CN101514224A - Method for extracting and purifying zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon - Google Patents
Method for extracting and purifying zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon Download PDFInfo
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- CN101514224A CN101514224A CNA2008100072254A CN200810007225A CN101514224A CN 101514224 A CN101514224 A CN 101514224A CN A2008100072254 A CNA2008100072254 A CN A2008100072254A CN 200810007225 A CN200810007225 A CN 200810007225A CN 101514224 A CN101514224 A CN 101514224A
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- aromatic hydrocarbon
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Abstract
The invention relates to a method for extracting and purifying zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbons (PAHS) and belongs to the fields of biological engineering technology and biodegradation technology of refractory organic pollutants. The method comprises the following steps: preparing a strain suspension from PAHS-degrading strains, performing anaerobic amplification culture on the strain suspension, and separating strains and a supernatant fluid through ultrasonic wave breaking; adding deionized water to the separated strains, performing the ultrasonic wave breaking to separate strains and a supernatant fluid, and repeating the step for twice; combining supernatant fluids collected by the three times to obtain a crude extract of the zymoprotein anaerobically degraded by the polycyclic aromatic hydrocarbons (PAHS); adding a ground (NH4)2SO4 solid to the zymoprotein crude extract in an anaerobic box until the saturation is between 45 and 85 percent, sealing the mixture, oscillating the mixture for 1 to 2 hours, and centrifugally separating precipitation; dissolving the precipitation into a small amount of buffer solution, and filling the mixture in a dialysis bag for dialysis for 24 hours; centrifugating the dialyzed sample to obtain a supernatant fluid; filtering the supernatant fluid on an anion-exchange column, collecting eluent with absorbance more than 0.01 on a 280 mm position; and determining the activity of the zymoprotein, and selecting an active part for standby. The method has the advantages of simple process, simple and convenient operation, low equipment requirement, low cost, high yield and the like.
Description
Technical field
The invention belongs to biotechnology and Persistent organic pollutants biologic treating technique field, be specifically related to a kind of extraction and purification method of zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon.
Background technology
Polycyclic aromatic hydrocarbons is the persistence organic pollutant of a quasi-representative, has carcinogenic, teratogenesis, mutagenesis, and ecological environment security and human health are caused very big threat.Polycyclic aromatic hydrocarbons contaminated in the environment in recent years have the trend that constantly increases the weight of, thereby polycyclic aromatic hydrocarbons contaminated control and recovery technique have become the important content of Chinese scholars research.Improvement technology to polycyclic aromatic hydrocarbons in the environment mainly contains physics method, chemical method and biological process at present.Mainly there is the high and halfway shortcoming of removal of processing costs in the physical treatment technology.In chemical method, there is the scholar to utilize polycyclic aromatic hydrocarbons in the wash-out environment such as organic solvent (as ethanol, normal hexane, methylene dichloride, trichloromethane, acetone) and tensio-active agent both at home and abroad, but can has secondary pollution problems.Simple to operate, advantages such as working cost is low, non-secondary pollution that biological process has, but in actual application, repairing efficiency is longer.Extracting enzyme from the degraded microorganism cells comes enhancing degradation to become a kind of important method of removing polycyclic aromatic hydrocarbons one-tenth.But,, have not yet to see the research report of relevant zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon extraction and purification because the polycyclic aromatic hydrocarbon anaerobic degradation microorganism is lacked deep research.Therefore, press for the extraction and purification method of the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon that a kind of simple and effective is provided,, thereby be widely used in the reparation and the improvement of polycyclic aromatic hydrocarbons contaminated environment for a large amount of preparation zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon provide new approach.
Summary of the invention
The extraction and purification method that the purpose of this invention is to provide a kind of zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon.Its concrete scheme is:
1. the polycyclic aromatic hydrocarbon anaerobic degradation bacterium that preserves in 4 ℃ of refrigerators being mixed with concentration is 1 * 10
8The bacteria suspension of individual/ml, inoculation 2ml bacteria suspension in the anaerobism bottle that adds 198ml inducing culture, 2.0ml trace metal liquid and 0.2ml vitamin c solution, for removing the oxygen in the substratum, inducing culture, trace metal liquid and vitamin solution were used high pure nitrogen aeration 3.5h before adding.Tighten then with Viton plug sealing bottleneck, and with screw top closure.With the polycyclic aromatic hydrocarbon anaerobic degradation bacterium mixed solution of anaerobism bottle after culture temperature is can obtain amplification cultivation after 25～30 ℃, rotating speed are to cultivate 25d in the vibrator of 100r/min.
