CN101845420A - Method of extracting crude enzyme preparation for degrading polycyclic aromatic hydrocarbons - Google Patents
Method of extracting crude enzyme preparation for degrading polycyclic aromatic hydrocarbons Download PDFInfo
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- CN101845420A CN101845420A CN 201010138364 CN201010138364A CN101845420A CN 101845420 A CN101845420 A CN 101845420A CN 201010138364 CN201010138364 CN 201010138364 CN 201010138364 A CN201010138364 A CN 201010138364A CN 101845420 A CN101845420 A CN 101845420A
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Abstract
The invention discloses a method of extracting a crude enzyme preparation for degrading polycyclic aromatic hydrocarbons, which comprises the following steps of: adding acidic buffer with pH of between 4 and 6 into fresh spent mushroom substrates; oscillating the mixed solution for 2 hours at the temperature of between 20 and 40 DEG C and at the rotating speed of between 100 and 200 rpm; and centrifuging the oscillated product at a high speed for 10 minutes with a centrifugal force of 11, 000 gravity to obtain supernatant which is the crude enzyme liquid. The invention provides a polycyclic aromatic hydrocarbon removing method which has good and quick effects, low cost and a great application value in the field of environmental remediation. Meanwhile, laccase contained in the crude enzyme preparation has various types of degradant substrates, so the method can be applied in the remediation of pollution caused by polycyclic aromatic hydrocarbon pollutants and the remediation of the pollution caused by other organic pollutants such as polychlorinated biphenyl and chlorophenol.
Description
One, technical field
The invention belongs to the environment remediation technical field, particularly a kind of extracting method that is used to remove polycyclic aromatic hydrocarbons contaminated zymin.
Two, background technology
Persistence organic pollutant (Persistent organic pollutants, POPs) be a class has toxicity, biological accumulation and refractory organics and the natural or synthetic that can exist lastingly in environment organic pollution materials (Lohmann R, Breivik K, Dachs J and Muir D.Global fate of POPs:Current and future research directions.EnvironmentalPollution.2007 (150): 150-165).Increase along with mankind's activity, POPs inevitably is dispersed to atmosphere, soil, in water and the settling, constitute risk (El-Shahawi MS to ecotope, Hamza A, Bashammakh AS andAl-Saggaf WT.An overview on the accumulation, distribution, transformations, toxicity andanalytical methods for the monitoring of persistent organic pollutants.Talanta.2010 (80): 1587-1597).(Polycyclic aromatic hydrocarbons PAHs) is a kind of typical POPs to polycyclic aromatic hydrocarbons, is condensed by 2 or 2 above phenyl ring to form.Along with the increase of number of rings, the toxicity of PAHs increases, and biological effectiveness reduces (Cerniglia CE.Biodegradation of polycyclic aromatic hydrocarbons.Biodegradation.1992:(3) 351-368).Up to the present, microorganism is repaired a kind of more satisfactory environment remediation technology that is regarded as.But, microorganism repair to the removal effect of epipodium PAHs such as benzo in the environment [a] pyrene not good (Juhasz A L and Naidu R.Bioremediation ofhigh molecular weight polycyclic aromatic hydrocarbons:a review of the microbial degradation ofbenzo[a] pyrene.International Biodeterioration and Biodegradation.2000 (45): 57-88).
