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Method for immobilizing protein by using biofilm

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

Active Publication Date: 2020-03-20
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the current amyloid-based enzyme immobilization method cannot meet the needs of fully utilizing biofilm materials, and it is urgent to develop a general-purpose method that can utilize various biofilms for enzyme immobilization.

Method used

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  • Method for immobilizing protein by using biofilm
  • Method for immobilizing protein by using biofilm
  • Method for immobilizing protein by using biofilm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: the cultivation of Clostridium acetobutylicum biofilm

[0044] Clostridium acetobutylicum preserved at -80°C was inoculated in P2 medium, cultured under anaerobic conditions at 37°C to prepare seed liquid, and then the seed liquid was inoculated into anaerobic culture bottles (P2 medium plus cotton towel), and Static culture and fermentation 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 using FITC-labeled PNA and UEA I, which specifically recognizes D(+)-galactose and UEA I specifically recognizes L(-)-trehalose, and PI was used to stain dead cells. The result of CLSM is as figure 1 , where green indicates lectin-labeled polysaccharides, red indicates PI-labeled dead cells, and yellow is the combined color of the two. The results showed that with the...

Embodiment 2

[0046] Embodiment 2: the modification of biofilm material

[0047]The above-mentioned biofilm was first oxidized with TEMPO by adding 10 g of the 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 the pH was adjusted to 7.0 with HCl solution. The oxidized biofilm material was subjected to high-speed centrifugation 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 an MES solution (100 mM, pH 6.0) containing 434 mM EDC and 53.2 mM NHS, and incubating with shaking at room temperature for 3 h. After the precipitate was collected by centrifugation, it was w...

Embodiment 3

[0050] Example 3: Preparation of Magnetic Nanoparticles

[0051] Preparation of Fe using chemical co-precipitation 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 of the mixture was controlled at 2:1, and the mixture was pre-stirred at 60°C under a 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 thoroughly with deionized water and ethanol.

[0052] Preparation of SiO using sol-gel method 2 Covered magnetic nanoparticles, the method is: take the above 0.145g Fe 3 o 4 Magnetic nanoparticles were dispersed in ethanol, and 6 mL of deionized water and 3 mL of ammonia water were added thereto. The reaction was started by adding 0.4 mL of TEOS thereto, and the mixture was incubated with stirring at room temperature. collect solid Fe 3 o 4 ...

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Abstract

The invention discloses a method for immobilizing a protein by using a biofilm. The microbial biofilm is used as an initial material, and chemical modification with a polysaccharide is performed to obtain a biofilm material for immobilization of a target object, wherein the target object comprises a free amino-containing protein and / or an amino-containing nanoparticle. The biofilm material disclosed by the invention can be used to immobilize proteins with catalytic activity or non-catalytic activity, and free amino-containing nanoparticles, and the stability of the immobilized protein can besignificantly improved. In addition, the method is universal and can be extended into the use of universal type biofilms, and the utilization range of biofilm materials is greatly improved.

Description

technical field [0001] The invention relates to the field of protein immobilization, in particular to a biofilm-based immobilized enzyme, an immobilized carrier and a preparation method thereof. Background technique [0002] Biofilm is a community of microorganisms in a natural state. It is formed by microorganisms attaching to the surface of materials and secreting extracellular polymers (EPS). The main components of EPS include water, proteins, polysaccharides, lipids, ions, etc. . The report pointed out that more than 99% of microorganisms in nature can form biofilms to resist the harsh environment of the outside world. [0003] In the field of biocatalysis, researchers have demonstrated that biofilms are good natural materials for the immobilization and stability of enzymes. The current methods for biofilm immobilization of enzymes mainly use the amyloid protein in the biofilm to achieve specific immobilization of enzymes, such as based on the curli system in Escherich...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/10C07K17/10C07K17/14C12P7/62C12R1/145
CPCC12N11/14C12N11/10C12N9/20C07K17/10C07K17/14C12P7/62C12Y301/01003
Inventor 王宜冰董浩王平张文学
Owner EAST CHINA UNIV OF SCI & TECH
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