CN107794250B - Extraction and activity determination of chlorella catechol 1, 2-dioxygenase - Google Patents
Extraction and activity determination of chlorella catechol 1, 2-dioxygenase Download PDFInfo
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- 108010066997 Catechol 1,2-dioxygenase Proteins 0.000 title claims abstract description 66
- 241000195649 Chlorella <Chlorellales> Species 0.000 title claims abstract description 36
- 238000000605 extraction Methods 0.000 title claims abstract description 20
- 230000000694 effects Effects 0.000 title abstract description 26
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- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 8
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
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- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 1
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
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- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses extraction and activity determination of chlorella catechol 1, 2-dioxygenase, which comprises the following specific steps: grinding the chlorella powder at low temperature, adding a Tris-HCl buffer solution, and centrifuging to obtain a crude enzyme solution of the intracellular enzyme; filtering the crude enzyme solution, concentrating, dialyzing, loading to a cellulose column, eluting, concentrating, dialyzing, concentrating, loading to a Mono-Q column, eluting, dialyzing, concentrating, and drying to obtain catechol 1, 2-dioxygenase. The enzyme activity determination method of the chlorella catechol 1, 2-dioxygenase comprises the following steps: the reaction system was reacted in a water bath, and the amount of the product produced was measured. The beneficial effects are that: the method for extracting the catechol 1, 2-dioxygenase has the advantages of high impurity removal rate, mild conditions and high purity, can well keep the activity of the enzyme, can maintain the interaction between the enzymes, and has great development potential; the activity of the enzyme can be simply and conveniently measured.
Description
Technical Field
The invention relates to catechol 1, 2-dioxygenase, in particular to extraction and activity determination of chlorella catechol 1, 2-dioxygenase.
Background
Catechol (C)6H6O2) Catechol is a compound formed by substituting two ortho-hydrogens of benzene with hydroxyl groups, is widely present in nature in the form of derivatives, and is an important intermediate product for degradation of aromatic substances. Catechol and its derivatives are important chemical products, and may be used widely in medicine, pesticide, food, perfume, chemical assistant, etc. Because of catecholThe method can be widely applied to various industries, the demand of catechol is increased along with the rapid development of economy, and the discharge amount and complexity of various waste water are increased along with the increase. Pyrocatechol belongs to a highly toxic substance, a small amount of pyrocatechol can cause eczematous dermatitis after being absorbed by skin, the pyrocatechol can enter organisms through skin, respiratory tract and digestive tract, and the wastewater containing the pyrocatechol has potential hazards of carcinogenesis, Kawasaki, mutagenesis and the like. Therefore, catechol released in the industry into soil and water not only pollutes the environment but also threatens the survival and development of human beings through the enrichment effect, has the hormone-like effect, and causes endocrine disturbance to endanger the biological health. With the increase of production and use amount, pyrocatechol entering the environment is correspondingly increased, and the pollution hazard of the pyrocatechol in the environment is gradually serious, so that the effective removal of the pyrocatechol in the environment has attracted people's attention.
At present, one of the most important routes in the biodegradation of catechol is: in the ortho-ring opening metabolic pathway (ortho-pathway), catechol 1, 2-dioxygenase catalyzes the cleavage of catechol to form a cis, cis-myfuroic acid intermediate, which is then degraded to succinic acid and acetyl-coa by the stepwise degradation of the respective enzymes. Therefore, how to prepare high-purity catechol 1, 2-dioxygenase is one of the effective means for treating water pollution.
Disclosure of Invention
The invention aims to provide extraction and activity determination of chlorella catechol 1, 2-dioxygenase, the extraction method has high impurity removal rate and mild conditions, the activity of the enzyme can be well maintained, the purity of the extracted catechol 1, 2-dioxygenase is high, and in addition, the activity determination of the enzyme is simple and feasible.
