CN102268414A - Coprinus comatus laccase with activity of inhibiting tumor cell proliferation and preparation method thereof - Google Patents
Coprinus comatus laccase with activity of inhibiting tumor cell proliferation and preparation method thereof Download PDFInfo
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Abstract
The invention discloses coprinus comatus laccase with activity of inhibiting tumor cell proliferation and a preparation method thereof. The coprinus comatus laccase provided in the invention is prepared by the following steps: 1) breaking up cells; 2) collecting a crude extract of proteins; 3) separating and purifying laccase from the crude extract of proteins collected in the step 2), wherein the obtained laccase is an extract of coprinus comatus, the molecular weight of the laccase is 64KD, and N terminal sequence of the laccase is described by sequence 1 in a sequence table. The coprinus comatus extract obtained through the preparation method in the invention has an obvious inhabiting effect on cancer cell proliferation and HIV reverse transcriptase activity and has an important value in the field of research on novel anti-cancer and anti-AIDS medicines.
Description
Technical field
The present invention relates to a kind of Shaggy Mane laccase and preparation method thereof, belong to biological technical field with anti-tumour cell proliferative activity.
Background technology
Shaggy Mane (Coprinus comatus) claims Coprinus comatus again, and its classification position is under the jurisdiction of Basidiomycota, Hymenomycetes, Agaricales, terrible umbrella section, Coprinus.Shaggy Mane be a kind of quality crisp and slide, the edible mushrooms of tasty mouthfeel, in China and European countries edible tradition is arranged all.Shaggy Mane has good characteristics such as high protein, lower fat, with the effect of food and medicine consangunity.According to one's analysis, contain crude protein 25.4g, fatty 3.3g, total reducing sugar 58.8g, fiber 7.3g, ash content 12.5g in every 100g dry product, contain 20 seed amino acids, wherein 8 seed amino acids of needed by human all possess, and Shaggy Mane also contains trace elements such as elements such as potassium, sodium, calcium, magnesium, phosphorus and iron, copper, manganese, zinc, molybdenum, cobalt and multivitamin, flavour substances, alkaloid etc.Shaggy Mane has been decided to be and has met that Food and Argriculture OrganizationFAO (FAO) and The World Health Organization (WHO) require, and collects natural, nutrition, three kinds of functions of health care are one of 16 kinds of rare edible mushroomss of one.It is reported, Shaggy Mane have beneficial stomach, clear refreshing, control Chinese medicine curative effect such as hemorrhoid; Analyze from medicinal function, Shaggy Mane has effects such as lowering blood glucose, nematicide, blood fat reducing, adjusting immunizing power, but it is also very insufficient for the research of its biological action both at home and abroad, never see the report that this bacterium suppresses the HIV reverse transcriptase activity, and this mycoprotein matter has the research of anti-tumor activity.
Because the toxic side effect of chemical anticarcinogenic drug is bigger, the anti-tumor activity of edible medicinal fungus has been subjected to paying close attention to widely, this class active metabolite is mainly based on macromolecular cpd, what the research report was more at present is edible fungi polysaccharide class material, their mainly immunologic function, activated macrophage, promotion T cell proliferation by regulating the host, induce the effect that produces effect performance anti-tumor in vivo such as immune factor, and less to the direct lethal effect of tumour cell.People such as Li Shipeng, Su Lei in 2000 find that Shaggy Mane Crude polysaccharides CcP can obviously suppress the activity of Kunming mouse S-180 solid tumor with the dosage abdominal injection of 12.5mg/kg, and prolong the survival time of ascitic tumor mouse.But the research for the external direct killing cancer cell of Shaggy Mane bioactive enzyme does not appear in the newspapers.
