CN102533682A - Enzyme capable of catalyzing and degrading indeno[1, 2, 3-cd]pyrene - Google Patents
Enzyme capable of catalyzing and degrading indeno[1, 2, 3-cd]pyrene Download PDFInfo
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- CN102533682A CN102533682A CN2011104358977A CN201110435897A CN102533682A CN 102533682 A CN102533682 A CN 102533682A CN 2011104358977 A CN2011104358977 A CN 2011104358977A CN 201110435897 A CN201110435897 A CN 201110435897A CN 102533682 A CN102533682 A CN 102533682A
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Abstract
The invention provides enzyme capable of catalyzing and degrading indeno[1, 2, 3-cd]pyrene, which is obtained from bacterium liquid of pandoraea sp. by extracting and purifying. The culture preservation number of the pandoraea sp. is CGMCC NO. 3615. When the enzyme catalyzes and degrades the indeno[1, 2, 3-cd]pyrene, the appropriate potential of hydrogen (pH) is 6.0 to 8.0, and the most appropriate pH is 7.0. When the pH is smaller than 4 or larger than 10, the enzyme is easy to cause degeneration and inactivation, appropriate temperature is 30 DEG C to 45 DEG C, and the most appropriate temperature is 35 DEG C. When temperature is higher than 50 DEG C, inactivation and denaturation are prone to happen. Cu (2+), Zn (2+) and Hg (2+) with the concentration of 1mmol/L has strong restrain on activity of the enzyme. The enzyme can effectively catalyze and degrade the indeno[1, 2, 3-cd]pyrene and has potentials to be applied to pollution of the indeno[1, 2, 3-cd]pyrene on soil and water.
Description
Technical field
The invention belongs to biotechnology and Persistent organic pollutants biologic treating technique field, particularly a kind of enzyme that can catalyzed degradation indeno [1,2,3-cd] pyrene.
Background technology
(Polycyclic Aromatic Hydrocarbons is one type of toxic organic pollutant that contains two or more phenyl ring PAHs) to polycyclic aromatic hydrocarbons, has intensive carcinogenesis, teratogenesis and mutagenesis.PAHs can cause very big harm to ecotope and HUMAN HEALTH through the transfer function of biological accumulation and food chain, has caused various countries environmentalist's great attention.EPA just confirms as the priority pollutant in the environment to 16 kinds with ramose PAHs as far back as the eighties, and China is also listed PAHs in the Black List of environmental pollution in.
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 the polycyclic aromatic hydrocarbons in the wash-out environment such as organic solvent (like 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.In general, along with the increase of polycyclic aromatic hydrocarbons phenyl ring quantity, its degradation rate reduces.Therefore, low-molecular-weight polycyclic aromatic hydrocarbons can comparatively fast be degraded in environment, and the time that in environment, exists is shorter, and the high-molecular weight polycyclic aromatic hydrocarbons, for example benzo [a] pyrene, indeno [1,2,3-cd] pyrene etc. then are difficult to degraded, and longer-term is present in the environment.From the degraded microorganism cells, extracting degrading enzyme comes enhancing degradation to become the polycyclic aromatic hydrocarbons contaminated a kind of important method of removal.Degrading enzyme has the degradation efficiency height in the application of degrading polycyclic aromatic hydrocarbons, the effect concentration of substrate hangs down and reaches advantages such as environmental compatibility is strong, has broad application prospects.But,, lack the research report of high ring polycyclic aromatic hydrocarbon degradation enzyme aspect both at home and abroad because the degrading polycyclic aromatic hydrocarbons mikrobe is lacked deep research.
Summary of the invention
The present invention provides a kind of enzyme that can catalyzed degradation indeno [1,2,3-cd] pyrene.Said enzyme that can catalyzed degradation indeno [1,2,3-cd] pyrene is obtained through extraction and purification by the bacterium liquid of Pandora bacterium Pandoraea sp..This enzyme is when carrying out catalyzed degradation indeno [1,2,3-cd] pyrene; The appropriate pH value is 6.0～8.0, and optimum pH value is 7.0, when the pH value less than 4 and easy inactivation sex change greater than 10 time; Suitable temperature of reaction is 30～45 ℃; Most suitable reaction temperature is 35 ℃, is easy to the inactivation sex change when temperature is higher than 50 ℃, and concentration is the Cu of 1mmol/L
2+, Zn
2+, Hg
2+The activity of ions enzyme has strong restraining effect.
