CN101906451A - Phanerochaete chrysosporium ecto polymer and extraction method and application thereof - Google Patents
Phanerochaete chrysosporium ecto polymer and extraction method and application thereof Download PDFInfo
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Abstract
The invention relates to a white rot fungi ecto polymer and extraction method and application thereof, in particular discloses an extraction method of phanerochaete chrysosporium ecto polymer. Firstly phanerochaete chrysosporium suspension is added into a liquid culture medium, oscillation culture is carried out under the conditions that the temperature is 35-37 DEG C and the rotating speed is 120-150r/min, and primary centrifugation is carried out under the condition that the temperature is 0-4 DEG C; then supernatant is filtered, filtrate is added with 4-5 volumes of absolute ethyl alcohol, standing is carried out at 0-4 DEG C, and secondary centrifugation is carried out; crude ecto polymer precipitated after centrifugation is subject to dialysis and then is sealed and stored, thus obtaining the phanerochaete chrysosporium ecto polymer. The phanerochaete chrysosporium ecto polymer extracted by the method of the invention has significance on researching the principle that phanerochaete chrysosporium degrades organic pollutant and/or treats heavy metal.
Description
Technical field
The present invention relates to a kind of sugar and proteinic mixture and its production and application of containing, relate in particular to a kind of white-rot fungi extracellular polymeric and extracting method and application.
Background technology
Remove the heavy metal ion in the trade effluent, for keeping aquatic environment, aquatic ecosystem and underground hydrosphere to have very important significance.Traditional treatment process mainly is that heavy metal ion is carried out ion-exchange with the hydroxide form precipitation or with synthetic resins.In recent years, because the cheap and higher loading capacity of cost, microbial technique begins to be used to more and more handle heavy metal wastewater thereby.
White-rot fungi also can be applied to the improvement of heavy metal contamination because the processing power to xenobiontics of its uniqueness can be handled the pollution of Persistent organic pollutants, and it becomes environmental organism Processing Technology focus gradually.Research white-rot fungi extracellular polymeric, its outer mechanism of action of born of the same parents of handling xenobiontics is significant for research.
White-rot fungi extracellular polymeric (EPS) is a class by white rot fungus secretion and attached to the saccharan protein matter on cell walls surface, secretion keeping fungal cell's biomorph, extracellular enzyme particularly plays an important role on the active defense mechanism to heavy metal ion.And in handling the heavy metal contamination process, saccharan in the white-rot fungi extracellular polymeric and protein substance contain a large amount of active function groups, and it plays an important role in surface adsorption complexing action, the effect of oxalate microdeposit and reductive action.Phanerochaete chrysosporium (Phanerochaete chrysosporium) is the white-rot fungi that a kind of application is wide and be easy to obtain, therefore, effectively, intactly extracting Phanerochaete chrysosporium ecto polymer is a prerequisite utilizing this material, and this is significant for advancing the outer Study on Mechanism of white-rot fungi born of the same parents.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, a kind of significant Phanerochaete chrysosporium ecto polymer of research to Phanerochaete chrysosporium degradable organic pollutant and absorption heavy metal is provided, and a kind of extracting method and application simple, reliable, effectively Phanerochaete chrysosporium ecto polymer are provided.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is a kind of extracting method of Phanerochaete chrysosporium ecto polymer, may further comprise the steps:
(1) culture of isolated: at first, the bacteria suspension of Phanerochaete chrysosporium is added in the liquid nutrient medium, and under 35 ℃～37 ℃ temperature, the speed conditions of 120r/min～150r/min, carry out shaking culture, culture after the shaking culture is carried out the centrifugation first time, temperature during centrifugation for the first time is controlled at 0 ℃～4 ℃, and rotating speed is controlled at 8000r/min～10000r/min;
(2) filtering separation: the supernatant liquid filtering film after the above-mentioned culture of isolated is carried out filtration treatment, in filtrate filtered, add 4～5 times of dehydrated alcohols then to its volume, mix, this mixture is being carried out the centrifugation second time after the standing over night under 0 ℃～4 ℃ conditions, temperature during centrifugation for the second time is controlled at 0 ℃～4 ℃, and rotating speed is controlled at 10000r/min～12000r/min;
(3) dialysis: the thick extracellular polymeric that above-mentioned filtering separation postprecipitation is got off is placed in the dialysis tubing dialyses, dialysis tubing molecular retention amount is 7000kDa～10000kDa, airtight being placed in the deionized water preserved, and obtains Phanerochaete chrysosporium ecto polymer.