2. the polycyclic aromatic hydrocarbon anaerobic degradation bacterium mixed solution of step in 1. carried out ultrasonic disruption 15min.Degradation bacteria mixed solution after the fragmentation centrifugal 15min under the 8000r/min condition isolates thalline and supernatant liquor in anaerobic box.
3. in anaerobic box, in 2. isolated thalline, add the deionized water after 30ml uses high pure nitrogen aeration 3.5h by step, proceed ultrasonic disruption after tightening with Viton plug sealing bottleneck and with screw top closure, centrifugal 15min under the 8000r/min condition isolates thalline and supernatant liquor in anaerobic box.
4. in anaerobic box, in 3. isolated thalline, add the deionized water after 30ml uses high pure nitrogen aeration 3.5h by step, proceed ultrasonic disruption after tightening with Viton plug sealing bottleneck and with screw top closure, centrifugal 15min under the 8000r/min condition isolates thalline and supernatant liquor in anaerobic box.
5. in anaerobic box, merge, can obtain the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon crude extract by 2., the 3. and 4. collected supernatant liquor of step.
6. in anaerobic box, in the zymoprotein crude extract that 5. obtains by step, add fine ground (NH
4)
2SO
4Solid to 45～85% saturation ratio is tightened the back 1～2h that vibrates with Viton plug sealing bottleneck and with screw top closure in rotating speed is the vibrator of 100r/min, centrifugal 15min under the 8000r/min condition isolates precipitation in anaerobic box then.
7. will be dissolved in a small amount of pH value by the precipitation that 6. step obtains is in 7.0～7.2 0.05mol/L trihydroxy methyl aminomethane (the Tris)-HCl damping fluid and the dialysis tubing of packing into, with the dialysis tubing 24h that dialyses in the pH value is 7.0～7.2 0.05mol/L Tris-HCl damping fluid.With dialyzed sample centrifugal 15min under the 8000r/min condition, in anaerobic box, isolate supernatant liquor.
8. on the supernatant liquor that in anaerobic box, will 7. obtain by step to being to filter on 7.0～7.2 the 0.05mol/L Tris-HCl damping fluid equilibrated Q Sepharose Fast Flow anion-exchange column with the pH value, adopting the pH value is the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon of 7.0～7.2 the linear gradient elution of bound of 0.05mol/LTris-HCl damping fluid (including 0.0～0.30mol/L sodium-chlor), and elution speed is 20ml/h.The utilization volume is that the anaerobism pipe of 10ml is collected elutriant, and wherein every anaerobism pipe is collected elutriant 5ml.Is that elutriant is detected at the 280nm place with ultraviolet spectrophotometer in absorbing wavelength, collects absorbancy greater than 0.01 elutriant.Measure the activity of zymoprotein, select active part standby.
Described step is consisting of of middle inducing culture 1.: NaNO
3: 2.0g/L, NH
4Cl:1.0gL
-1, KH
2PO
4: 1.0gL
-1, MgCl
2: 0.1gL
-1, CaCl
22H
2O:0.05gL
-1, PAHs:0.03gL
-1
Described step is consisting of of middle trace metal liquid 1.: CoCl
26H
2O:35mgL
-1, CuCl
2: 0.25mgL
-1, H
3BO
3: 6.0mgL
-1, MnCl
24H
2O:30mgL
-1, Na
2MoO
42H
2O:3.0mgL
-1, NiCl
22H
2O:2.5mgL
-1, ZnCl
2: 2.5mgL
-1
The described step 2. condition of middle ultrasonic disruption is: electric current 35%, pulse 9.0s, treatment time 15min.
6. middle (the NH that adds of described step
4)
2SO
4The solid amount is preferably to 60～70% saturation ratios.
The present invention has that technology is simple, easy and simple to handle, low for equipment requirements, cost is low, the yield advantages of higher.
Embodiment
Further specify the present invention below in conjunction with example.