Acquired immune deficiency syndrome (AIDS) is the viral infectious of serious threat human health.Hiv virus HIV belongs to the lentiviridae of Retroviridae.Reverse transcription is one of HIV virus most important characteristic, and process is exactly to be the process of the synthetic viral DNA of template with the viral RNA under the effect of ThermoScript II in virosome.In transcriptive process,reversed, ThermoScript II plays an important role, and this also is one of best target spot of research anti-AIDS drug, and most of medicine of developing at present all is that reverse transcription with HIV is a target spot.Mitsuya etc. find that at first deoxythymidine class AZT has the effect of significant inhibition hiv virus, research subsequently finds successively that again deoxynucleoside, acyclic nucleotide, nucleoside analog can effectively suppress duplicating of HIV, but the resistance problem of these inhibitor becomes increasingly conspicuous, and has serious toxic side effect.
Summary of the invention
The purpose of this invention is to provide a kind of Shaggy Mane laccase and preparation method thereof with anti-tumour cell proliferative activity.
Shaggy Mane laccase provided by the present invention prepares according to the method that may further comprise the steps: 1) smudge cells; 2) collect protein crude extract administration; 3) from step 2) separation and purification laccase in the protein crude extract administration collected.The molecular weight of described laccase is that 64KD and N-terminal sequence are seen sequence table sequence 1.
Described step 2) in, adopt the centrifugal mode to collect protein crude extract administration, employed centrifugal force is 6010-15151g, as 12000g.
From step 2) the separation and purification laccase comprises following a)～d) four steps the protein crude extract administration collected:
A) will be from step 2) protein crude extract administration collected carries out anion exchange chromatography, collects the elution peak with laccase activity; The anion exchange groups that is adopted in the described anion exchange chromatography is DEAE, and the elution program that is adopted comprises following three step wash-outs:
The first step wash-out phosphate buffer solution wash-out A of pH6.8-7.5; Second goes on foot the following eluant solution of wash-out with pH6.8-7.5: solute is 0.1M NaCl, and solvent is described phosphate buffer solution A; The 3rd goes on foot the following eluant solution of wash-out with pH6.8-7.5: solute is 0.2M NaCl, and solvent is described phosphate buffer solution A; The solute of described phosphate buffer solution A is 3.5-4.5mM NaH
2PO
4With 5.5-6.5mM Na
2HPO
4, solvent is a water;
B) will carry out cation exchange column chromatography from the elution peak that step a) obtains, collect elution peak with laccase activity; The cation exchange group that is adopted in the described cation exchange column chromatography is CM, and the elution program that is adopted comprises following three step wash-outs:
The first step wash-out phosphate buffer solution B wash-out of pH6.1-6.8; Second goes on foot the following eluant solution of wash-out with pH6.1-6.8: solute is 0.1M NaCl, and solvent is described phosphate buffer solution B; The 3rd goes on foot the following eluant solution of wash-out with pH6.1-6.8: solute is 0.15M NaCl, and solvent is described phosphate buffer solution B; The solute of described phosphate buffer solution B is 5.75-6.75mM NaH
2PO
4With 3.2-4.2mM Na
2HPO
4, solvent is a water;
C) will carry out anion exchange chromatography from the elution peak that step b) obtains, collect elution peak with laccase activity; The anion exchange groups that is adopted in the described anion exchange chromatography is Q; The elution program that is adopted is for carrying out the NaCl linear elution, and the pH value of used NaCl linear elution liquid is 3.5-4.5 in the described NaCl linear elution, and solute is NaCl, and solvent is a hac buffer; The time of described linear elution is 200 minutes, and the concentration of NaCl rose to 0.15M by the 0M linearity in the described linear elution in 200 minutes; The solute of described hac buffer is 7.7-8.7mM acetic acid and 1.3-2.3mM sodium-acetate, and solvent is a water;
D) will carry out gel permeation chromatography from the elution peak that step c) obtains, obtain having the elution peak of laccase activity; The separating ranges of the chromatography media that described gel permeation chromatography is used comprises 64KD, and the column length of used chromatography column and column internal diameter ratio are 25: 1～50: 1.
The carrier of described step a) anion exchange chromatography is Cellulose, the carrier of described step b) cation exchange column chromatography is Cellulose, the carrier of described step c) anion exchange chromatography is Sepharose, and the medium of described step d) gel permeation chromatography is Superdex 75.
The first step wash-out described phosphate buffer solution A wash-out of pH7.0 in the described step a); Second goes on foot the following eluant solution of wash-out with pH7.0: solute is 0.1M NaCl, and solvent is described phosphate buffer solution A; The 3rd goes on foot the following eluant solution of wash-out with pH7.0: solute is 0.2M NaCl, and solvent is described phosphate buffer solution A; The solute of described phosphate buffer solution A is 3.9mM NaH
2PO
4With 6.1mM Na
2HPO
4, solvent is a water;
The first step wash-out described phosphate buffer solution B wash-out of pH6.6 in the described step b); Second goes on foot the following eluant solution of wash-out with pH6.6: solute is 0.1M NaCl, and solvent is described phosphate buffer solution B; The 3rd goes on foot the following eluant solution of wash-out with pH6.6: solute is 0.15M NaCl, and solvent is described phosphate buffer solution B; The solute of described phosphate buffer solution B is 6.25mM NaH
2PO
4With 3.7mM Na
2HPO
4, solvent is a water;
Described in the described step c) in the NaCl linear elution pH value of used NaCl linear elution liquid be 4.0, the solute of described hac buffer is 8.2mM acetic acid and 1.8mM sodium-acetate, solvent is a water;
The elution peak that obtains having laccase activity described in the described step d) obtains with following eluant solution: the pH value is the NH of 8.5 0.2M
4HCO
3The aqueous solution.
The column length of the chromatography column that described step d) is used and column internal diameter ratio are 30: 1.
Another object of the present invention provides a kind of Shaggy Mane extract, and the molecular weight of this extract is that 64KD and N-terminal sequence are seen sequence table sequence 1.
The application of described extract in the following arbitrary product of preparation also is protection scope of the present invention:
A) suppress the product that cancer cell is bred; B) improve the product of cancer patients's healthy state; C) product of inhibition HIV-1 reverse transcriptase activity; D) improve the product of AIDS patient's healthy state.
According to the Shaggy Mane laccase that preparation method of the present invention obtained cancer cell propagation and HIV reverse transcriptase activity all there is the obvious suppression effect, be a kind of novel antivirus inhibitor, in cancer drug and anti-AIDS new drug research field, have important value.
Description of drawings
Fig. 1 is a DEAE-Cellulose weak anionic exchange column chromatography wash-out collection of illustrative plates.
Fig. 2 is a CM-Cellulose weak cation exchange column chromatography wash-out collection of illustrative plates.
Fig. 3 is a Q-Sepharose reinforcing yin essence ion-exchange chromatography wash-out collection of illustrative plates.
Fig. 4 is FPLC-Superdex 75 gel permeation chromatography wash-out collection of illustrative plates.
Fig. 5 is that Shaggy Mane laccase SDS-PAGE electrophoresis is identified collection of illustrative plates.Standard molecular weight (available from GE Healthcare company) is followed successively by from top to bottom: phosphorylase b (94KD), bovine serum albumin (67KD), ovalbumin (43KD), carbonic anhydrase (30KD), soybean insulin inhibitor (20KD), opalescin (14.4KD).
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The separation and purification and the evaluation of embodiment 1, Shaggy Mane (Coprinus comatus) laccase
Starting material of the present invention are by from Shaggy Mane (Coprinus comatus) (Chinese common micro-organisms preservation administrative center, numbering CGMCC 5.615) separates in the sporophore, utilize PD substratum (potato 200g, glucose 20g, add water to 1000ml), fermentation obtains mycelium under the following conditions: the Shaggy Mane bacterial classification is inoculated into comprehensive PDA (potato 200g, glucose 20g, agar 20g, add water to 1000ml) activate on the flat board, 25 ℃ of constant temperature culture, treat after mycelia is covered with flat board it to be inoculated in the PD substratum, 25 ℃ of 160rpm shake-flask culture are after 7 days, and the liquid shaking bottle of transferring is again cultivated after 7 days and collected mycelium, is stored in-20 ℃ of refrigerators stand-by.
1, extraction separates with thick level
NaCl solution suspension Shaggy Mane mycelium with 0.15M utilizes tissue mashing machine to carry out historrhexis to mycelium and is pasty state, and in 4 ℃ place 12 hours after, the centrifugal 15min of 8000rpm (12000g) collects supernatant solution, obtains the mycelia crude extract; In the mycelia crude extract, add (NH
4)
2SO
4To 80% saturation ratio, under 4 ℃ of conditions, left standstill 4 hours, the centrifugal 15min of 8000rpm (12000g), collecting precipitation obtains protein crude extract administration, and dissolved in distilled water protein precipitation crude extract was also dialysed 12-16 hour in distilled water.
2, ion-exchange chromatography separation and purification
Behind the corresponding buffered soln of chromatography column utilization 8-10 column volume of flow velocity balance with 4ml/min, whether balance is good with definite chromatography column to utilize pH meter to measure the pH value of chromatography column effluent liquid, albumen crude samples after the dialysis is at the uniform velocity gone up sample with the flow velocity of 1.5ml/min, after treating to finish on the sample, flow velocity is modulated 2.5ml/min (the Q-Sepharose chromatography is 1ml/min) carry out wash-out, each elution peak utilizes the laccase activity detection method to determine whether to be active peak.
Select for use interpolation 0,0.1M, 0.2M and 1M NaCl to carry out stepwise elution during DEAE-Cellulose chromatography column wash-out; Select for use interpolation 0,0.1M, 0.15M and 1M NaCl to carry out stepwise elution during CM-Cellulose chromatography column wash-out; Select for use interpolation 0-0.15M NaCl to carry out linear elution during Q-Sepharose chromatography column wash-out.
(1) DEAE-Cellulose weak anionic exchange column chromatography
(solute is 3.9mM NaH to the phosphate buffer solution of protein crude extract administration process pH7.0 after the dialysis
2PO
4With 6.1mM Na
2HPO
4, solvent is a water) and after the balance, through DEAE-Cellulose (Sigma company, D0909) weak anionic exchange column chromatography.The column volume of this ion exchange column is 100ml.Carry out four step wash-outs, flow velocity 2.5ml/min.(solute is 3.9mM NaH to the first step wash-out with the phosphate buffer solution of pH7.0
2PO
4With 6.1mM Na
2HPO
4, solvent is a water) and 2.58 column volumes of wash-out; Second goes on foot wash-out 3.29 column volumes of following eluant solution with pH7.0: solute is 0.1M NaCl, and solvent is that (solute is 3.9mM NaH to phosphate buffer solution
2PO
4With 6.1mM Na
2HPO
4, solvent is a water); The 3rd goes on foot wash-out 2.13 column volumes of following eluant solution with pH7.0: solute is 0.2MNaCl, and solvent is that (solute is 3.9mM NaH to phosphate buffer solution
2PO
4With 6.1mM Na
2HPO
4, solvent is a water); The 4th goes on foot wash-out 2.87 column volumes of following eluant solution with pH7.0: solute is 1.0M NaCl, and solvent is that (solute is 3.9mM NaH to phosphate buffer solution
2PO
4With 6.1mM Na
2HPO
4, solvent is a water).
The elution peak that collection obtains carries out laccase activity and detects, the result shows that (elution volume is 588-721ml to the 3rd step the 3rd elution peak D3 that obtain of wash-out among Fig. 1, i.e. 5.88-7.21 column volume) be the active peak of target extract (its laccase activity is 95.1U/mg), dialysed in distilled water 10-12 hour in the active peak of D3.
(2) CM-Cellulose weak cation exchange column chromatography
(solute is 6.25mM NaH to the phosphate buffer solution of D3 component process pH6.6
2PO
4With 3.7mM Na
2HPO
4, solvent is a water) and after the balance, through CM-Cellulose (Sigma company, C0805) weak cation exchange column chromatography.The column volume of this ion exchange column is 50ml.Carry out four step wash-outs, flow velocity 2.5ml/min.(solute is 6.25mM NaH to the first step wash-out with the phosphate buffer solution of pH6.6
2PO
4With 3.7mM Na
2HPO
4, solvent is a water) and 7.14 column volumes of wash-out; Second goes on foot wash-out 4.2 column volumes of following eluant solution with pH6.6: solute is 0.1M NaCl, and solvent is that (solute is 6.25mM NaH to phosphate buffer solution
2PO
4With 3.7mM Na
2HPO
4, solvent is a water); The 3rd goes on foot wash-out 2.38 column volumes of following eluant solution with pH6.6: solute is 0.15M NaCl, and solvent is that (solute is 6.25mM NaH to phosphate buffer solution
2PO
4With 3.7mM Na
2HPO
4, solvent is a water); The 4th goes on foot wash-out 1.68 column volumes of following eluant solution with pH6.6: solute is 1.0M NaCl, and solvent is that (solute is 6.25mM NaH to phosphate buffer solution
2PO
4With 3.7mM Na
2HPO
4, solvent is a water).
The elution peak that collection obtains carries out laccase activity and detects, the result shows that (elution volume is 568-630ml to the 3rd step the 3rd elution peak D3C3 that obtain of wash-out among Fig. 2, i.e. 11.34-12.6 column volume) be the active peak of target extract (its laccase activity is 646.8U/mg), dialysed in distilled water 10-12 hour in the active peak of D3C3.
(3) Q-Sepharose reinforcing yin essence ion-exchange chromatography
The D3C3 component is through after hac buffer (solute is 8.2mM acetic acid and 1.8mM sodium-acetate, and solvent the is a water) balance of pH4.0, through Q-Sepharose (GE Healthcare company, 17-0510-10) reinforcing yin essence ion-exchange chromatography.The column volume of this ion exchange column is 10ml.Carry out 200 minutes NaCl linear elution, flow velocity 1ml/min.This NaCl linear elution pH value of solution value is 4.0, and solute is NaCl, and solvent is that (solute is 8.2mM acetic acid and 1.8mM sodium-acetate to hac buffer, and solvent is a water, and the concentration of NaCl rose to 0.15M by the 0M linearity in 200 minutes.
The elution peak that collection obtains carries out laccase activity and detects, the result shows that (elution volume is 121-200ml to the second elution peak D3C3Q2 that obtains among Fig. 3, i.e. 13-20 column volume) be the active peak of target extract (laccase activity at this activity peak is 721.1U/mg), dialysed in distilled water 10-12 hour in the active peak of D3C3Q2.
3, gel permeation chromatography
(GE Healthcare company, 17-5174-01) gel permeation chromatography, elutriant are pH 8.50.2M NH to the D3C3Q2 component through FPLC-Superdex 75
4HCO
3Solution, chromatography column specification 30cm (column length) * 1cm (internal diameter), last sample volume is 0.2ml, flow velocity is 0.8ml/min.The elution peak that collection obtains carries out laccase activity and detects, the result shows that the first elution peak D3C3Q2SU1 (elution volume is 8.8-11.2ml among Fig. 4, i.e. 0.37-0.47 column volume) that obtains is the active peak of target extract (laccase activity at this activity peak is 790.9U/mg).
Above-mentioned laccase activity all detects as follows:
Collect elution peak, utilize BCA protein quantification test kit (the rich Deco skill company that steps in Beijing, PP0103) protein content of mensuration elution peak utilizes following laccase activity measuring method to determine the activity of elution peak, by calculating the enzyme activity unit number that exists in every milligram of albumen.