In the extracting method of above-mentioned Phanerochaete chrysosporium ecto polymer, described culture preferably is meant the culture when Phanerochaete chrysosporium has entered logarithmic phase after shaking culture.Under one situation, the Phanerochaete chrysosporium behind the shaking culture 40h promptly begins to enter logarithmic phase, and it is also more relatively to enter the culture excretory extracellular polymeric of logarithmic phase, more helps follow-up extraction, observation, research and application.
In the extracting method of above-mentioned Phanerochaete chrysosporium ecto polymer, the time of the described centrifugation first time is preferably 12min～15min, and the time of the described centrifugation second time is preferably 10min～12min.
In the extracting method of above-mentioned Phanerochaete chrysosporium ecto polymer, the aperture of described filter membrane is 0.45 μ m.The filter membrane in this aperture is some bigger particulate matter of filtering and the other biological matter that breaks away from thalline in centrifugal process better.
As a total technical conceive, the present invention also provides the Phanerochaete chrysosporium ecto polymer that extraction obtains in a kind of above-mentioned each extracting method.This extracellular polymeric is meant attached to the hypha,hyphae surface or is centered around mycelium saccharan protein matter on every side, plays an important role on external interferential defense mechanisms such as secretion that keeps fungal cell's biomorph, extracellular enzyme and antagonism heavy metal ion.
As a total technical conceive, the present invention also provides a kind of above-mentioned application of Phanerochaete chrysosporium ecto polymer in degradable organic pollutant and/or processing heavy metal.Change by analyzing the protein component of Phanerochaete chrysosporium ecto polymer in degradable organic pollutant and/or processing heavy metal process, thereby find out the specific enzyme that plays an important role in degradation process, inquiring into for the practical application of Phanerochaete chrysosporium and mechanism provides theoretical and supports.
Compared with prior art, the invention has the advantages that: broken through existing microorganism extracellular polymeric and extracted difficult technologies bottleneck (existing extracellular polymeric Study on Extraction Method mainly concentrates on microorganisms such as active sludge, bacterium), a kind of simple, reliable, effective extracting method of Phanerochaete chrysosporium ecto polymer is provided, has also obtained a kind of Phanerochaete chrysosporium ecto polymer simultaneously by this extracting method preparation.Change by analyzing the protein component of Phanerochaete chrysosporium ecto polymer in degradable organic pollutant and/or processing heavy metal process, can find out the specific enzyme that in degradation process, plays an important role, for Phanerochaete chrysosporium is provided fundamental basis and technical support in the practical application and the mechanism discussion in Pollutant Treatment field from now on, also significant to the related science researchs such as the outer mechanism of action of born of the same parents of carrying out Phanerochaete chrysosporium degradable organic pollutant and absorption heavy metal simultaneously.
Description of drawings
Fig. 1 is the Phanerochaete chrysosporium mycelium dry weight and the broken line graph of its extracellular polymeric content with the incubation time variation of embodiment of the invention preparation.
Fig. 2 is the microtexture stereoscan photograph of Phanerochaete chrysosporium ecto polymer before extraction in the embodiment of the invention.
Fig. 3 is the microtexture stereoscan photograph of Phanerochaete chrysosporium ecto polymer after extraction in the embodiment of the invention.
Fig. 4 is the histogram that contents of saccharide and protein content change with incubation time in the Phanerochaete chrysosporium ecto polymer in the embodiment of the invention.
Fig. 5 be in the embodiment of the invention Phanerochaete chrysosporium at the later microtexture stereoscan photograph of adsorpting lead ion.
Fig. 6 is Phanerochaete chrysosporium particulate energy spectrum analysis figure shown in the arrow of thalline surface after adsorpting lead ion in the embodiment of the invention.