Separation can catalyzed degradation indeno [1; 2; 3-cd] the used bacterial strain Pandora bacterium Pandoraea sp. of enzyme of pyrene was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 01 28th, 2010; This is centered close to Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and culture presevation number is CGMCC NO.3615.From Pandora bacterium Pandoraea sp., separate can catalyzed degradation indeno [1,2,3-cd] pyrene the concrete scheme of enzyme be:
1. in the culturing bottle that adds 198ml inducing culture, 2.0ml trace metal liquid and 0.2ml vitamin c solution, inoculate the bacterial strain Pandora bacterium Pandoraea sp. that is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; Seal bottleneck with air-permeable envelope then, with the bacterium liquid of culturing bottle after culture temperature is can obtain amplification cultivation after 25～28 ℃, rotating speed are to cultivate 40d in the vibrator of 100r/min;
2. the bacterium liquid of step in is 1. carried out ultrasonic disruption 15min; Bacterium liquid after the fragmentation centrifugal 15min under the 8000r/min condition isolates thalline and supernatant;
3. in the 2. isolated thalline by step, add the 30ml deionized water, proceed ultrasonic disruption 15min, centrifugal 15min under the 8000r/min condition isolates thalline and supernatant;
4. in the 3. isolated thalline by step, add the 30ml deionized water, proceed ultrasonic disruption 15min, centrifugal 15min under the 8000r/min condition isolates thalline and supernatant;
5. merge by 2., the 3. and 4. collected supernatant of step, can obtain enzyme extract;
6. in the enzyme extract that 5. obtains by step, add fine ground (NH
4)
2SO
4Solid to 65～70% saturation ratio, the 1～2h that in rotating speed is the vibrator of 100r/min, vibrates, centrifugal 15min under the 8000r/min condition isolates deposition then;
7. will be dissolved in a small amount of pH value by the deposition that 6. step obtains is in 7.0～7.2 the 0.05mol/L trihydroxy methyl aminomethane-HCl damping fluid and the dialysis tubing of packing into, with the dialysis tubing 24h that in the pH value is 0.05mol/L trihydroxy methyl aminomethane-HCl damping fluid of 7.0～7.2, dialyses; With dialyzed sample centrifugal 15min under the 8000r/min condition, isolate supernatant;
Be to filter on 0.05mol/L trihydroxy methyl aminomethane-HCl damping fluid equilibrated Q Sepharose Fast Flow anion-exchange column of 7.0～7.2 to using the pH value on the supernatant that 8. will 7. obtain by step; Adopting the pH value is 7.0～7.2 to include the enzyme of the 0.05mol/L trihydroxy methyl aminomethane-linear gradient elution of bound of HCl damping fluid of 0.25mol/L sodium-chlor, and elution speed is 20ml/h; The utilization volume is that the test tube of 10ml is collected elutriant, and wherein every test tube is collected elutriant 5ml; Use ultraviolet spectrophotometer to be that in absorbing wavelength the 280nm place detects elutriant, collect absorbancy greater than 0.01 elutriant; Measure the activity of enzyme, select active part subsequent use.
Said step is consisting of of middle inducing culture 1.: NaNO
3: 2.5g/L, NH
4Cl:1.5gL
-1, KH
2PO
4: 1.5gL
-1, MgCl
2: 0.2gL
-1, CaCl
22H
2O:0.15gL
-1, indeno [1,2,3-cd] pyrene: 0.01gL
-1
Said 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
Said step 2., the 3. and 4. condition of middle ultrasonic disruption is: electric current 35%, pulse 9.0s, treatment time 15min.
The enzyme that the present invention obtains is catalyzed degradation indeno [1,2,3-cd] pyrene effectively, for polycyclic aromatic hydrocarbons indeno [1,2,3-cd] pyrene contaminated soil and water body application potential is arranged.
Embodiment
Further specify the present invention below in conjunction with instance.
