10min, and keeping the temperature at (4 + -1) deg.C under 10000 r.min -1 Centrifuging for 20min, and collecting supernatant to obtain enzyme extractive solution B.
And secondly, adding solid ammonium sulfate into the collected enzyme extracting solution B under the condition of slowly stirring by a digital display constant temperature magnetic stirrer at the temperature of (4 +/-1) DEG C to ensure that the saturation degree of the ammonium sulfate in the enzyme extracting solution B is 90 percent, finishing the adding within 30min, and then continuously stirring for 10 min. Standing at (4 + -1) deg.C12h, (4 +/-1) DEG C, 10000r min -1 Centrifuging for 30min, discarding supernatant, collecting precipitate and oily floating substance, and dissolving the precipitate and oily floating substance at saturation with 25ml of phosphate buffer solution with pH8.5 to obtain enzyme extract C.
3) Dialysis treatment of enzyme extract
Adding the enzyme extract C into the pretreated dialysis bag, clamping with dialysis clamp, and completely immersing in 500mL of 0.05 mol.L -1 The resulting mixture was allowed to stand at (4. + -. 1) ° C, and the buffer was replaced every 4 hours until BaCl was used 2 Detecting whether the dialyzed external solution has no precipitate by using the solution, and transferring the liquid from the dialysis bag to obtain enzyme extract D.
4) Chromatographic purification of enzyme extract
DEAE cellulose anion exchange chromatography
Equilibration, phosphate buffer at pH8.5 was poured into the pretreated column until the pH of the effluent from the lower port of the column was consistent with the pH of the buffer.
Adding enzyme extractive solution C into the balanced chromatographic column for several times, adding 5mL once, adding 25mL totally, after adding enzyme extractive solution each time, closing the lower outlet of the chromatographic column, standing for 10min, opening the lower outlet of the chromatographic column, collecting effluent with 1.5mL small centrifuge tube at flow rate of 0.5 mL/min -1 One tube was taken 1.5mL, i.e., one tube was taken 3 min.
Respectively using prepared 0.1, 0.2, 0.3, 0.4 and 0.5 mol.L -1 The NaCl solution is subjected to gradient elution from low concentration to high concentration, 15mL of the NaCl solution is eluted at each concentration, and the flow rate is controlled to be 0.5 mL/min -1 One tube is connected with 1.5mL, namely one tube is connected in 3min, and the whole process is finished at the temperature of (4 +/-1) ° C.
Detecting the protein collection effect of each tube by using an ultraviolet spectrophotometer, measuring OD280, and collecting a plurality of tubes of enzyme liquid adjacent to an active peak to obtain enzyme extract E.
② Sephadex G-100 chromatography of Sephadex
After the gel chromatographic column is pretreated, the gel chromatographic column is balanced by a buffer solution until the pH of the effluent liquid at the lower end of the chromatographic column is consistent with that of the buffer solution. Then, the sample loading was started, and the collected enzyme extract E was added to the column in its entirety, and 0.5mL of the enzyme extract E was taken every minute, and one tube was 1.5mL, i.e., one tube was connected for three minutes.
Eluting with buffer solution at flow rate of 0.5 mL/min -1 The whole process is carried out at (4 +/-1) DEG C.
Detecting the OD of each tube of eluent by using an ultraviolet spectrophotometer 280 And collecting a plurality of tubes of enzyme liquid near the activity peak to obtain an enzyme extracting solution F, and measuring the PPO activity of the shiitake mushrooms.
Example 40
1) Extraction of mushroom polyphenol oxidase crude enzyme liquid
Taking 50g of fresh shiitake mushroom, and mixing according to the material-liquid ratio (g.mL) -1 )1:2, phosphate buffer mixture (PBS: pH7, 12% (g.mL) -1 ) PVP)100mL is put into a precooled homogenizer for homogenate, leached for 15min and transferred into a centrifugal tube for low-temperature centrifugation (8000 r.min) -1 )15min, the obtained supernatant is enzyme extract A, and the whole process is carried out at (4 + -1) deg.C.
2) Fractional precipitation of ammonium sulfate in crude enzyme liquid
Firstly, adding 8.2g of solid ammonium sulfate into 50mL of crude enzyme extracting solution A stirred by a digital display constant temperature magnetic stirrer at the temperature of (4 +/-1) DEG C, namely, the saturation of the ammonium sulfate in the crude enzyme extracting solution A is 30%, and then continuously stirring for 10min within 20 min. Standing the crude enzyme extractive solution for 10min, and keeping the temperature at (4 + -1) deg.C under 10000 r.min -1 Centrifuging for 20min, and collecting supernatant to obtain enzyme extractive solution B.