Lignin-degrading enzymes is that a class can and have the general name of the enzyme of lignin degradation function by the white-rot fungi exocytosis, mainly comprise lignin peroxidase (Lignin peroxidase, LiP), manganese peroxidase (Manganese peroxidase, MnP) and laccase (Laccase) (Pointing SB, Feasibility of bioremediation by white-rot fungi.Appl MicrobiolBiotechnol.2001 (57): 20-33).Since Bumpus in 1985 utilizes the lignoenzyme of white-rot fungi to be tied to form a series of POPs (lindanes of degraded of merit, dichlorodiphenyl trichloroethane, benzo [a] pyrene etc.) (Bumpus JA since, Tien M, Wright D, and Aust SD.Oxidation of persistent environmental pollutants by a white rot fungus.Science.1985 (228): 1434-1436), utilize zymin to come degradation of contaminant extensively to be subjected to people and pay close attention to.Compare with traditional repairing method of microorganism, that the enzyme reparation has is efficient, easy, the degraded substrate is extensively with to advantage (Dur á n N and Esposito E such as envrionment conditions are less demanding, Potential applications of oxidative enzymes and phenoloxidase-like compounds in wastewater andsoil treatment:a review, Applied Catalysis B:Environmental.2000 (28): 83-99).Laccase is a kind of copper bearing polyphenoloxidase, can the oxidation polycyclic aromatic hydrocarbons, multiple POPs such as polychlorobiphenyl, chlorophenol, therefore the environment remediation function of laccase is subjected to people day by day and payes attention to (Dur á n N and Esposito E, Potential applications of oxidative enzymes andphenoloxidase-like compounds in wastewater and soil treatment:a review, Applied Catalysis B:Environmental.2000 (28): 83-99).Laccase has also reduced O2 and has generated H2O in conversion of substrate, therefore also have eco-friendly characteristics (Riva S.Laccases:blue enzymes for green chemistry, TRENDS in Biotechnology.2006 (24): 219-226).But,, thereby limited the large-scale application of laccase in the environment remediation field because the cost of production of laccase at present and purifying is very high.
(Spent mushroom substrate SMS) claims the bacterium chaff again to the mushroom slag, is meant the culture material after edible mushrooms is gathered in the crops.China planting edible mushroom industry mushroom slag output in 2010 will reach 3,600,000 tons (Wang Dehan, a money is refined, Chen Guangyin. the ecological higher value application progress of mushroom residue resource, nonferrous metallurgy design and research .2007 (27): 262-266).Studies show that, many edible mushroomss such as flat mushroom, Coprinus comatus etc. can secretion laccase etc. oxydase (Ko HG, Park SH, Kim SH, Park HG and Park WM.Detection and recovery of hydrolytic enzymes from spent compost of four mushroom species.FoliaMicrobiol.2005 (50): 103-106), after the sporophore of edible mushrooms was gathered in the crops, these lignin-degrading enzymes still remained in the mushroom slag in a large number.All the time, most of mushroom slag is arbitrarily abandoned, and brings certain risk to ecotope.
From above data as can be seen, the too high large-scale application of laccase that limited of one side cost in the environment remediation field; Mushroom industry has all been made and a large amount of are rich in the mushroom slag of degrading enzyme such as laccase and arbitrarily abandon every year on the other hand, causes risk to ecotope, therefore, inquires into and how to extract oxydase to be used for environment remediation from the mushroom slag, has higher using value.
Three, summary of the invention
Goal of the invention: this patent is at the too high problem of laccase cost in environment remediation, proposed a kind of oxidasic method such as laccase of extracting in the mushroom slag, and this crude zyme preparation can be used for the degraded of polycyclic aromatic hydrocarbons efficiently.This method is simple to operate, and cost is lower, and the remediation efficiency height has been realized the utilization again of waste resource simultaneously, therefore has environment protection and changing waste into resources double meaning.
Technical scheme: a kind of extracting method that is used for the degrading polycyclic aromatic hydrocarbons crude zyme preparation, extraction step is: adding pH is 4～6 acidic buffer in the fresh mushroom slag, at 20～40 ℃, 2h vibrates under the condition of rotating speed 100～200rpm, 11,000g high speed centrifugation 10min, the gained supernatant liquor is crude enzyme liquid.
The fresh mushroom slag is bisporous mushroom, Pleurotus eryngii, flat mushroom or drumstick mushroom slag.
Described acidic buffer is acetic acid, phosphoric acid or citric acid.
Described acidic buffer is pH 5, the 50mM acetate buffer solution.