Embodiment
Embodiment:
Select a Phanerochaete chrysosporium (Phanerochaete chrysosporium BKMF-1767 for example for use, available from Wuhan University China typical culture center), Phanerochaete chrysosporium spore on the flat board is scraped in the sterilized water, form the spore suspension of Phanerochaete chrysosporium, then quantitative suspension is added in the liquid nutrient medium (liquid nutrient medium carries out the 30min sterilising treatment under 105 ℃ of conditions), and in 35 ℃ of temperature, shaking culture under the condition of 120r/min rotating speed, respectively in 40～140h scope, the timed interval every 20～24h takes a sample, the incubation time of sampling spot is respectively 41h, 65h, 89h, 113h, 137h obtains the Phanerochaete chrysosporium culture under each incubation time respectively; With each culture filter respectively, dry (in 80 ℃ the baking oven) to constant weight, obtains the Phanerochaete chrysosporium mycelium, then the mycelial dry weight of the Phanerochaete chrysosporium under each incubation time is measured, measuring result is as shown in table 1 below.
Meanwhile, other gets the culture that above-mentioned cultivation 41h, 65h, 89h, 113h, 137h obtain, Phanerochaete chrysosporium culture after above-mentioned each parallel cultivation is carried out the centrifugation first time under high speed freezing centrifuge, temperature during centrifugation for the first time is controlled at 4 ℃, rotating speed is controlled at 10000r/min, and the time of centrifugation for the first time is 15min.
The filter membrane of supernatant liquor after the above-mentioned centrifugation first time being crossed 0.45 μ m filters, in filtrate filtered, add 5 times of dehydrated alcohols (volume fraction is 95%) then to its volume, behind the uniform mixing, with this mixture standing over night (preserving 12h) under 4 ℃ of conditions, carry out the centrifugation second time then, temperature during centrifugation for the second time is controlled at 4 ℃, and rotating speed is controlled at 10000r/min, and the time of centrifugation for the second time is 10min.
The thick extracellular polymeric that centrifugation second time postprecipitation is got off is put into dialysis tubing and is dialysed, the molecular retention amount of dialysis tubing is 7000kDa, airtight being placed in the beaker that fills deionized water under the room temperature finally obtains purified Phanerochaete chrysosporium ecto polymer.
With different incubation times each Phanerochaete chrysosporium ecto polymer down of dissolved in distilled water present embodiment preparation, and with TOC automatic analyser mensuration extracellular polymeric content, its measurement result is as shown in table 1 below.
Table 1: the measuring result that Phanerochaete chrysosporium mycelium dry weight and its extracellular polymeric change with incubation time
| Incubation time (h) | 41 | 65 | 89 | 113 | 137 |
| Mycelium dry weight (g/L) | 0.828 | 0.922 | 1.114 | 1.612 | 1.356 |
| Extracellular polymeric content (mg/L) | 94.5 | 109.9 | 119.3 | 125.5 | 118.6 |
As can be seen from Table 1, along with the prolongation of incubation time, the content of Phanerochaete chrysosporium mycelium dry weight and its extracellular polymeric all presents ascendant trend, reaches maximum value when cultivating 113h, and after this weight begins to descend.The broken line graph that Phanerochaete chrysosporium mycelium dry weight and its extracellular polymeric content change with incubation time as shown in Figure 1.
Under environmental scanning electronic microscope, the Phanerochaete chrysosporium ecto polymer of observing behind the above-mentioned cultivation 65h is extracting forward and backward microtexture, and its observations respectively as shown in Figures 2 and 3.As seen from Figure 2, the Phanerochaete chrysosporium mycelium that does not extract extracellular polymeric is to be twined mutually, be interweaved and formed by a lot of mycelia, and it has a lot of holes and mycelia tip, and the surface is the pelletizing shape, and the extracellular polymeric content of surface viscosity is abundant.As seen from Figure 3, the Phanerochaete chrysosporium mycelium surface after extracellular polymeric extracts is block, and mycelia is thinner, and smooth surface, and the extracellular polymeric content of viscosity reduces in a large number.This has further confirmed the validity of extracting method of the present invention.