Material: (1) is preserved in the bacterial strain Pandora bacterium Pandoraea sp at China Committee for Culture Collection of Microorganisms common micro-organisms center, and culture presevation number is respectively CGMCC NO.3615;
(2) indeno [1,2,3-cd] pyrene (purity >=99%, Sigma Aldrich);
(3) NaCl, (NH
4)
2SO
4: analytical pure;
(4) the pH value is 0.05mol/L trihydroxy methyl aminomethane-HCl damping fluid of 7.0～7.2, and the pH value is 3.0,4.0,5.0,6.0,6.5,7.0,7.5,8.0,9.0,10.0 phosphoric acid buffer;
(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, indeno [1,2,3-cd] pyrene: 0.01gL
-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 detection method of enzymic activity: the activity of confirming enzyme with indeno [1,2,3-cd] the pyrene amount of enzyme and indeno [1,2,3-cd] pyrene reaction consumes.Get 0.2mL enzyme and 4.8mL and contain indeno [1; 2,3-cd] pyrene concentration is that 0.2 μ g/L pH value is that 7.5 phosphoric acid buffer mixes, and is to react 24h under 35 ℃ of conditions in temperature; Adding 0.3mL concentration is the HCl termination enzyme reaction of 2.0mol/L; Measure the variation that degraded finishes back indeno [1,2,3-cd] pyrene concentration.
Embodiment
Inoculation is preserved in the bacterial strain Pandora bacterium Pandoraea sp. at China Committee for Culture Collection of Microorganisms common micro-organisms center in the culturing bottle that adds 198ml inducing culture, 2.0ml trace metal liquid and 0.2ml vitamin c solution; Be that 25～28 ℃, rotating speed are to cultivate 40d in the vibrator of 100r/min with culturing bottle in culture temperature then, obtain the bacterium liquid after the amplification cultivation.
With behind the bacterium liquid ultrasonic disruption 15min that obtains under the 8000r/min condition centrifugal 15min, isolate thalline and supernatant.In isolated thalline, add the 30ml deionized water, proceed ultrasonic disruption 15min, centrifugal 15min under the 8000r/min condition isolates thalline and supernatant, repeats 2 times.Merge 3 times collected supernatant, obtain enzyme extract.
In enzyme extract, add fine ground (NH
4)
2SO
4Solid to 65% saturation ratio, the 1～2h that in rotating speed is the vibrator of 100r/min, vibrates, centrifugal 15min under the 8000r/min condition isolates deposition then.Then deposition being dissolved in a small amount of pH value is in 7.0～7.2 the 0.05mol/L trihydroxy methyl aminomethane-HCl damping fluid and the dialysis tubing of packing into, with the dialysis tubing 24h that in the pH value is 0.05mol/L trihydroxy methyl aminomethane-HCl damping fluid of 7.0～7.2, dialyses.With dialyzed sample centrifugal 15min under the 8000r/min condition, isolate supernatant.With being to filter on 0.05mol/L trihydroxy methyl aminomethane-HCl damping fluid equilibrated Q Sepharose Fast Flow anion-exchange column of 7.0～7.2 to using the pH value on the supernatant; Adopting the pH value is 7.0～7.2 to include the enzyme of the 0.05mol/L trihydroxy methyl aminomethane-linear gradient elution of bound of HCl damping fluid of 0.25mol/L sodium-chlor, and elution speed is 20ml/h.The utilization volume is that the test tube of 10ml is collected elutriant, and wherein every test tube is collected elutriant 5ml.Use ultraviolet spectrophotometer to be that in absorbing wavelength the 280nm place detects elutriant, collect absorbancy greater than 0.01 elutriant.
Use respectively the pH value be 3.0,4.0,5.0,6.0,6.5,7.0,7.5,8.0,9.0,10.0 contain indeno [1; 2; 3-cd] pyrene concentration is that the enzyme of 0.2 μ g/L phosphoric acid buffer and 0.2mL is made into the reaction system that TV is 5.0mL; Reaction is 24 hours under 30 ℃ of conditions, and adding 0.3mL concentration is the HCl termination enzyme reaction of 2.0mol/L.Set not enzyme-added control test simultaneously respectively, the peak of getting degradation rate is each relative enzyme activity of handling of 100 calculating, to confirm the optimum pH value of enzyme reaction.The result shows that enzyme is to indeno [1,2 between 3.0～10.0 in the pH value; 3-cd] pyrene all has degraded in various degree, and its suitable pH value is 6.0～8.0, and optimum pH value is 7.0; When the pH value less than 4 and easy inactivation sex change greater than 10 time; 24 hours degradation rates to indeno [1,2,3-cd] pyrene of reaction are 82.9% under optimum pH value condition.