Aiming at the problems mentioned in the background technology, the invention adopts the technical scheme that: the extraction of chlorella catechol 1, 2-dioxygenase comprises the steps of crude enzyme liquid preparation of intracellular enzyme, concentration dialysis, primary purification and secondary purification, and the extraction comprises the following specific steps:
preparing crude enzyme solution of intracellular enzyme: performing suction filtration on chlorella liquid in a logarithmic phase, drying to obtain chlorella powder, then grinding for 5-15min at 2-5 ℃, adding Tris-HCl buffer solution with pH of 7.8-8.2, wherein the weight-volume ratio of the chlorella powder to the Tris-HCl buffer solution is 1:1.8-2.3, uniformly mixing, centrifuging for 10-20min at the temperature of 2-5 ℃ and the rotating speed of 8000-12000r/min, and obtaining supernatant, namely crude enzyme liquid of intracellular enzyme, grinding the chlorella powder in a low-temperature state, rapidly breaking cell walls, rapidly releasing enzyme in chlorella cells, and having strong operability, and meanwhile, the buffer solution in the step has high solubility on the enzyme, can prevent the severe fluctuation of the pH value, protects the biological activity of intracellular catechol 1, 2-dioxygenase from being damaged, and is beneficial to the extraction of catechol 1, 2-dioxygenase, can effectively inhibit the degradation of enzymes and maintain the interaction between the enzymes;
concentration and dialysis: filtering the crude enzyme solution of the intracellular enzyme through a 0.15-0.3 mu m microporous filter membrane, concentrating by using polyethylene glycol PEG20000, dialyzing for 12-18h by using a phosphate buffer solution with the concentration of 9-11mmol/L, pH of 6.8-7.2 to obtain a crude product of the catechol 1, 2-dioxygenase, wherein the microporous filter membrane can filter and remove redundant thalli and reduce impurities in the crude enzyme product, an absorbent only absorbs a micromolecular solvent leaked from a dialysis bag during concentration, and a water absorbent can be continuously and repeatedly used after being dried;
primary purification: firstly, a phosphate buffer solution with the concentration of 13-17mmol/L, pH of 7.0-7.5 is used for balancing a DEAE cellulose column, then a catechol 1, 2-dioxygenase crude product is loaded on the cellulose column, the phosphate buffer solution with the concentration of 24-27mmol/L, pH of 7.3-7.7 is used for elution, after being concentrated by polyethylene glycol PEG20000, the phosphate buffer solution with the concentration of 9-11mmol/L, pH of 6.8-7.2 is used for dialysis for 12-24h, finally, an ultrafiltration membrane is used for secondary concentration, the step utilizes the physical adsorption and ion exchange of DEAE cellulose and the difference of each component in the enzyme crude product on the electrostatic force of DEAE cellulose to separate the catechol 1, 2-dioxygenase crude product, can remove impurities such as foreign protein and the like, thereby achieving the purposes of coarse refining and primary separation, and having no influence on the biological activity of the enzyme, in addition, the purity of the enzyme can be further improved by combining dialysis and an ultrafiltration membrane, and impurities can be better removed by combining phosphate buffer solutions with different concentrations;
and (3) secondary purification: after a Mono-Q column is balanced by a phosphate buffer solution containing 0.11-0.13mmol/L hydrazine hydrate and having a concentration of 4-6mmol/L, pH of 7.3-7.7, crude catechol 1, 2-dioxygenase after primary purification is loaded, is eluted by a phosphate buffer solution having a concentration of 24-27mmol/L, pH of 7.3-7.7, is dialyzed for 12-24h by a phosphate buffer solution having a concentration of 9-11mmol/L, pH of 6.8-7.2, is finally concentrated by an ultrafiltration membrane and dried to obtain the catechol 1, 2-dioxygenase, the hydrazine hydrate can enhance the binding force, resolution and stability of the Mono-Q column, further improve the speed and efficiency of the Mono-Q column for purifying the catechol 1, 2-dioxygenase, and can shorten the balancing time of the Mono-Q column, the flow rate is improved, the denaturation or inactivation of the enzyme cannot be caused, the crude catechol 1, 2-dioxygenase is purified again in the step, the product purity can be effectively improved, the purity of the obtained catechol 1, 2-dioxygenase is high, and the activity of the enzyme is further improved.
The enzyme activity of chlorella catechol 1, 2-dioxygenase is determined by the following steps: reacting the reaction system in a water bath at the temperature of 20-30 ℃ for 30min, sampling every 5min, and determining the generation amount of a product, wherein the reaction system comprises the following steps: 0.18-0.22mL of EDTA (ethylene diamine tetraacetic acid) of 30-50mmol/L, 0.019-0.021mL of catechol of 20-40mmol/L, 0.3-0.5mg of chlorella catechol 1, 2-dioxygenase, 0.04-0.06mol/L and 1.35-1.40mL of phosphate buffer with pH of 6.8-7.2.