And secondly, adding solid ammonium sulfate into the collected enzyme extracting solution B under the condition of slowly stirring by a digital display constant-temperature magnetic stirrer at the temperature of (4 +/-1) DEG C to ensure that the saturation of the ammonium sulfate in the enzyme extracting solution B is 60 percent, finishing the addition within 30min, and then continuously stirring for 10 min. Standing at (4 + -1) deg.C for 12 hr, and standing at (4 + -1) deg.C under 10000r min -1 Centrifuging for 30min, discarding supernatant, collecting precipitate and oily floating substance, and dissolving the precipitate and oily floating substance at saturation with phosphate buffer solution of pH7 to obtain enzyme extract C.
3) Dialysis treatment of enzyme extract
Adding the enzyme extract C into the pretreated dialysis bag, and clamping with dialysis clamp to obtain a complete solutionImmersed in 500mL of 0.05 moL. L -1 The resulting mixture was allowed to stand at (4. + -. 1) ° C, and the buffer was replaced every 4 hours until BaCl was used 2 Detecting whether the dialyzed external solution has no precipitate by using the solution, and transferring the liquid from the dialysis bag to obtain enzyme extract D.
4) Chromatographic purification of enzyme extract
(ii) DEAE cellulose anion exchange chromatography
Equilibration, phosphate buffer at pH7 was poured into the pretreated column until the pH of the effluent from the lower port of the column was consistent with the pH of the buffer.
Adding enzyme extractive solution D into the balanced chromatographic column for several times, adding 5mL once, adding 25mL totally, closing the outlet at the lower end of the chromatographic column after adding enzyme extractive solution D each time, standing for 10min, opening the outlet at the lower end of the chromatographic column, collecting the effluent with 1.5mL small centrifuge tube at flow rate of 0.5 mL/min -1 One tube was taken 1.5mL, i.e., one tube was taken 3 min.
Respectively using prepared 0.1, 0.2, 0.3, 0.4 and 0.5 mol.L -1 The NaCl solution is subjected to gradient elution from low concentration to high concentration, 15mL of the NaCl solution is eluted at each concentration, and the flow rate is controlled to be 0.5 mL/min -1 One tube is connected with 1.5mL, namely one tube is connected in 3min, and the whole process is finished at the temperature of (4 +/-1) ° C.
Detecting the protein collection effect of each tube by using an ultraviolet spectrophotometer to determine OD 280 Several tubes of enzyme solution with adjacent active peaks are collected to obtain enzyme extract E.
② Sephadex G-100 chromatography of Sephadex
After the gel chromatography column is pretreated, phosphate buffer solution with pH7 is used for balancing until the pH of effluent liquid at the lower end of the chromatography column is consistent with the pH of the buffer solution. Then, the sample loading was started, and the collected enzyme extract E was added to the column in its entirety, and 0.5mL of the enzyme extract E was taken every minute, and one tube was 1.5mL, i.e., one tube was connected for three minutes.
Eluting with buffer solution at flow rate of 0.5 mL/min -1 The whole process is carried out at (4 +/-1) DEG C.
Detecting the OD of each tube of eluent by using an ultraviolet spectrophotometer 280 Collecting several tubes of enzyme solution near the active peak as enzyme extract F,and the PPO activity of the mushroom is measured.
Experiments show that most of the active polyphenol oxidase is eluted, and most of the foreign proteins are adsorbed by the anion column, so that the good effect is achieved, and the OD is the OD 280 Several peaks near the peak were collected after the lower test, and the collected peaks were obtained as crude enzyme extract E. From 0.1 mol. L -1 ～0.5mol·L -1 For a total of 5 concentrations, 10 tubes were collected for each concentration, and 1.5mL were collected for each tube, thus a total of 50 tubes of eluate were collected. As can be seen from FIG. 5, the DEAE cellulose DE-52 elution peak appeared in the 23 rd tube, i.e., 0.3 mol. L was used -1 When the NaCl eluate is eluted, the collection from the 22 nd tube is started, and the collection to the 29 th tube is ended.
The crude enzyme extract E was subjected to Sephadex G-100 gel column chromatography to measure the enzymatic activity of polyphenol oxidase, as shown in FIG. 6, and the protein peak appeared in the first 3 collected tubes, so that the first 3 collected tubes were the enzyme extract F.
The enzyme activity U and the specific activity U/mg of the enzyme extract obtained by the extraction and purification steps in 32 were calculated, and the results are shown in table 2, which indicates that the specific activity of the enzyme extract after chromatography purification is significantly improved.
The enzyme extracts F obtained in examples 32 to 40 were examined by SDS-PAGE electrophoresis, and a uniform protein band appeared as shown in FIG. 7, indicating that the purified lentinus edodes polyphenol oxidase of the present invention has an extremely high purity and can be used as a polyphenol oxidase standard.