In the pH value is under 7.0 conditions; To contain indeno [1; 2; 3-cd] pyrene concentration is that the enzyme of 0.2 μ g/L phosphoric acid buffer and 0.2mL is made into the reaction system that volume is 5.0mL, reaction 24 hours under 10 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ conditions respectively, the HCl that adds 0.3mL concentration then and be 2.0mol/L stops enzyme reaction.Set not enzyme-added control test simultaneously respectively, the peak of getting degradation rate is each relative enzyme activity of handling of 100 calculating, to confirm the optimal temperature of enzyme reaction.The result shows that enzyme is to indeno [1,2 between 10～50 ℃ in temperature; 3-cd] pyrene all has degraded in various degree, and its suitable temperature is 30～45 ℃, and optimal temperature is 35 ℃; When being higher than 50 ℃, temperature is easy to the inactivation sex change; 24 hours degradation rates to indeno [1,2,3-cd] pyrene of reaction are 88.7% under the optimal temperature condition.
Be that 7.0 to contain indeno [1,2,3-cd] pyrene concentration be to add Cu in the 0.2 μ g/L phosphoric acid buffer to 8 parts of pH values respectively
2+, Zn
2+, Fe
2+, Pb
2+, Co
2+, Ni
2+, Mg
2+, Hg
2+, making ionic concentration is 1mmol/L, and the enzyme that adds 0.2mL then is made into the reaction system that TV is 5.0mL, and reaction is 24 hours under 30 ℃ of conditions, and adding 0.3mL concentration is the HCl termination enzyme reaction of 2.0mol/L.Set not enzyme-added and the control test that does not add metals ion simultaneously respectively, the peak of getting degradation rate is 100 to calculate relative enzyme activities that each is handled, with definite active influence of each ions enzyme.The result shows that concentration is the Cu of 1mmol/L
2+, Zn
2+, Hg
2+The ions enzyme activity has stronger restraining effect.
Claims (2)
- One kind can catalyzed degradation indeno [1,2,3-cd] pyrene enzyme, it is characterized in that this enzyme is obtained through extraction and purification by the bacterium liquid of Pandora bacterium Pandoraea sp., the culture presevation of Pandora bacterium Pandoraea sp. number is CGMCC NO.3615.
- 2. according to the said enzyme that can catalyzed degradation indeno [1,2,3-cd] pyrene of claim 1, it is characterized in that; This enzyme is falling catalysis when separating indeno [1,2,3-cd] pyrene, and the appropriate pH value is 6.0～8.0; Optimum pH value is 7.0, when the pH value less than 4 and easy inactivation sex change greater than 10 time, suitable temperature of reaction is 30～45 ℃; Most suitable reaction temperature is 35 ℃, is easy to the inactivation sex change when temperature is higher than 50 ℃, and concentration is the Cu of 1mmol/L 2+, Zn 2+, Hg 2+The pair ion degrading enzymatic activity has stronger restraining effect.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421774A (en) * | 2013-04-26 | 2013-12-04 | 北京师范大学 | Degradative plasmid containing indene [1,2,3-cd] pyrene degradation gene |
| CN103756928A (en) * | 2013-11-26 | 2014-04-30 | 浙江大学 | Bacterial strain for degradation of p-xylene and culture method and application thereof |
| CN104673763A (en) * | 2015-03-05 | 2015-06-03 | 北京师范大学 | Functional protein capable of degrading chrysene |
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2011
- 2011-12-23 CN CN2011104358977A patent/CN102533682A/en active Pending
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421774A (en) * | 2013-04-26 | 2013-12-04 | 北京师范大学 | Degradative plasmid containing indene [1,2,3-cd] pyrene degradation gene |
| CN103756928A (en) * | 2013-11-26 | 2014-04-30 | 浙江大学 | Bacterial strain for degradation of p-xylene and culture method and application thereof |
| 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 |
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Application publication date: 20120704 |