Material: (1) polycyclic aromatic hydrocarbon anaerobic degradation bacterium (obtaining) by the domestication separation of own laboratory;
(2) PAHs: naphthalene, phenanthrene, anthracene, pyrene (purity 〉=99%, Sigma Aldrich);
(3) NaCl, (NH
4)
2SO
4: analytical pure;
(4) the 0.05mol/L trihydroxy methyl aminomethane-HCl damping fluid of pH value 7.0～7.2;
(5) inducing culture 1L, its composition and each component concentrations are: NaNO
3: 2.0g/L, NH
4Cl:1.0gL
-1, KH
2PO
4: 1.0gL
-1, MgCl
2: 0.1gL
-1, CaCl
22H
2O:0.05gL
-1, PAHs:0.03gL
-1
(6) trace metal liquid 1L, its composition and each component concentrations are: CoCl
26H
2O:35mgL
-1, CuCl
2: 0.25mgL
-1, H
3BO
3: 6.0mgL
-1, MnCl
24H
2O:30mgL
-1, Na
2MoO
42H
2O:3.0mgL
-1, NiCl
22H
2O:2.5mgL
-1, ZnCl
2: 2.5mgL
-1
The active detection method of zymoprotein: the activity of determining zymoprotein with the naphthalene amount of zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon and naphthalene reaction consumes.Getting a certain amount of zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon and 40ml concentration is the naphthalene solution mixing of 30mg/L, is anaerobic reaction 24h under 30 ℃ of conditions in temperature, measures the variation of naphthalene concentration in the anaerobic degradation process.
Embodiment 1
It is 1 * 10 that the polycyclic aromatic hydrocarbon anaerobic degradation bacterium that preserves in 4 ℃ of refrigerators is mixed with concentration
8The bacteria suspension of individual/ml, inoculation 2ml bacteria suspension in the anaerobism bottle that adds 198ml inducing culture, 2.0ml trace metal liquid and 0.2ml vitamin c solution, wherein inducing culture, trace metal liquid and vitamin solution were used high pure nitrogen aeration 3.5h before adding.Be that 25～30 ℃, rotating speed are to cultivate 25d in the vibrator of 100r/min with the anaerobism bottle of good seal in culture temperature then, obtain the polycyclic aromatic hydrocarbon anaerobic degradation bacterium mixed solution after the amplification cultivation.
With behind the degradation bacteria mixed solution ultrasonic disruption 15min that obtains under the 8000r/min condition centrifugal 15min, in anaerobic box, isolate thalline and supernatant liquor.Add the deionized water after 30ml uses high pure nitrogen aeration 3.5h in isolated thalline, proceed ultrasonic disruption after the sealing, centrifugal 15min under the 8000r/min condition isolates thalline and supernatant liquor in anaerobic box, repeat 2 times.Merge 3 times collected supernatant liquor, obtain the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon crude extract.
In anaerobic box, in the zymoprotein crude extract, add fine ground (NH
4)
2SO
4Solid to 45% saturation ratio, the sealing back 1～2h that vibrates in rotating speed is the vibrator of 100r/min, centrifugal 15min under the 8000r/min condition isolates precipitation in anaerobic box then.Then precipitation is dissolved in a small amount of pH value and is in 7.0～7.2 the 0.05mol/L Tris-HCl damping fluid and the dialysis tubing of packing into, with the dialysis tubing 24h that in the pH value is 7.0～7.2 0.05mol/L Tris-HCl damping fluid, dialyses.With dialyzed sample centrifugal 15min under the 8000r/min condition, in anaerobic box, isolate supernatant liquor.With on the supernatant liquor to being to filter on 7.0～7.2 the 0.05mol/L Tris-HCl damping fluid equilibrated Q Sepharose Fast Flow anion-exchange column with the pH value, adopting the pH value is the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon of 7.0～7.2 the linear gradient elution of bound of 0.05mol/L Tris-HCl damping fluid (including 0.0～0.30mol/L sodium-chlor), and elution speed is 20ml/h.The utilization volume is that the anaerobism pipe of 10ml is collected elutriant, and wherein every anaerobism pipe is collected elutriant 5ml.Is that elutriant is detected at the 280nm place with ultraviolet spectrophotometer in absorbing wavelength, collects absorbancy greater than 0.01 elutriant.
It is that the solution of 29.4mg/L carries out the anaerobic degradation test to the naphthalene starting point concentration that the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon that is purified into divide is extracted in utilization, and the result shows that the concentration of naphthalene is reduced to 2.2mg/L behind the 24h, and clearance is 92.5%.