At last, with anthrone-H
2SO
4Colorimetry, with glucose preparation standard curve, the content of glucide in each Phanerochaete chrysosporium ecto polymer under the different incubation times of mensuration present embodiment preparation, measuring result is as shown in table 2 below; Simultaneously, use the Lowry method, with bovine serum albumin preparation standard curve, Protein content in each Phanerochaete chrysosporium ecto polymer under the different incubation times of mensuration present embodiment preparation, measuring result is as shown in table 2 below.
Table 2: Phanerochaete chrysosporium ecto polymer is formed the measuring result that changes along with incubation time
| Incubation time (h) | 41 | 65 | 89 | 113 | 137 |
| Contents of saccharide (%) | 46.6 | 52.7 | 54.2 | 53.7 | 54.3 |
| Protein content (%) | 31.2 | 33.5 | 34.8 | 34.7 | 35.1 |
By last table 2 as can be seen, along with the prolongation of incubation time, carbohydrate and protein content all present ascendant trend in the Phanerochaete chrysosporium ecto polymer, and after cultivating 89h, its content tends towards stability.The histogram that contents of saccharide and protein content change with incubation time in the Phanerochaete chrysosporium ecto polymer as shown in Figure 4.
According to our experimental studies results, Phanerochaete chrysosporium ecto polymer may be relevant with the effect of the specific enzymes that promotes to relate to lignin degradation, and our Phanerochaete chrysosporium ecto polymer of discovering can be by the metallic throw out of extracellular polymeric reaction formation.When xenobionticses such as processing Persistent organic pollutants and heavy metal, Phanerochaete chrysosporium can secrete special protein and enzyme promotes the decomposition of Persistent organic pollutants, and improves the tolerance to heavy metal.As seen, the Phanerochaete chrysosporium ecto polymer that utilizes the inventive method to extract, help us to further investigate the outer mechanism of born of the same parents of Phanerochaete chrysosporium, quickening and raising are to the application of Phanerochaete chrysosporium (or even white-rot fungi) on Environmental Biotechnology.Our preliminary study result shows, when Phanerochaete chrysosporium is handled lead waste water, can form the spheroidal particle of a large amount of rules as shown in Figure 5 on its thalline surface, by particle shown in the arrow among Fig. 5 is carried out energy spectrum analysis, analytical results as shown in Figure 6, this particle contains the absorption peak of C, O, P, K, Pb, Mg, N and S as seen from Figure 6, this explanation Phanerochaete chrysosporium forms protein-Pb particle by secretion extracellular polymeric parcel heavy metal ion, therefore the N peak is an amino acid whose absorption peak in the protein, and the S peak may be the chemical bond SH in the protein.
Claims (6)
1. the extracting method of a Phanerochaete chrysosporium ecto polymer may further comprise the steps:
(1) culture of isolated: at first, the bacteria suspension of Phanerochaete chrysosporium (Phanerochaete chrysosporium) is added in the liquid nutrient medium, and under 35 ℃～37 ℃ temperature, the speed conditions of 120r/min～150r/min, carry out shaking culture, culture after the shaking culture is carried out the centrifugation first time, temperature during centrifugation for the first time is controlled at 0 ℃～4 ℃, and rotating speed is controlled at 8000r/min～10000r/min;
(2) filtering separation: the supernatant liquid filtering film after the above-mentioned culture of isolated is carried out filtration treatment, in filtrate filtered, add 4～5 times of dehydrated alcohols then to its volume, mix, this mixture is being carried out the centrifugation second time after the standing over night under 0 ℃～4 ℃ conditions, temperature during centrifugation for the second time is controlled at 0 ℃～4 ℃, and rotating speed is controlled at 10000r/min～12000r/min;
(3) dialysis: the thick extracellular polymeric that above-mentioned filtering separation postprecipitation is got off is placed in the dialysis tubing dialyses, dialysis tubing molecular retention amount is 7000kDa～10000kDa, airtight being placed in the deionized water preserved, and obtains Phanerochaete chrysosporium ecto polymer.
