Immobilized cholinesterase and preparation and application thereof
A technology of cholinesterase and solid materials, which is applied in the direction of immobilization on/in organic carriers, biochemical equipment and methods, and measurement/testing of microorganisms, and can solve the problem of reduced enzyme activity and poor stability of cholinesterase , Inability to carry out homogeneous reaction and other problems, to achieve good catalytic efficiency and good suppression sensitivity
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Embodiment 1
[0020] (1) Immobilization of acetylcholinesterase
[0021] (1) Prepare 10mM pH 8 phosphate buffer saline (PBS).
[0022] (2) Dilute the purchased commercial pure acetylcholinesterase with phosphate buffer solution containing 1-10 mg / mL bovine serum albumin to a concentration of 1 U / mL.
[0023] (3) Weigh the polyvinylpyrrolidone, dissolve it in water, and prepare a 1-10% polyvinylpyrrolidone solution by weight and volume percentage.
[0024] (4) Mix the enzyme solution and the polyvinylpyrrolidone solution according to the volume ratio of 1:1, and mix well.
[0025] (5) Pipette 60 μl dropwise onto a plastic sheet to dry to form a film.
[0026] (2) Acetylcholinesterase-polyvinylpyrrolidone water-soluble enzyme membrane for the detection of organophosphorus poisons
[0027] Weigh the vegetable samples to be tested, shake 50 times with 10mM pH 8 buffer solution according to the ratio of 2ml / g to extract the residual pesticides. Put the enzyme membrane in the centrifuge tube,...
Embodiment 2
[0029] (1) Preparation of blank liposomes
[0030] (1) Prepare 10mM pH 8 phosphate buffer saline (PBS).
[0031] (2) Weigh 0.9g of phospholipid and 0.3g of cholesterol in a 50ml small beaker, add 1-2ml of absolute ethanol, place in a water bath at 65-70°C, stir to dissolve, rotate the small beaker to make the ethanol solution of phospholipid in the cup A film was formed on the wall and dried with nitrogen.
[0032] (3) Take another 30ml of PBS in a small beaker, put them in a water bath at 65-70°C, keep warm, and set aside.
[0033] (4) Take 30ml of preheated phosphate buffer solution, add it to a small beaker containing phospholipids and cholesterol lipid film, stir and hydrate in a water bath at 65-70°C for 10 minutes. Then place the small beaker on a magnetic stirrer and stir at room temperature for 30-60 minutes. If the volume of the solution decreases, add water to 30ml and mix well to obtain the product.
[0034] (2) Immobilization of butyrylcholinesterase-liposome me...
Embodiment 3
[0042] (1) Extraction of cholinesterase from earthworms
[0043] (1) Prepare 10mM pH 8 phosphate buffer saline (PBS).
[0044] (2) Fresh earthworm isolated tissues were homogenized by adding equal mass of PBS for 2 minutes, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was used as cholinesterase.
[0045] (2) Cholinesterase immobilization
[0046] (1) Weigh the gum arabic, dissolve it in PBS, and prepare a gum arabic aqueous solution containing 400-500 grams of gum arabic per liter.
[0047] (4) Mix the enzyme solution and the gum arabic solution according to the volume ratio of 4:1, mix thoroughly, draw 200 μl dropwise into a plastic mold and dry it into enzyme granules.
[0048] (3) Cholinesterase Water-Soluble Enzyme Granules Used in the Detection of Carbamate Pesticides
[0049] Weigh the vegetable sample to be tested, shake 50 times with 10mM pH 8 buffer solution according to the ratio of 2ml / g to extract residual pesticides, take 880μl extract, add enz...
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