Embodiment 2
It is 1 * 10 that the polycyclic aromatic hydrocarbon anaerobic degradation bacterium that preserves in 4 ℃ of refrigerators is mixed with concentration
8The bacteria suspension of individual/ml, inoculation 2ml bacteria suspension in the anaerobism bottle that adds 198ml inducing culture, 2.0ml trace metal liquid and 0.2ml vitamin c solution, wherein inducing culture, trace metal liquid and vitamin solution were used high pure nitrogen aeration 3.5h before adding.Be that 25～30 ℃, rotating speed are to cultivate 25d in the vibrator of 100r/min with the anaerobism bottle of good seal in culture temperature then, obtain the polycyclic aromatic hydrocarbon anaerobic degradation bacterium mixed solution after the amplification cultivation.
With behind the degradation bacteria mixed solution ultrasonic disruption 15min that obtains under the 8000r/min condition centrifugal 15min, in anaerobic box, isolate thalline and supernatant liquor.Add the deionized water after 30ml uses high pure nitrogen aeration 3.5h in isolated thalline, proceed ultrasonic disruption after the sealing, centrifugal 15min under the 8000r/min condition isolates thalline and supernatant liquor in anaerobic box, repeat 2 times.Merge 3 times collected supernatant liquor, obtain the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon crude extract.
In anaerobic box, in the zymoprotein crude extract, add fine ground (NH
4)
2SO
4Solid to 85% saturation ratio, the sealing back 1～2h that vibrates in rotating speed is the vibrator of 100r/min, centrifugal 15min under the 8000r/min condition isolates precipitation in anaerobic box then.Then precipitation is dissolved in a small amount of pH value and is in 7.0～7.2 the 0.05mol/L Tris-HCl damping fluid and the dialysis tubing of packing into, with the dialysis tubing 24h that in the pH value is 7.0～7.2 0.05mol/L Tris-HCl damping fluid, dialyses.With dialyzed sample centrifugal 15min under the 8000r/min condition, in anaerobic box, isolate supernatant liquor.With on the supernatant liquor to being to filter on 7.0～7.2 the 0.05mol/L Tris-HCl damping fluid equilibrated Q Sepharose Fast Flow anion-exchange column with the pH value, adopting the pH value is the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon of 7.0～7.2 the linear gradient elution of bound of 0.05mol/L Tris-HCl damping fluid (including 0.0～0.30mol/L sodium-chlor), and elution speed is 20ml/h.The utilization volume is that the anaerobism pipe of 10ml is collected elutriant, and wherein every anaerobism pipe is collected elutriant 5ml.Is that elutriant is detected at the 280nm place with ultraviolet spectrophotometer in absorbing wavelength, collects absorbancy greater than 0.01 elutriant.
It is that the solution of 28.6mg/L carries out the anaerobic degradation test to the naphthalene starting point concentration that the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon that is purified into divide is extracted in utilization, and the result shows that the concentration of naphthalene is reduced to 1.6mg/L behind the 24h, and clearance is 94.4%.
Embodiment 3
It is 1 * 10 that the polycyclic aromatic hydrocarbon anaerobic degradation bacterium that preserves in 4 ℃ of refrigerators is mixed with concentration
8The bacteria suspension of individual/ml, inoculation 2ml bacteria suspension in the anaerobism bottle that adds 198ml inducing culture, 2.0ml trace metal liquid and 0.2ml vitamin c solution, wherein inducing culture, trace metal liquid and vitamin solution were used high pure nitrogen aeration 3.5h before adding.Be that 25～30 ℃, rotating speed are to cultivate 25d in the vibrator of 100r/min with the anaerobism bottle of good seal in culture temperature then, obtain the polycyclic aromatic hydrocarbon anaerobic degradation bacterium mixed solution after the amplification cultivation.
With behind the degradation bacteria mixed solution ultrasonic disruption 15min that obtains under the 8000r/min condition centrifugal 15min, in anaerobic box, isolate thalline and supernatant liquor.Add the deionized water after 30ml uses high pure nitrogen aeration 3.5h in isolated thalline, proceed ultrasonic disruption after the sealing, centrifugal 15min under the 8000r/min condition isolates thalline and supernatant liquor in anaerobic box, repeat 2 times.Merge 3 times collected supernatant liquor, obtain the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon crude extract.