Laccase activity measuring method: with 2,2-connection nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) is a substrate, it is configured to the acidic solution (the pH value is 2.8) of 0.34mg/ml, the above-mentioned substrate that adds 190 μ l in the 10 μ l samples, 37 ℃ of reactions are after 5 minutes, measure light absorption value at wavelength 400nm place, replace enzyme liquid to react in contrast with distilled water.Enzyme activity unit is defined as: every milliliter of reaction system of per minute produces 1 needed enzyme amount of light absorption value in the time of 37 ℃ at the 400nm place.
4, ultrafiltration and concentration
D3C3Q2SU1 dialysed in distilled water 10-12 hour at active peak; 4 ℃ carry out ultrafiltration and concentration that molecular weight cut-off is 3KD after, down to freezing fully, it is standby that freezing sample is lyophilized into powder in-80 ℃ of conditions; this dry powder is the pure product of laccase, also is the Shaggy Mane extract that the present invention protects.The laccase activity of the pure product of this laccase is 790.9U/mg (the laccase activity measuring method is the same).The pure product of this laccase are carried out the SDS-PAGE electrophoresis identify, and attached gel filtration chromatography elution curve determines that the size of this extract is the 64KD (see figure 5).
5, determined amino acid sequence
Adopt automatization EDMAN edman degradation Edman that the N-terminal aminoacid sequence of the pure product of laccase is measured, be furnished with the protein sequence determinator of HPLC system with Hewlett Packard 1000A and measure the N-terminal sequence, record sequence and see sequence table sequence 1.
The Function detection of embodiment 2, Shaggy Mane (Coprinus comatus) laccase
1, the Function detection of extracorporeal suppression tumor cell propagation
1) accurately takes by weighing the pure product of laccase that embodiment 1 obtains, be configured to various dose solution (seeing Table 1), to detect the activity of vitro inhibition tumor cell proliferation with serum-free medium;
2) choose human hepatoma cell strain HepG2 (ATCC company, HB-8065) and human breast cancer cell strain MCF-7 (ATCC company HTB-22) be detected object, and cultivation HepG2 and MCF-7 are 8 * 10 with cell according to density when cell enters logarithmic phase
3The ratio in individual/hole is inoculated in 96 orifice plates, and cultivates 6 hours with the nutrient solution that contains serum, so that cell is all adherent;
3) remove the serum nutrient solution, and usefulness PBS (the prosperous Bioisystech Co., Ltd of Beijing ancient cooking vessel state, BF-0013) washed cell changes to serum-free medium, and adds the Shaggy Mane laccase solution of various dose, cultivates altogether 48 hours;
4) remove cell culture fluid, utilize mtt assay to detect the propagation situation of cell, calculate inhibiting rate.
Inhibiting rate (%)=A
540nm (sample of laccase treatment))/ A
540nm (untreated contrast)* 100%
Table 1 various dose Shaggy Mane laccase is to the tumor cell proliferation inhibition rate
Wherein, IC50 is the concentration that inhibiting rate reaches 50% o'clock laccase.
2, the Function detection that suppresses the HIV-1 reverse transcriptase activity
1) accurately takes by weighing the pure product of laccase that embodiment 1 obtains, be configured to various dose solution (seeing Table 2), to detect the activity that suppresses the HIV-1 ThermoScript II;
2) use Non-radioactive reverse transcriptase ELISA test kit (Roche company, 11468120910) the Shaggy Mane laccase to be suppressed the determination of activity of HIV-1 ThermoScript II (HIV-1RT).
Inhibiting rate (%)=(A405
Over against photograph-A405
Sample)/(A405
Over against photograph-A405
Negative contrast) * 100%
A405
Over against photograph: do not contain the Shaggy Mane laccase in the reaction system, contain the HIV-1 ThermoScript II
A405
Negative contrast: do not contain HIV-1 ThermoScript II and Shaggy Mane laccase in the reaction system
Table 2 various dose Shaggy Mane laccase is to the inhibiting rate of HIV-1 ThermoScript II
Claims (9)
1. Shaggy Mane (Coprinus comatus) preparation method of extract may further comprise the steps:
1) smudge cells;
2) collect protein crude extract administration;
3) from step 2) separation and purification laccase the protein crude extract administration collected, the laccase that obtains is the Shaggy Mane extract, and the molecular weight of described laccase is that 64KD and N-terminal sequence are seen sequence 1 in the sequence table.