Compared with the prior art, the invention has the advantages that: the extraction method has high impurity removal rate and mild conditions, can better keep the activity of the enzyme, and the extracted catechol 1, 2-dioxygenase has high purity; the extraction method can rapidly release enzyme in Chlorella cell, has strong operability, can protect the bioactivity of intracellular enzyme from being damaged, is beneficial to the extraction of catechol 1, 2-dioxygenase, and can maintain the interaction between enzymes; the catechol 1, 2-dioxygenase crude product obtained by extraction is purified twice, the purity is high, the activity of the enzyme is high, and the activity of the enzyme is simple and feasible to measure.
Detailed Description
The scheme of the invention is further illustrated by the following examples:
example 1:
the extraction of chlorella catechol 1, 2-dioxygenase comprises the steps of crude enzyme liquid preparation of intracellular enzyme, concentration dialysis, primary purification and secondary purification, and the extraction comprises the following specific steps:
1) preparing crude enzyme solution of intracellular enzyme: filtering the chlorella solution at the later logarithmic phase, drying to obtain chlorella powder, grinding at 3 deg.C for 14min, adding Tris-HCl buffer solution with pH of 7.8 at a weight-to-volume ratio of 1:2.3, mixing, centrifuging at 5 deg.C and 8000r/min for 20min to obtain supernatant as crude enzyme solution of intracellular enzyme, the step grinds the chlorella powder at low temperature, can quickly break cell walls, enables enzymes in the chlorella cells to be quickly released, has strong operability, meanwhile, the buffer solution in the step has high solubility on the enzyme, can prevent the severe fluctuation of the pH value, protects the bioactivity of the intracellular catechol 1, 2-dioxygenase from being damaged, is beneficial to the extraction of the catechol 1, 2-dioxygenase, can effectively inhibit the degradation of the enzyme and can maintain the interaction between the enzymes;
2) concentration and dialysis: filtering the crude enzyme solution of the intracellular enzyme through a 0.15 mu m microporous filter membrane, concentrating by using polyethylene glycol PEG20000, and dialyzing for 15h by using a phosphate buffer solution with the concentration of 9mmol/L, pH of 7.2 to obtain a crude product of the catechol 1, 2-dioxygenase, wherein the microporous filter membrane can filter and remove redundant thalli and reduce impurities in the crude enzyme product, an absorbent only absorbs a small molecular solvent exuded from a dialysis bag during concentration, and a water absorbent can be continuously and repeatedly used after being dried;
3) primary purification: firstly, a phosphoric acid buffer solution with the concentration of 13mmol/L, pH of 7.5 is used for balancing a DEAE cellulose column, then a catechol 1, 2-dioxygenase crude product is loaded on the cellulose column, eluted by a phosphoric acid buffer solution with the concentration of 24mmol/L, pH of 7.7, concentrated by polyethylene glycol PEG20000, dialyzed for 15 hours by a phosphoric acid buffer solution with the concentration of 9mmol/L, pH of 7.2, and finally concentrated again by an ultrafiltration membrane, the step utilizes the physical adsorption and ion exchange action of the DEAE cellulose and the difference of electrostatic force of each component in the enzyme crude product on the DEAE cellulose to separate the catechol 1, 2-dioxygenase crude product, can preliminarily remove impurities such as foreign protein and the like, thereby achieving the purposes of coarse extraction and fine separation, simultaneously has no influence on the biological activity of the enzyme, and in addition, the combination of dialysis and the ultrafiltration membrane can further improve the purity of the enzyme, meanwhile, phosphate buffer solutions with different concentrations are combined, so that impurities can be better removed;
4) and (3) secondary purification: after a Mono-Q column is balanced by a phosphate buffer solution containing 0.13mmol/L hydrazine hydrate and having a concentration of 4mmol/L, pH of 7.7, crude catechol 1, 2-dioxygenase after primary purification is loaded, the phosphate buffer solution having a concentration of 24mmol/L, pH of 7.7 is used for elution, the phosphate buffer solution having a concentration of 9mmol/L, pH of 7.2 is used for dialysis for 15 hours, and finally an ultrafiltration membrane is used for concentration and drying to obtain the catechol 1, 2-dioxygenase, wherein the hydrazine hydrate can enhance the binding force, the resolution and the stability of the Mono-Q column, further improve the speed and the efficiency of the Mono-Q column for purifying the catechol 1, 2-dioxygenase, in addition, the hydrazine hydrate can also shorten the balancing time of the Mono-Q column, improve the flow rate, and cannot cause the denaturation or inactivation of enzyme, the crude catechol 1, 2-dioxygenase is purified again in the step, can effectively improve the purity of the product, ensure that the purity of the obtained catechol 1, 2-dioxygenase is high, and further improve the activity of the enzyme.