Described extraction conditions is 25 ℃, vibration rotating speed 150rpm.
Beneficial effect: the crude zyme preparation that the present invention the extracted multiple polycyclic aromatic hydrocarbonss such as acenaphthene, fluorenes, anthracene, benzo [a] anthracene and benzo [a] pyrene of to degrade efficiently.The crude zyme preparation that is extracted from Pleurotus eryngii mushroom slag even can within 48 hours, can remove 1 μ gl fully
-1Anthracene, to 1 μ gl
-1The clearance of benzo [a] pyrene also reach about 90%, effect is very remarkable.Simultaneously and since this zymin be from planting edible mushroom industry waste---the mushroom slag extracts and obtains, thereby greatly reduce rehabilitation cost.Therefore, the present invention is a kind of effective, instant effect, the method that the low polycyclic aromatic hydrocarbons of cost is removed, in the environment remediation field to having very big using value.Simultaneously, owing to the degraded substrate kind of the contained laccase of crude zyme preparation is a lot, therefore, the present invention not only can be used for polycyclic aromatic hydrocarbons contaminated reparation, also can be used for other organic pollutant such as polychlorobiphenyl, the reparation that chlorophenol etc. pollute.
Fig. 1 is the crude enzyme liquid that the extracts degradation rate synoptic diagram to degrading polycyclic aromatic hydrocarbons from bisporous mushroom, Pleurotus eryngii, flat mushroom and Coprinus comatus.
Four, embodiment
Embodiment 1: the extraction of crude enzyme liquid
1.1 for examination mushroom slag
Bisporous mushroom, this laboratory culture of Pleurotus eryngii mushroom slag system, flat mushroom, drumstick mushroom slag specimen product are purchased in the academy of agricultural sciences, Henan.Sample obtains the back and preserves stand-by under 4 ℃ of conditions.
1.2 the extraction of crude enzyme liquid
Extraction step is: adding pH is 4～6 acidic buffer in bisporous mushroom, Pleurotus eryngii, flat mushroom or drumstick mushroom slag, described acidic buffer is acetic acid, phosphoric acid or citric acid (preferred pH 5, the 50mM acetate buffer solution), at 20～40 ℃, 2h vibrates under the condition of rotating speed 100～200rpm, 11,000g high speed centrifugation 10min, the gained supernatant liquor is crude enzyme liquid.
Or, get fresh mushroom slag 50g and place the 500mL triangular flask, add 250mL sodium-acetate (50mM, pH 5) damping fluid, vibration 2h (150rpm, 25 ℃), 11,000g high speed centrifugation (10min), supernatant liquor is crude enzyme liquid.
Embodiment 2: crude enzyme liquid is to the degraded kind of 15 kinds of PAHs
2.1 test method
The crude enzyme liquid 4.5mL that gets step 1.2 acquisition places the brown reagent bottle of 15mL tool plug, adding 0.5mL contains the acetonitrile solution (the PAHs starting point concentration sees Table 1 in the reaction system) of 15 kinds of PAHs, tightens bottle stopper, shakes up, place the darkroom to cultivate 48h (25 ℃), add 5mL acetonitrile termination reaction.Shake up, leave standstill 1h (25 ℃, 150rpm), high speed centrifugation (11,000g), cross 0.22 μ m filter membrane, high performance liquid phase is measured.Contrast adds with deactivation (boiling 30min) form for crude enzyme liquid, and each handles 3 repetitions.
2.2 sample analysis
Adopt Shimadzu UFLC-20 ultra-high voltage liquid phase analysis sample.Analytical column is that PAH analyzes special-purpose macropore alkyl C18 post (3 * 150mm, internal diameter 2.2 μ m); Detector PF-10AXL fluorimetric detector.Working conditions: moving phase is acetonitrile/water (70: 30), gradient elution, flow velocity 0.8mL/min, 50 ℃ of column temperatures.
2.3 data statistics
Adopt the One-way ANOVA check of SPSS11.0 software to carry out data processing.
Embodiment 3: crude enzyme liquid is to the degraded of the anthracene and benzo [a] pyrene of high density
3.1 test method
The crude enzyme liquid 4.5mL that gets step 1.2 acquisition places the brown reagent bottle of 15mL tool plug, adds the acetonitrile solution that 0.5mL contains anthracene and benzo [a] pyrene, makes anthracene and benzo [a] pyrene in the reaction system reach 1000 μ gl
-1, other step is with 2.1.
3.2 sample analysis
With 2.2.
3.3 data statistics
With 2.3.
Table 1 is Chinese, number of rings, solubleness and the starting point concentration in reaction system of 15 kinds of polycyclic aromatic hydrocarbonss to be measured.
The starting point concentration of 15 kinds of polycyclic aromatic hydrocarbonss of table 1
Table 2 is to extract the crude enzyme liquid the obtain degradation rate to 15 kinds of PAHs.Numerical value is in the table: mean+SD has the representative of same letter not have significant difference (p＞0.05) with delegation's acceptance of the bid.As can be seen from the table, 4 kinds of crude enzyme liquids all have in various degree degradation capability to polycyclic aromatic hydrocarbons, wherein almost can degrade fully anthracene and benzo [a] pyrene of the crude enzyme liquid of bisporous mushroom, Pleurotus eryngii and flat mushroom mushroom slag; Acenaphthene, fluorenes and benzo [a] anthracene also there is stronger degradation capability.Reaction is under 25 ℃ of conditions, cultivates to carry out in 24h hour, and crude enzyme liquid is described, and not only efficient is very high to the degraded of PAHs, and very rapid.Take all factors into consideration, the mushroom slag crude enzyme liquid of bisporous mushroom, Pleurotus eryngii and flat mushroom all can be used as and is used for the zymin that polycyclic aromatic hydrocarbons is removed.
Table 2 crude enzyme liquid is to the degradation rate of 15 kinds of polycyclic aromatic hydrocarbonss
Claims (5)
1. extracting method that is used for the degrading polycyclic aromatic hydrocarbons crude zyme preparation, it is characterized in that extraction step is: adding pH is 4～6 acidic buffer in the fresh mushroom slag, at 20～40 ℃, 2h vibrates under the condition of rotating speed 100～200rpm, 11,000g high speed centrifugation 10min, the gained supernatant liquor is crude enzyme liquid.
2. the extracting method that is used for the crude zyme preparation of degrading polycyclic aromatic hydrocarbons according to claim 1 is characterized in that the fresh mushroom slag is bisporous mushroom, Pleurotus eryngii, flat mushroom or drumstick mushroom slag.
3. the extracting method that is used for the crude zyme preparation of degrading polycyclic aromatic hydrocarbons according to claim 1 is characterized in that described acidic buffer is acetic acid, phosphoric acid or citric acid.
4. the extracting method that is used for the crude zyme preparation of degrading polycyclic aromatic hydrocarbons according to claim 1 is characterized in that described acidic buffer is pH 5, the 50mM acetate buffer solution.