2. preparation method according to claim 1 is characterized in that: described from step 2) the separation and purification laccase comprises the steps: the protein crude extract administration collected
A) will be from step 2) protein crude extract administration collected carries out anion exchange chromatography, collects the elution peak with laccase activity; The anion exchange groups that is adopted in the described anion exchange chromatography is DEAE, and the elution program that is adopted comprises following three step wash-outs:
The first step wash-out phosphate buffer solution A wash-out of pH6.8-7.5; Second goes on foot the following eluant solution of wash-out with pH6.8-7.5: solute is 0.1M NaCl, and solvent is described phosphate buffer solution A; The 3rd goes on foot the following eluant solution of wash-out with pH6.8-7.5: solute is 0.2M NaCl, and solvent is described phosphate buffer solution A; The solute of described phosphate buffer solution A is 3.5-4.5mM NaH
2PO
4With 5.5-6.5mM Na
2HPO
4, solvent is a water;
B) will carry out cation exchange column chromatography from the elution peak that step a) obtains, collect elution peak with laccase activity; The cation exchange group that is adopted in the described cation exchange column chromatography is CM, and the elution program that is adopted comprises following three step wash-outs:
The first step wash-out phosphate buffer solution B wash-out of pH6.1-6.8; Second goes on foot the following eluant solution of wash-out with pH6.1-6.8: solute is 0.1M NaCl, and solvent is described phosphate buffer solution B; The 3rd goes on foot the following eluant solution of wash-out with pH6.1-6.8: solute is 0.15M NaCl, and solvent is described phosphate buffer solution B; The solute of described phosphate buffer solution B is 5.75-6.75mM NaH
2PO
4With 3.2-4.2mM Na
2HPO
4, solvent is a water;
C) will carry out anion exchange chromatography from the elution peak that step b) obtains, collect elution peak with laccase activity; The anion exchange groups that is adopted in the described anion exchange chromatography is Q; The elution program that is adopted is for carrying out the NaCl linear elution, and the pH value of used NaCl linear elution liquid is 3.5-4.5 in the described NaCl linear elution, and solute is NaCl, and solvent is a hac buffer; The time of described linear elution is 200 minutes, and the concentration of NaCl rose to 0.15M by the 0M linearity in the described linear elution in 200 minutes; The solute of described hac buffer is 7.7-8.7mM acetic acid and 1.3-2.3mM sodium-acetate, and solvent is a water;
D) will carry out gel permeation chromatography from the elution peak that step c) obtains, obtain having the elution peak of laccase activity; The separating ranges of the chromatography media that described gel permeation chromatography is used comprises 64KD, and the column length of used chromatography column and column internal diameter ratio are 25: 1～50: 1.
3. preparation method according to claim 2, it is characterized in that: the carrier of described step a) anion exchange chromatography is Cellulose, the carrier of described step b) cation exchange column chromatography is Cellulose, the carrier of described step c) anion exchange chromatography is Sepharose, and the medium of described step d) gel permeation chromatography is Superdex75.