TABLE 2 calculation of enzyme Activity and specific Activity
Claims (3)
1. The extraction and purification method of the lentinus edodes polyphenol oxidase is characterized by comprising the following steps of:
(1) extraction of mushroom polyphenol oxidase crude enzyme liquid
a) Homogenizing fresh mushrooms and a phosphate mixed buffer solution added with PVP, leaching for 15min, and centrifuging to obtain a supernatant, namely a mushroom polyphenol oxidase crude enzyme solution A, wherein the whole process is carried out at 0-4 ℃;
feed-liquid ratio g.mL of fresh mushroom and phosphate mixed buffer solution added with PVP -1 The ratio is 1:3, the pH value of the phosphate mixed buffer solution added with PVP is 7.5, and the addition amount of PVP in the phosphate mixed buffer solution added with PVP is 10%;
or b), taking fresh mushrooms and a phosphate mixed buffer solution added with PVP for homogenizing, leaching for 20min, centrifuging to obtain a supernatant, namely a mushroom polyphenol oxidase crude enzyme solution A, and carrying out the whole process at 0-4 ℃;
feed-liquid ratio g.mL of fresh mushroom and phosphate mixed buffer solution added with PVP -1 The ratio of the PVP to the phosphate mixed buffer solution is 1:4, the pH value of the phosphate mixed buffer solution added with PVP is 8, and the addition amount of the PVP in the phosphate mixed buffer solution added with PVP is 16%;
(2) fractional precipitation of ammonium sulfate in crude enzyme liquid
Firstly, adding ammonium sulfate into a crude enzyme solution A, stirring until the ammonium sulfate is completely dissolved, so that the saturation degree of the ammonium sulfate in the crude enzyme solution A is 20-50%, standing and centrifuging, wherein the whole process is carried out at 0-4 ℃, and the centrifuged supernatant is a lentinus edodes polyphenol oxidase extracting solution B;
secondly, adding ammonium sulfate into the extracting solution B, stirring until the ammonium sulfate is completely dissolved, keeping the saturation degree of the ammonium sulfate in the extracting solution B at 60-90%, standing, centrifuging, removing supernate, collecting precipitated precipitate and oily floating substances, performing the whole process at 0-4 ℃, and dissolving the collected precipitate and oily floating substances by using a phosphate buffer solution with the pH of 6-8.5 to obtain a lentinus edodes polyphenol oxidase extracting solution C;
(3) dialysis treatment of enzyme extract
Adding the extracting solution C into a pretreated dialysis bag, sealing the dialysis bag, completely immersing the sealed dialysis bag into phosphate buffer solution with the pH value of 6-8.5, standing, and replacing the buffer solution in due time until BaCl is used 2 Detecting whether the dialyzed external solution has precipitate by using the solution, transferring the liquid out of the dialysis bag to obtain a lentinus edodes polyphenol oxidase extracting solution D, and carrying out the whole process at 0-4 ℃;
(4) chromatographic purification of enzyme extract
Ion exchange chromatography: adding the enzyme extracting solution D into an ion exchange resin column balanced by phosphate buffer solution, standing, and respectively using 0.1-0.5 mol.L -1 Carrying out gradient elution on the NaCl solution from low concentration to high concentration, wherein the whole process is carried out at 0-4 ℃; collecting enzyme solution adjacent to the activity peak to obtain lentinus edodes polyphenol oxidase extracting solution E;
gel chromatography: adding the extracting solution E into a gel chromatography column balanced by a phosphate buffer solution, eluting by using the buffer solution, carrying out the whole process at 0-4 ℃, and collecting enzyme solution near an active peak to obtain a lentinus edodes polyphenol oxidase extracting solution F;
the ion exchange resin column is DEAE cellulose DE-52, and the gel column is Sephadex G-100;
activity peaks OD determination by UV Spectrophotometer 280 And (4) determining.
2. The extraction and purification method of lentinan polyphenol oxidase as claimed in claim 1, which is characterized in that: the first step of the fractional precipitation of ammonium sulfate in the crude enzyme solution is carried out at 1-4 ℃, and the saturation degree of the ammonium sulfate in the crude enzyme solution A is 30-40%; the second step of the fractional precipitation of the crude enzyme liquid by ammonium sulfate is carried out at 1-4 ℃, and the saturation degree of the ammonium sulfate in the extracting solution B is 60-70%.
3. The extraction and purification method of lentinan polyphenol oxidase as claimed in claim 1, which is characterized in that: ion exchange chromatography: adding the enzyme extracting solution D into an ion exchange resin column balanced by phosphate buffer solution, standing, and respectively using 0.1-0.5 mol.L -1 The NaCl solution is subjected to gradient elution from low concentration to high concentration, and the whole process is carried out at 1-4 ℃; collecting enzyme liquid adjacent to an active peak, namely lentinus edodes polyphenol oxidase extracting solution E; gel chromatography: and adding the extracting solution E into a gel chromatography column balanced by a phosphate buffer solution, eluting by using the buffer solution, carrying out the whole process at 1-4 ℃, and collecting enzyme solution near an active peak to obtain the lentinus edodes polyphenol oxidase extracting solution F.
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| CN104498448A (en) * | 2014-12-31 | 2015-04-08 | 湖南农业大学 | Tea polyphenol oxidase isozyme monomers PP01 and PP02 and preparation method thereof |
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