Example 2:
the extraction of chlorella catechol 1, 2-dioxygenase comprises the following specific steps:
1) carrying out suction filtration on the chlorella solution in the later logarithmic phase, drying to obtain chlorella powder, then grinding for 7min at 5 ℃, adding Tris-HCl buffer solution with pH of 8.2, wherein the weight-volume ratio of the chlorella powder to the Tris-HCl buffer solution is 1:1.8, uniformly mixing, and then centrifuging for 10min at the temperature of 5 ℃ and the rotating speed of 12000r/min, wherein the supernatant is crude enzyme solution of intracellular enzyme;
2) filtering the crude enzyme solution of the intracellular enzyme with a 0.3 mu m microporous membrane, concentrating with polyethylene glycol PEG20000, and dialyzing with phosphate buffer solution with concentration of 11mmol/L, pH of 6.8 for 18h to obtain crude product of catechol 1, 2-dioxygenase;
3) firstly, a phosphoric acid buffer solution with the concentration of 17mmol/L, pH of 7.0 is used for balancing a DEAE cellulose column, then a crude product of catechol 1, 2-dioxygenase is loaded on the cellulose column, is eluted by a phosphoric acid buffer solution with the concentration of 27mmol/L, pH of 7.3, is concentrated by polyethylene glycol PEG20000, is dialyzed by a phosphoric acid buffer solution with the concentration of 11mmol/L, pH of 6.8 for 18 hours, and is finally concentrated again by an ultrafiltration membrane;
4) after the Mono-Q column is balanced by phosphate buffer solution containing 0.11mmol/L hydrazine hydrate and having the concentration of 6mmol/L, pH of 7.3, crude catechol 1, 2-dioxygenase after primary purification is loaded, the crude catechol 1, 2-dioxygenase is eluted by phosphate buffer solution having the concentration of 27mmol/L, pH of 7.3, then phosphate buffer solution having the concentration of 11mmol/L, pH of 6.8 is dialyzed for 18h, and finally, the catechol 1, 2-dioxygenase is obtained by concentrating and drying the crude catechol by using an ultrafiltration membrane.
Example 3:
the extraction of chlorella catechol 1, 2-dioxygenase comprises the following specific steps:
preparing crude enzyme solution of intracellular enzyme: carrying out suction filtration on the chlorella solution at the later logarithmic phase, drying to obtain chlorella powder, then grinding for 10min at 4 ℃, then adding a Tris-HCl buffer solution with the concentration of 50mmol/L, pH of 8.0, wherein the weight-volume ratio of the chlorella powder to the Tris-HCl buffer solution is 1:2, uniformly mixing, and then centrifuging for 15min at the temperature of 4 ℃ and the rotating speed of 10000r/min, wherein the supernatant is crude enzyme solution of intracellular enzyme;
concentration and dialysis: filtering the crude enzyme solution of the intracellular enzyme by a 0.22 mu m microporous membrane, concentrating by using polyethylene glycol PEG20000, and dialyzing for 15h by using a phosphate buffer solution with the concentration of 10mmol/L, pH of 7.0 to obtain a crude product of the catechol 1, 2-dioxygenase;
primary purification: firstly, a phosphoric acid buffer solution with the concentration of 15mmol/L, pH of 7.3 is used for balancing a DEAE cellulose column, then a crude product of catechol 1, 2-dioxygenase is loaded on the cellulose column, is eluted by a phosphoric acid buffer solution with the concentration of 26mmol/L, pH of 7.5, is concentrated by polyethylene glycol PEG20000, is dialyzed by a phosphoric acid buffer solution with the concentration of 10mmol/L, pH of 7.0 for 18 hours, and is finally concentrated again by an ultrafiltration membrane;
and (3) secondary purification: after the MonoQ column is balanced by phosphate buffer solution containing 0.12mmol/L hydrazine hydrate and having the concentration of 5mmol/L, pH of 7.5, crude catechol 1, 2-dioxygenase after the first purification is loaded, the crude catechol 1, 2-dioxygenase is eluted by phosphate buffer solution having the concentration of 26mmol/L, pH of 7.5, then the crude catechol 1, 2-dioxygenase is dialyzed by phosphate buffer solution having the concentration of 10mmol/L, pH of 7.0 for 18h, and finally the crude catechol 1, 2-dioxygenase is concentrated by an ultrafiltration membrane and dried to obtain the finished product.