In anaerobic box, in the zymoprotein crude extract, add fine ground (NH
4)
2SO
4Solid to 65% saturation ratio, the sealing back 1～2h that vibrates in rotating speed is the vibrator of 100r/min, centrifugal 15min under the 8000r/min condition isolates precipitation in anaerobic box then.Then precipitation is dissolved in a small amount of pH value and is in 7.0～7.2 the 0.05mol/L Tris-HCl damping fluid and the dialysis tubing of packing into, with the dialysis tubing 24h that in the pH value is 7.0～7.2 0.05mol/L Tris-HCl damping fluid, dialyses.With dialyzed sample centrifugal 15min under the 8000r/min condition, in anaerobic box, isolate supernatant liquor.With on the supernatant liquor to being to filter on 7.0～7.2 the 0.05mol/L Tris-HCl damping fluid equilibrated Q Sepharose Fast Flow anion-exchange column with the pH value, adopting the pH value is the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon of 7.0～7.2 the linear gradient elution of bound of 0.05mol/L Tris-HCl damping fluid (including 0.0～0.30mol/L sodium-chlor), and elution speed is 20ml/h.The utilization volume is that the anaerobism pipe of 10ml is collected elutriant, and wherein every anaerobism pipe is collected elutriant 5ml.Is that elutriant is detected at the 280nm place with ultraviolet spectrophotometer in absorbing wavelength, collects absorbancy greater than 0.01 elutriant.
It is that the solution of 31.2mg/L carries out the anaerobic degradation test to the naphthalene starting point concentration that the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon that is purified into divide is extracted in utilization, and the result shows that the concentration of naphthalene is reduced to 0.6mg/L behind the 24h, and clearance is 98.1%.
Claims (5)
1. the extraction and purification method of a zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon is characterized in that, the concrete steps of this method are as follows:
1. the polycyclic aromatic hydrocarbon anaerobic degradation bacterium that preserves in 4 ℃ of refrigerators being mixed with concentration is 1 * 10
8The bacteria suspension of individual/ml, inoculation 2ml bacteria suspension in the anaerobism bottle that adds 198ml inducing culture, 2.0ml trace metal liquid and 0.2ml vitamin c solution, for removing the oxygen in the substratum, inducing culture, trace metal liquid and vitamin solution were used high pure nitrogen aeration 3.5h before adding.Tighten then with Viton plug sealing bottleneck, and with screw top closure.With the polycyclic aromatic hydrocarbon anaerobic degradation bacterium mixed solution of anaerobism bottle after culture temperature is can obtain amplification cultivation after 25～30 ℃, rotating speed are to cultivate 25d in the vibrator of 100r/min.
2. the polycyclic aromatic hydrocarbon anaerobic degradation bacterium mixed solution of step in 1. carried out ultrasonic disruption 15min.Degradation bacteria mixed solution after the fragmentation centrifugal 15min under the 8000r/min condition isolates thalline and supernatant liquor in anaerobic box.
3. in anaerobic box, in 2. isolated thalline, add the deionized water after 30ml uses high pure nitrogen aeration 3.5h by step, proceed ultrasonic disruption after tightening with Viton plug sealing bottleneck and with screw top closure, centrifugal 15min under the 8000r/min condition isolates thalline and supernatant liquor in anaerobic box.
4. in anaerobic box, in 3. isolated thalline, add the deionized water after 30ml uses high pure nitrogen aeration 3.5h by step, proceed ultrasonic disruption after tightening with Viton plug sealing bottleneck and with screw top closure, centrifugal 15min under the 8000r/min condition isolates thalline and supernatant liquor in anaerobic box.
5. in anaerobic box, merge, can obtain the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon crude extract by 2., the 3. and 4. collected supernatant liquor of step.
6. in anaerobic box, in the zymoprotein crude extract that 5. obtains by step, add fine ground (NH
4)
2SO
4Solid to 45～85% saturation ratio is tightened the back 1～2h that vibrates with Viton plug sealing bottleneck and with screw top closure in rotating speed is the vibrator of 100r/min, centrifugal 15min under the 8000r/min condition isolates precipitation in anaerobic box then.
7. will be dissolved in a small amount of pH value by the precipitation that 6. step obtains is in 7.0～7.2 0.05mol/L trihydroxy methyl aminomethane (the Tris)-HCl damping fluid and the dialysis tubing of packing into, with the dialysis tubing 24h that dialyses in the pH value is 7.0～7.2 0.05mol/L Tris-HCl damping fluid.With dialyzed sample centrifugal 15min under the 8000r/min condition, in anaerobic box, isolate supernatant liquor.
