A method for immobilizing proteins using biofilms
A biofilm and protein technology, applied in the field of protein immobilization, can solve the problems of not being able to fully utilize biofilm materials, and achieve the effect of increasing the scope of use and improving stability
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Embodiment 1
[0043] Example 1: Cultivation of Clostridium acetobutylicum biofilms
[0044] The Clostridium acetobutylicum stored at -80°C was inoculated in the P2 medium, and the seed liquid was prepared by culturing under anaerobic conditions at 37°C, and then the seed liquid was inoculated into the anaerobic culture flask (P2 medium plus cotton towel), and The fermentation was carried out in static culture at 37°C. Biofilms grown on cotton towels were sampled at 3, 5, and 7 days. The obtained biofilm was washed with PBS and used for polysaccharide staining and subsequent preparation of immobilized materials.
[0045] Biofilm polysaccharides were stained with FITC-labeled PNA and UEA I, and dead cells were stained with PI, where PNA specifically recognizes D(+)-galactose and UEA I specifically recognizes L(-)-trehalose. The results of CLSM are as figure 1 shown, where green represents lectin-labeled polysaccharides, red represents PI-labeled dead cells, and yellow is the color of the c...
Embodiment 2
[0046] Example 2: Modification of Biofilm Materials
[0047]The above biofilm was first oxidized with TEMPO by adding 10 g of biofilm to a solution containing 1 mM TEMPO and 10 mM NaBr, and then adding NaClO solution to the mixture to start the reaction. During the reaction, NaOH solution was continuously added to the system to control the pH of the system at 10, and when the pH no longer changed, a small amount of ethanol was added to terminate the reaction, and HCl solution was used to adjust the pH to 7.0. The oxidized biofilm material was centrifuged at high speed to collect the precipitate and washed three times with deionized water.
[0048] The oxidized biofilm material was further modified with EDC / NHS by immersing the oxidized biofilm material in MES solution (100 mM, pH 6.0) containing 434 mM EDC and 53.2 mM NHS, and shaking at room temperature for 3 h. After the precipitate was collected by centrifugation, washed three times with deionized water to obtain the final...
Embodiment 3
[0050] Example 3: Preparation of Magnetic Nanoparticles
[0051] Preparation of Fe using chemical coprecipitation 3 O 4 Magnetic nanoparticles by adding 1.25 g of FeCl to 100 mL of deionized water 2 ·4H 2 O and 3.40 g FeCl 3 ·6H 2 O, making Fe 3+ and Fe 2+ The ratio was controlled at 2:1, and the mixture was pre-stirred at 60°C under nitrogen atmosphere. Then 6 mL of ammonia water was added thereto, and stirring was continued for 30-40 minutes. The black precipitate was collected by centrifugation and washed extensively with deionized water and ethanol.
[0052] Preparation of SiO using the sol-gel method 2 Covered magnetic nanoparticles, the method is: take the above 0.145g Fe 3 O 4 Magnetic nanoparticles, dispersed in ethanol, to which 6 mL of deionized water and 3 mL of ammonia water were added. The reaction was started by adding 0.4 mL of TEOS to it, and the mixture was incubated at room temperature with stirring. Collect solid Fe 3 O 4 -SiO 2 , and washed ...
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