5. the extracting method that is used for the crude zyme preparation of degrading polycyclic aromatic hydrocarbons according to claim 1 is characterized in that described extraction conditions is 25 ℃, vibration rotating speed 150rpm.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710363A (en) * | 2013-12-16 | 2014-04-09 | 南京林业大学 | Laccase gene Lac6 and expression protein and application thereof |
| CN103710364A (en) * | 2013-12-16 | 2014-04-09 | 南京林业大学 | Laccase gene Lac7 and expression protein and application thereof |
| CN103736241A (en) * | 2013-12-23 | 2014-04-23 | 丽水学院 | Method for biodegrading chlorophenols |
| CN104495971A (en) * | 2014-11-27 | 2015-04-08 | 福建农林大学 | Method for processing aniline-blue-containing pollution wastewater by using cultivation material of harvested pleurotus eryngii |
| CN104974991A (en) * | 2015-07-09 | 2015-10-14 | 福建农林大学 | Method for preparing solid laccase through pleurotus eryngii waste fungus chaff |
| CN105505809A (en) * | 2015-07-21 | 2016-04-20 | 南开大学 | Microbial agent for cleaning oil extraction wastewater filtering membrane |
| CN107267473A (en) * | 2017-06-29 | 2017-10-20 | 商丘师范学院 | The method that laccase is extracted from pleurotus eryngii bacteria residue |
| 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 |
| US10907143B2 (en) | 2014-09-08 | 2021-02-02 | Battelle Memorial Institute | Enzyme formulation and method for degradation |
| CN114105714A (en) * | 2021-12-15 | 2022-03-01 | 桂润环境科技股份有限公司 | Soil remediation agent, preparation method and application thereof, and soil remediation method |
| CN115011587A (en) * | 2022-07-03 | 2022-09-06 | 北京建筑大学 | Immobilized crude enzyme for degrading composite polycyclic aromatic hydrocarbon in soil and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101208424A (en) * | 2005-04-26 | 2008-06-25 | 迈科酶有限公司 | Wood-rotting basidiomycetes for production of ligninolytic enzymes |
-
2010
- 2010-03-31 CN CN 201010138364 patent/CN101845420A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101208424A (en) * | 2005-04-26 | 2008-06-25 | 迈科酶有限公司 | Wood-rotting basidiomycetes for production of ligninolytic enzymes |
Non-Patent Citations (2)
| Title |
|---|
| 《Curr Microbiol》 20091119 Xuanzhen Li et al. Degradation of Polycyclic Aromatic Hydrocarbons by Crude Extracts from Spent Mushroom Substrate and its Possible Mechanisms 336-342 1－5 , 第60期 2 * |
| 《有色冶金设计与研究》 20070331 王德汉等 蘑菇渣资源的生态高值化利用研究进展 262-266 1－5 第28卷, 第23期 2 * |
Cited By (14)
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| CN103710364A (en) * | 2013-12-16 | 2014-04-09 | 南京林业大学 | Laccase gene Lac7 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 |
| CN103710363B (en) * | 2013-12-16 | 2015-06-10 | 南京林业大学 | Laccase gene Lac6 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 |
| CN103736241A (en) * | 2013-12-23 | 2014-04-23 | 丽水学院 | Method for biodegrading chlorophenols |
| US10907143B2 (en) | 2014-09-08 | 2021-02-02 | Battelle Memorial Institute | Enzyme formulation and method for degradation |
| CN104495971A (en) * | 2014-11-27 | 2015-04-08 | 福建农林大学 | Method for processing aniline-blue-containing pollution wastewater by using cultivation material of harvested pleurotus eryngii |
| CN104974991A (en) * | 2015-07-09 | 2015-10-14 | 福建农林大学 | Method for preparing solid laccase through pleurotus eryngii waste fungus chaff |
| CN105505809A (en) * | 2015-07-21 | 2016-04-20 | 南开大学 | Microbial agent for cleaning oil extraction wastewater filtering membrane |
| CN107267473A (en) * | 2017-06-29 | 2017-10-20 | 商丘师范学院 | The method that laccase is extracted from pleurotus eryngii bacteria residue |
| 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 |
| CN114105714A (en) * | 2021-12-15 | 2022-03-01 | 桂润环境科技股份有限公司 | Soil remediation agent, preparation method and application thereof, and soil remediation method |
| CN115011587A (en) * | 2022-07-03 | 2022-09-06 | 北京建筑大学 | Immobilized crude enzyme for degrading composite polycyclic aromatic hydrocarbon in soil and preparation method thereof |
| CN115011587B (en) * | 2022-07-03 | 2023-04-25 | 北京建筑大学 | Immobilized crude enzyme for degrading compound polycyclic aromatic hydrocarbon in soil and preparation method thereof |
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Application publication date: 20100929 |