4. according to claim 2 or 3 described preparation methods, it is characterized in that:
The first step wash-out described phosphate buffer solution A wash-out of pH7.0 in the described step a); Second goes on foot the following eluant solution of wash-out with pH7.0: solute is 0.1M NaCl, and solvent is described phosphate buffer solution A; The 3rd goes on foot the following eluant solution of wash-out with pH7.0: solute is 0.2M NaCl, and solvent is described phosphate buffer solution A; The solute of described phosphate buffer solution A is 3.9mM NaH
2PO
4With 6.1mM Na
2HPO
4, solvent is a water;
The first step wash-out described phosphate buffer solution B wash-out of pH6.6 in the described step b); Second goes on foot the following eluant solution of wash-out with pH6.6: solute is 0.1M NaCl, and solvent is described phosphate buffer solution B; The 3rd goes on foot the following eluant solution of wash-out with pH6.6: solute is 0.15M NaCl, and solvent is described phosphate buffer solution B; The solute of described phosphate buffer solution B is 6.25mM NaH
2PO
4With 3.7mM Na
2HPO
4, solvent is a water;
Described in the described step c) in the NaCl linear elution pH value of used NaCl linear elution liquid be 4.0, the solute of described hac buffer is 8.2mM acetic acid and 1.8mM sodium-acetate, solvent is a water;
The elution peak that obtains having laccase activity described in the described step d) obtains with following eluant solution: the pH value is the NH of 8.5 0.2M
4HCO
3The aqueous solution.
5. according to arbitrary described preparation method among the claim 2-4, it is characterized in that: the column length of the chromatography column that described step d) is used and column internal diameter ratio are 30: 1.
6. the extract that obtains by arbitrary described preparation method among the claim 1-5.
7. extract according to claim 6 is characterized in that: the molecular weight of described extract is that 64KD and N-terminal sequence are seen sequence table sequence 1.
8. claim 6 or the 7 described extracts application in the following arbitrary product of preparation:
A) suppress the product that cancer cell is bred;
B) improve the product of cancer patients's healthy state;
C) product of inhibition HIV-1 reverse transcriptase activity;
D) improve the product of AIDS patient's healthy state.
9. the product that contains claim 6 or 7 described extracts, described product are following arbitrary product:
A) suppress the product that cancer cell is bred;
B) improve the product of cancer patients's healthy state;
C) product of inhibition HIV-1 reverse transcriptase activity;
D) improve the product of AIDS patient's healthy state.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110162219 CN102268414B (en) | 2011-06-16 | 2011-06-16 | Coprinus comatus laccase with activity of inhibiting tumor cell proliferation and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710364A (en) * | 2013-12-16 | 2014-04-09 | 南京林业大学 | Laccase gene Lac7 and expression protein and application thereof |
| CN103710363A (en) * | 2013-12-16 | 2014-04-09 | 南京林业大学 | Laccase gene Lac6 and expression protein and application thereof |
| CN108220258A (en) * | 2016-12-15 | 2018-06-29 | 天津市林业果树研究所 | A kind of preparation method of the Stropharia rugoso-annulata activated protein with laccase activity |
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2011
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Non-Patent Citations (3)
| Title |
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| 《生物加工过程》 20110331 周雪，朱启忠 白腐真菌漆酶的纯化及性质 53－57 1－9 第9卷, 第2期 * |
| 《西昌学院学报·自然科学版》 20080630 韩洪波 鸡腿菇漆酶基本酶学性质研究 49－54 1－9 第22卷, 第2期 * |
| 《食用菌学报》 20101231 马茜茜 等 云芝漆酶的分离纯化和理化性质及抗增殖活性 43－47 1－9 第17卷, 第2期 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710364A (en) * | 2013-12-16 | 2014-04-09 | 南京林业大学 | Laccase gene Lac7 and expression protein and application thereof |
| CN103710363A (en) * | 2013-12-16 | 2014-04-09 | 南京林业大学 | Laccase gene Lac6 and expression protein and application thereof |
| CN103710363B (en) * | 2013-12-16 | 2015-06-10 | 南京林业大学 | Laccase gene Lac6 and expression protein and application thereof |
| CN103710364B (en) * | 2013-12-16 | 2015-06-10 | 南京林业大学 | Laccase gene Lac7 and expression protein and application thereof |
| CN108220258A (en) * | 2016-12-15 | 2018-06-29 | 天津市林业果树研究所 | A kind of preparation method of the Stropharia rugoso-annulata activated protein with laccase activity |
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| CN102268414B (en) | 2013-01-23 |
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