8. on the supernatant liquor that in anaerobic box, will 7. obtain by step to being to filter on 7.0～7.2 the 0.05mol/L Tris-HCl damping fluid equilibrated Q Sepharose Fast Flow anion-exchange column with the pH value, adopting the pH value is the zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon of 7.0～7.2 the linear gradient elution of bound of 0.05mol/LTris-HCl damping fluid (including 0.0～0.30mol/L sodium-chlor), and elution speed is 20ml/h.The utilization volume is that the anaerobism pipe of 10ml is collected elutriant, and wherein every anaerobism pipe is collected elutriant 5ml.Is that elutriant is detected at the 280nm place with ultraviolet spectrophotometer in absorbing wavelength, collects absorbancy greater than 0.01 elutriant.Measure the activity of zymoprotein, select active part standby.
2. according to the extraction and purification method of the described a kind of zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon of claim 1, it is characterized in that step is consisting of of middle inducing culture 1.: NaNO
3: 2.0g/L, NH
4Cl:1.0gL
-1, KH
2PO
4: 1.0gL
-1, MgCl
2: 0.1gL
-1, CaCl
22H
2O:0.05gL
-1, PAHs:0.03gL
-1
3. according to the extraction and purification method of the described a kind of zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon of claim 1, it is characterized in that step is consisting of of middle trace metal liquid 1.: CoCl
26H
2O:35mgL
-1, CuCl
2: 0.25mgL
-1, H
3BO
3: 6.0mgL
-1, MnCl
24H
2O:30mgL
-1, Na
2MoO
42H
2O:3.0mgL
-1, NiCl
22H
2O:2.5mgL
-1, ZnCl
2: 2.5mgL
-1
4. according to the extraction and purification method of the described a kind of zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon of claim 1, it is characterized in that the step 2. condition of middle ultrasonic disruption is: electric current 35%, pulse 9.0s, treatment time 15min.
5. according to the extraction and purification method of the described a kind of zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon of claim 1, it is characterized in that the 6. middle (NH that adds of step
4)
2SO
4The solid amount is preferably to 60～70% saturation ratios.
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|---|---|---|---|
| CNA2008100072254A CN101514224A (en) | 2008-02-20 | 2008-02-20 | Method for extracting and purifying zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon |
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|---|---|---|---|
| CNA2008100072254A CN101514224A (en) | 2008-02-20 | 2008-02-20 | Method for extracting and purifying zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon |
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ID=41038854
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533682A (en) * | 2011-12-23 | 2012-07-04 | 北京师范大学 | Enzyme capable of catalyzing and degrading indeno[1, 2, 3-cd]pyrene |
| CN103232999A (en) * | 2013-04-26 | 2013-08-07 | 北京师范大学 | Degradative plasmid containing functional gene capable of degradating benz(a)anthracene |
| CN104673763A (en) * | 2015-03-05 | 2015-06-03 | 北京师范大学 | Functional protein capable of degrading chrysene |
| CN108042968A (en) * | 2017-11-23 | 2018-05-18 | 浙江海洋大学 | A kind of preparation method of reagent for polycyclic aromatic hydrocarbon selective degradation |
| CN110294801A (en) * | 2019-06-24 | 2019-10-01 | 中国水产科学研究院 | A method of extracting two kinds of metallothioneins from blood clam |
-
2008
- 2008-02-20 CN CNA2008100072254A patent/CN101514224A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533682A (en) * | 2011-12-23 | 2012-07-04 | 北京师范大学 | Enzyme capable of catalyzing and degrading indeno[1, 2, 3-cd]pyrene |
| CN103232999A (en) * | 2013-04-26 | 2013-08-07 | 北京师范大学 | Degradative plasmid containing functional gene capable of degradating benz(a)anthracene |
| CN103232999B (en) * | 2013-04-26 | 2015-02-18 | 北京师范大学 | Degradative plasmid containing functional gene capable of degradating benz(a)anthracene |
| CN104673763A (en) * | 2015-03-05 | 2015-06-03 | 北京师范大学 | Functional protein capable of degrading chrysene |
| CN104673763B (en) * | 2015-03-05 | 2018-10-26 | 北京师范大学 | A kind of functional protein for the * that can degrade |
| CN108042968A (en) * | 2017-11-23 | 2018-05-18 | 浙江海洋大学 | A kind of preparation method of reagent for polycyclic aromatic hydrocarbon selective degradation |
| CN110294801A (en) * | 2019-06-24 | 2019-10-01 | 中国水产科学研究院 | A method of extracting two kinds of metallothioneins from blood clam |
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