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

Inactive Publication Date: 2009-11-25
HANGZHOU EVERLONG BIOTECHNICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention aims at problems such as poor stability of cholinesterase, reduced enzyme activity after immobilization, i

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0019] Example 1:

[0020] (1) Acetylcholinesterase fixation

[0021] (1) Prepare 10mM pH 8 phosphate buffered saline (PBS).

[0022] (2) Dilute the purchased commercial pure acetylcholinesterase with bovine albumin phosphate buffer containing 1-10 mg / mL, with a concentration of 1 U / mL.

[0023] (3) Weigh polyvinylpyrrolidone, dissolve it in water, and prepare 1-10% polyvinylpyrrolidone solution according to weight and volume percentage.

[0024] (4) Mix the enzyme solution and the polyvinylpyrrolidone solution in a volume ratio of 1:1, and mix well.

[0025] (5) Pipette 60 μl and drop it onto the plastic sheet to dry and form a film.

[0026] (2) Acetylcholinesterase-polyvinylpyrrolidone water-soluble enzyme membrane for the detection of organophosphate poisons

[0027] Weigh the vegetable sample to be tested, and shake 50 times with 10mM pH 8 buffer solution at a ratio of 2ml / g to extract the residual pesticide. Put the enzyme membrane in the centrifuge tube, add 50μl extract so...

Example Embodiment

[0028] Example 2:

[0029] (1) Preparation of blank liposomes

[0030] (1) Prepare 10mM pH 8 phosphate buffered saline (PBS).

[0031] (2) Weigh 0.9g phospholipid and 0.3g cholesterol in a 50ml small beaker, add 1~2ml absolute ethanol, place in a 65~70℃ water bath, stir to dissolve, rotate the small beaker to make the ethanol solution of phospholipid in the cup A film is formed on the wall and dried with nitrogen.

[0032] (3) Take another 30ml of PBS in a small beaker, and place it in a water bath at 65~70℃, keep it warm, and set aside.

[0033] (4) Take 30ml of preheated phosphate buffer, add it to a small beaker containing phospholipid and cholesterol lipid membrane, stir and hydrate in a water bath at 65~70℃ for 10min. Then place the small beaker on a magnetic stirrer, and stir for 30-60 minutes at room temperature. If the volume of the solution decreases, add water to 30ml and mix well to get it.

[0034] (2) Immobilization of butyrylcholinesterase-liposome membrane

[0035] ...

Example Embodiment

[0041] Example 3:

[0042] (1) Extraction of cholinesterase from earthworms

[0043] (1) Prepare 10mM pH 8 phosphate buffered saline (PBS).

[0044] (2) The freshly isolated earthworm tissue was homogenized with equal quality PBS for 2 minutes, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was taken as cholinesterase.

[0045] (2) Cholinesterase fixation

[0046] (1) Weigh the gum arabic, dissolve it in PBS, and prepare an aqueous solution of gum arabic containing 400 to 500 grams of gum arabic per liter.

[0047] (4) Mix the enzyme solution and the gum arabic solution in a volume ratio of 4:1, mix well, and add 200 μl dropwise to a plastic mold to dry into enzyme granules.

[0048] (3) Cholinesterase water-soluble enzyme granules are used for the detection of carbamate pesticides

[0049] Weigh the vegetable sample to be tested, shake 50 times with 10mM pH 8 buffer solution at a ratio of 2ml / g to extract residual pesticides, take 880μl extract, add enzyme granules...

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PUM

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Abstract

An immobilized cholinesterase and preparation and application thereof are disclosed, the designated carrier is water-soluble material such as polyvinyl alcohol, polyvinyl pyrrolidone or Arabic gum, capable of film-forming by drying and of being dissolved rapidly in aqueous solution; the carrier also comprises composite material formed by the water-soluble material and lipoplast. 1-10mg/mL of bovine serum albumin phosphate buffer solution is used as a protective agent; the water-soluble material is used to immobilize cholinesterase, cholinesterase water-soluble material solution is either directly adhered to solid material by drip or soakage and then dried to form a thin film or dripped into a mould and then dried to form enzyme granules, when the material is put into the solution, enzyme molecules therein are dissolved and released immediately to catalytically function in homogeneous phase with better catalytic efficiency and inhibitive sensitivity; the invention can be applied to rapid detection of pesticide residue or screening of cholinesterase inhibitor.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an enzyme product and its processing and application, specifically a cholinesterase immobilized with a water-soluble material and its preparation, and rapid detection of pesticide residues or screening of cholinesterase inhibitors Applications. Background technique [0002] Cholinesterase is an important enzyme in humans and animals, including acetylcholinesterase and butyrylcholinesterase. Organophosphorus, carbamate pesticides or poisons can inhibit the activity of cholinesterase. Many biological toxins or active ingredients also inhibit cholinesterase activity, which can be applied to the treatment of Alzheimer's disease and many neurological diseases. The inhibitory effect of cholinesterase can be used to detect pesticide residues in food or environment, and can also be used for drug screening of neurological diseases. [0003] However, cholinesterase is not easy to preserve in ...

Claims

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Application Information

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IPC IPC(8): C12N11/08C12N11/02C12Q1/46
Inventor 缪煜清马倩倩
Owner HANGZHOU EVERLONG BIOTECHNICS
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