Purification method of acetylcholinesterase from earthworm serum

An acetylcholinesterase and earthworm technology, applied in the directions of hydrolase and the like, can solve the problems of not realizing the separation of esterase, low purification multiple, weakening enzyme activity, etc., and achieve the advantages of cheap and extensive enzyme sources, simple and stable preparation process, and low production cost. Effect

Active Publication Date: 2013-08-14
武汉中科志康生物科技有限公司
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Problems solved by technology

The method is complicated, the purification factor is low, and the separation of esterase is not realized, which will weaken the activity of the enzyme

Method used

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  • Purification method of acetylcholinesterase from earthworm serum
  • Purification method of acetylcholinesterase from earthworm serum

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Embodiment Construction

[0014] see figure 1 , a method for purifying acetylcholinesterase from earthworm serum:

[0015] 1) Earthworm pretreatment: wash the purchased earthworms and put them in clean water for 24 hours to make them discharge the feces in the body, and store them in a refrigerator at 4°C for later use;

[0016] 2) Crude extraction of earthworm serum: use phosphate buffer (pH8.0, 0.1mol / L) as the solution to prepare the crude extract, and the homogenate ratio is 1:1 (weight of earthworm (g) / volume of phosphate buffer ( mL)), centrifuge at 4°C (10000r / min) for 20min, discard the supernatant, and obtain the crude enzyme extract, which is stored in the refrigerator. ;

[0017] 3) Cloud point extraction process: Take a 10mL centrifuge tube, then add 100μL of crude earthworm serum extract, that is, crude enzyme extract, then add surfactant Triton X-114 (0.09%, w / v) solution and 0.6mL pH4.1 citric acid buffer solution to adjust the pH value of the solution, dilute to 10mL with secondary w...

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Abstract

The invention provides a purification method of acetylcholinesterase from earthworm serum. The method comprises the following steps: placing earthworm in clean water for 24 hours to eliminate excrement in the earthworm body, and storing the earthworm in a refrigerator for later use; homogenizing with phosphoric acid buffer solution in an ice-water bath condition to prepare homogenate, centrifuging to obtain crude enzyme extract, and storing in the refrigerator; adding surfactant Triton X-114 solution and citric acid buffer solution into 100mu L of crude extract, and diluting with secondary water until the volume is 10mL, sufficiently oscillating and shaking uniformly; placing the solution in a constant-temperature water bath with the water bath temperature of 35 DEG C to balance for 9 minutes; and centrifuging for layering to obtain a lower product which is the purified acetylcholinesterase product. The purification extraction method of the acetylcholinesterase from earthworm serum has wide enzyme sources and low price, high sensitivity, simple and stable preparation process and low production cost.

Description

technical field [0001] The invention relates to an extraction method of acetylcholinesterase, in particular to a method for purifying acetylcholinesterase from earthworm serum. Background technique [0002] Acetylcholinesterase is a key enzyme in biological nerve conduction and plays a vital function in nerve conduction. This enzyme degrades acetylcholine, terminates the stimulating effect of neurotransmitters on the post-synaptic membrane, and ensures the normal conduction of nerve signal molecules in the organism. Acetylcholinesterase has the action sites of organic phosphorus and carbamate compounds. When these two types of compounds are combined with acetylcholinesterase, it will cause phosphorylation and carbamylation of the catalytic site of the enzyme, making it sensitive to acetylcholine The acetylation of the protein cannot be carried out, thereby destroying the normal nerve conduction. The inactivation of enzymes can cause biological poisoning, and this principle...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18
Inventor 陈建荣张改梅仲淑贤潘小欧李娜
Owner 武汉中科志康生物科技有限公司
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