Example 4:
immobilization of chlorella catechol 1, 2-dioxygenase: ultrasonically dispersing the nano material into water according to the weight ratio of 1.8-2.2:1, then mixing the nano material with the catechol 1, 2-dioxygenase solution obtained in the example 3 to ensure that the pH of the solution is 7.8-8.2 and the enzyme concentration is 80-100 mu g/mL, then placing the solution in a shaking table with the temperature of 25-35 ℃ and the enzyme concentration of 130-3O4The weight ratio of the mixture of graphene oxide and pyrimidinamine is 1:0.13-0.17:0.02-0.03, the addition of pyrimidinamine can uniformly and stably disperse the nano material in water, so that the nano material can keep the highest specific surface area, the contact area between the nano material and enzyme is greatly increased, the loading capacity of the nano material to the enzyme is greatly improved, the activity of the immobilized enzyme is similar to that of the original enzyme, and simultaneously Fe3O4Can be effectively recovered through the action of magnetic force, avoids the pollution of enzyme and materials, improves the purity of the product, well retains the activity of the catechol 1, 2-dioxygenase, obviously improves the stability and the activity, has relatively high loading capacity and ensures that the relative activity is realized after the immobilized catechol 1, 2-dioxygenase is repeatedly used for 9 times56% remain and better stability can be maintained in the high concentration catechol solution.
Example 5:
the enzyme activity of chlorella catechol 1, 2-dioxygenase is determined by the following steps: reacting the reaction system in a water bath at 25 ℃ for 30min, sampling every 5min, and determining the increase value of absorbance within 30min at a wavelength corresponding to a substrate by taking the reaction system without adding an enzyme solution as a control, wherein the reaction system comprises the following steps: 0.2mL of 40mmol/L EDTA, 0.02mL of 30mmol/L catechol, 0.4mg of enzyme solution, 0.05mol/L and 1.37mL of phosphate buffer solution having a pH of 7.0.
The test groups are the products obtained in example 1, example 2, example 3 and example 4, the control group is pure catechol 1, 2-dioxygenase, the enzyme activity is set as 100%, and the enzyme activities of the products obtained in example 1, example 2, example 3 and example 4 are 84.31%, 83.49%, 88.20% and 88.02%, which shows that the catechol 1, 2-dioxygenase extracted in example 1, example 2 and example 3 of the present invention has high purity, and the immobilization treatment of catechol 1, 2-dioxygenase in example 4 has little influence on the enzyme activity.
Example 6:
determination of degradation of petroleum hydrocarbon pollutants by chlorella catechol 1, 2-dioxygenase:
the soil to be tested is taken from farmland soil without petroleum hydrocarbon pollution, plant residues are removed, the farmland soil is dried in the air and then is sieved by a 2mm sieve, petroleum is dissolved in petroleum ether, then the petroleum ether and the soil are uniformly mixed according to the volume-to-mass ratio of 1:10, and the mixture is placed in a ventilation position for 4 weeks to completely volatilize the petroleum ether, so that the petroleum-polluted soil with 50000-80000mg/kg is prepared. The petroleum-polluted soil and the product in the example 3 are uniformly mixed according to the proportion of 10:0.5, and the action is carried out for 12-24h, and the removal rate of the product in the example 3 to the petroleum hydrocarbon in the soil is found to be 35-60%, which shows that the product in the example 3 has good catalytic oxidation degradation effect on the petroleum hydrocarbon and can be used for repairing the petroleum-polluted soil.
Conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims (1)
1. The extraction of chlorella catechol 1, 2-dioxygenase comprises the steps of preparing crude enzyme liquid of intracellular enzyme, concentrating and dialyzing, purifying for the first time and purifying for the second time, and is characterized in that:
preparing crude enzyme solution of intracellular enzyme: filtering the chlorella liquid in the later logarithmic phase, drying to obtain chlorella powder, grinding for 5-15min at 2-5 ℃, adding Tris-HCl buffer solution with pH of 7.8-8.2, wherein the weight-volume ratio of the chlorella powder to the Tris-HCl buffer solution is 1:1.8-2.3, uniformly mixing, centrifuging for 10-20min at the temperature of 2-5 ℃ and the rotation speed of 8000-12000r/min, and obtaining the supernatant which is crude enzyme liquid of intracellular enzymes;
concentration and dialysis: filtering the crude enzyme solution of the intracellular enzyme with 0.15-0.3 μm microporous membrane, concentrating with polyethylene glycol PEG20000, and dialyzing with phosphate buffer solution with concentration of 9-11mmol/L, pH of 6.8-7.2 for 12-18h to obtain crude product of catechol 1, 2-dioxygenase;
primary purification: firstly, a phosphate buffer solution with the concentration of 13-17mmol/L, pH of 7.0-7.5 is used for balancing a DEAE cellulose column, then a crude product of catechol 1, 2-dioxygenase is loaded on the cellulose column, the crude product is eluted by the phosphate buffer solution with the concentration of 24-27mmol/L, pH of 7.3-7.7, after being concentrated by polyethylene glycol PEG20000, the phosphate buffer solution with the concentration of 9-11mmol/L, pH of 6.8-7.2 is used for dialysis for 12-24h, and finally, the phosphate buffer solution is concentrated again by an ultrafiltration membrane;
and (3) secondary purification: after the Mono-Q column is balanced by a phosphate buffer solution containing 0.11-0.13mmol/L hydrazine hydrate and having a concentration of 4-6mmol/L, pH of 7.3-7.7, crude catechol 1, 2-dioxygenase after primary purification is loaded, is eluted by a phosphate buffer solution having a concentration of 24-27mmol/L, pH of 7.3-7.7, is dialyzed for 12-24h by a phosphate buffer solution having a concentration of 9-11mmol/L, pH of 6.8-7.2, and is finally concentrated by an ultrafiltration membrane and dried to obtain the catechol 1, 2-dioxygenase.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105567651A (en) * | 2016-03-22 | 2016-05-11 | 云南师范大学 | Heat-stable catechol 1,2-dioxygenase derived from animal manure metagenome and coding gene thereof, and preparation method of heat-stable catechol 1,2-dioxygenase |
| CN106222147A (en) * | 2016-08-03 | 2016-12-14 | 浙江海洋大学 | Microalgae is used for the preparation method of the cytochrome P 450 enzymes of petroleum hydrocarbon degradation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567651A (en) * | 2016-03-22 | 2016-05-11 | 云南师范大学 | Heat-stable catechol 1,2-dioxygenase derived from animal manure metagenome and coding gene thereof, and preparation method of heat-stable catechol 1,2-dioxygenase |
| CN106222147A (en) * | 2016-08-03 | 2016-12-14 | 浙江海洋大学 | Microalgae is used for the preparation method of the cytochrome P 450 enzymes of petroleum hydrocarbon degradation |
Non-Patent Citations (4)
| Title |
|---|
| Catechol 1,2-Dioxygenase from Acinetobacter calcoaceticus:Purification and Properties;RAMESH N. PATEL等;《JOURNAL OF BACTERIOLOGY》;19760731;第127卷(第1期);536-544 * |
| 海洋苯酚降解菌 Candidasp.P5的分离鉴定及其降解特性;胡忠等;《应用与环境生物学报》;20071231;第13卷(第2期);243-247 * |
| 邻苯二酚1 , 2-双加氧酶(C12O)基因的定位、克隆和表达;罗如新等;《应用与环境生物学报》;19991231;第5卷(第2期);208-211 * |
| 邻苯二酚-1，2-双加氧酶的催化性质;李丽等;《生物化学与生物物理进展》;19901231;第17卷(第2期);157-158,160 * |
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