2. the extracting method of Phanerochaete chrysosporium ecto polymer according to claim 1, it is characterized in that: described culture is meant the culture when Phanerochaete chrysosporium has entered logarithmic phase after shaking culture.
3. the extracting method of Phanerochaete chrysosporium ecto polymer according to claim 1 and 2, it is characterized in that: the time of the described centrifugation first time is 12min～15min, the time of the described centrifugation second time is 10min～12min.
4. the extracting method of Phanerochaete chrysosporium ecto polymer according to claim 1 and 2, it is characterized in that: the aperture of described filter membrane is 0.45 μ m.
5. one kind is extracted the Phanerochaete chrysosporium ecto polymer that obtains as each extracting method in the claim 1～4.
6. the application of Phanerochaete chrysosporium ecto polymer as claimed in claim 5 in degradable organic pollutant and/or processing heavy metal.
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| CN103196847A (en) * | 2013-03-21 | 2013-07-10 | 湖南大学 | Quantitative detection method for intracellular active sulfhydryl compound of white-rot fungi under heavy metal stress |
| CN103554214A (en) * | 2013-10-23 | 2014-02-05 | 湖南大学 | Extraction method of extracellular polymeric substance for keeping mycelial morphology of phanerochaete chrysosporium |
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| CN103196847B (en) * | 2013-03-21 | 2015-09-30 | 湖南大学 | The quantitative detecting method of sulfhydryl-group activity compound in white-rot fungi born of the same parents under heavy metal stress |
| CN103196847A (en) * | 2013-03-21 | 2013-07-10 | 湖南大学 | Quantitative detection method for intracellular active sulfhydryl compound of white-rot fungi under heavy metal stress |
| CN103554214A (en) * | 2013-10-23 | 2014-02-05 | 湖南大学 | Extraction method of extracellular polymeric substance for keeping mycelial morphology of phanerochaete chrysosporium |
| CN103554214B (en) * | 2013-10-23 | 2016-06-01 | 湖南大学 | Keep the extracting method of the extracellular polymeric of Phanerochaete chrysosporium thalli morphology |
| CN103784479A (en) * | 2014-01-20 | 2014-05-14 | 湖南大学 | Phanerochaete chrysosporium antioxidant extractive solution and preparation method thereof |
| CN103789279A (en) * | 2014-01-20 | 2014-05-14 | 湖南大学 | Panerochaete chrysosporium extracting solution with antioxidant enzyme and preparation method |
| CN103789279B (en) * | 2014-01-20 | 2016-08-17 | 湖南大学 | Phanerochaete chrysosporium extracting solution containing antioxidase and preparation method |
| CN103784479B (en) * | 2014-01-20 | 2017-01-04 | 湖南大学 | Phanerochaete chrysosporium antioxidation extracting solution and preparation method thereof |
| CN105233458A (en) * | 2015-10-19 | 2016-01-13 | 桂林理工大学 | Method for degrading bisphenol A through white-rot fungus crude enzyme |
| CN105233458B (en) * | 2015-10-19 | 2018-11-06 | 桂林理工大学 | A method of utilizing white-rot fungi crude enzyme liquid degradation bisphenol-A |
| CN109926029A (en) * | 2017-12-18 | 2019-06-25 | 武汉理工大学 | The extracting method of a kind of extracellular polymeric substances from activated sludge and its in uranium polluted-water/soil adsorption applications |
| CN108569831A (en) * | 2018-04-25 | 2018-09-25 | 中原工学院 | A kind of extraction separation method of effective activated sludge exoprotein and application |
| CN108504695A (en) * | 2018-06-04 | 2018-09-07 | 广东工业大学 | A kind of extracellular polymeric and preparation method thereof, heavy metal treatment agent and process for treating heavy-metal waste water |
| CN108504695B (en) * | 2018-06-04 | 2021-08-13 | 广东工业大学 | Extracellular polymer and preparation method thereof, heavy metal treatment agent and heavy metal wastewater treatment method |
| CN111334305A (en) * | 2020-04-13 | 2020-06-26 | 潍坊友容实业有限公司 | Saline-alkali soil heavy metal ion remover and preparation method thereof |
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