A kind of organophosphorus degrading active nanoparticle and its preparation method and application
A technology for degrading active, nano-particles, applied in biochemical equipment and methods, chemical instruments and methods, microorganism-based methods, etc., can solve the problems of cumbersome operation, high cost, etc. Reuse works well
Active Publication Date: 2020-10-09
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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AI Technical Summary
Problems solved by technology
The immobilization of conventional enzymes requires the preparation of free enzyme proteins and immobilized carriers separately, which is cumbersome and relatively expensive
Method used
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Embodiment 1
[0038] 1.1 Extraction of Cupriavidus necator genomic DNA
[0039] (1) Cultivate 5mL actinomycetes culture, take 1mL culture and centrifuge at 12000rpm for 2min;
[0040] (2) Add 567μL of TE buffer to the precipitate and resuspend it by pipetting repeatedly. Add 30μL of 20mg / mL lysozyme and bath in 37℃ water for 1h;
[0041] (3) Add 300μL of 10% SDS and 30μL of 20mg / mL proteinase K, mix well, and bath in 37℃ water for 1h;
[0042] (4) Add 100μL of 5M NaCl, mix well, then add 80μL of CTAB / NaCl solution, mix well, and bath in 65°C for 10min;
[0043] (5) Add an equal volume of phenol / chloroform and extract once with the chloroform solution;
[0044] (6) Add 0.6 times volume of isopropanol, mix gently, centrifuge at 12000 rpm for 5 min, discard the supernatant, wash twice with 70% ethanol, and dry in ultra-clean typhoon;
[0045] (7) Add 50μL of TE buffer and dissolve DNA at 4°C overnight.
[0046] 1.2 Amplification of the target gene
[0047] Using Cupriavidus necator genomic DNA as template,...
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Abstract
The invention discloses active nano-particles for degrading organophosphorus, as well as a preparation method and applications of the active nano-particles. According to the technical scheme, by constructing engineering bacteria expressing beta-keto-thiolase, acetoacetyl CoA reductase, fusion protein of organophosphorus acid anhydride hydrolase and polyhydroxyalkanoate synthetase, and fusion protein of organophosphorus acid anhydride hydrolase and polyhydroxyalkanoate binding protein, the obtained engineered organophosphorus acid anhydride hydrolase is fixed to the surfaces of the polyhydroxyalkanoate nano-particles in a combined manner through two methods including covalence and non-covalence, the nano-particles with the activity of degrading the organophosphorus is efficiently produced at one step through a fermentation method, and the high stability and recycling effect are achieved. Compared with the traditional enzyme immobilization method, the method provided by the invention hasthe advantages that the zymoprotein and the carrier for immobilization do not need to be respectively prepared, the production of the immobilization particles can be completed through one step, and the production cost is low. The active nano-particles have wide applications in the aspects of the purification for pesticide residue of fruits and vegetables and the treatment for the environmental pollutants.
Description
Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an organic phosphorus degradation active nanoparticle, a production engineering strain thereof, a fermentation preparation method and application. Background technique [0002] Human production activities discharge large amounts of organic pollutants, causing environmental pollution and threatening ecological security and human health. Organophosphorus compounds are a class of phosphonic acid or phosphoramidate esters or thiol derivatives, which exert virulence by covalently binding with serine, the active center of acetylcholinesterase. Since it was first synthesized in 1937, it has been widely used in wars and production activities against humans. During World War II, Type G and Type V organophosphorus nerve agents were important chemical weapons. Organophosphorus was widely used as pesticides, flame retardants, petroleum additives and plasticizers in modern industrial and a...
Claims
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IPC IPC(8): C12N1/21A23L5/20C12R1/19
CPCA23L5/27C07K14/00C07K2319/00C12N9/001C12N9/14
Inventor 龙丽娟杨键李茹
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI