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Glycerophosphoryl choline degradation or detection enzymologic method, product and application thereof

A technology of glycerophosphocholine and glycerophosphocholine phosphodiesterase, which is applied in the field of biotechnology and enzyme engineering, and can solve the problems of many interfering substances, low specificity, and the need for special personnel to operate

Active Publication Date: 2016-07-20
益海嘉里(连云港)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0021] Among these methods, the first method has disadvantages such as long time-consuming, many interfering substances, and low specificity.
The third type of method has the disadvantages of needing expensive experimental equipment to carry out, and requiring special personnel to operate.
Quantification of choline, the hydrolysis product of another GPC, requires the use of choline oxidase, 4-aminopyrine, and peroxidase to complete a multi-step reaction, which is the same as the 3-phosphoglycerol quantification method in terms of time and experimental cost Disadvantages such as high cost and long time consumption

Method used

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  • Glycerophosphoryl choline degradation or detection enzymologic method, product and application thereof
  • Glycerophosphoryl choline degradation or detection enzymologic method, product and application thereof
  • Glycerophosphoryl choline degradation or detection enzymologic method, product and application thereof

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Effect test

Embodiment 1

[0114] Embodiment 1. Preparation of glycerophosphocholine phosphodiesterase

[0115] (1) Construction of Glycerophosphocholine Phosphodiesterase Expression Vector

[0116] The glycerophosphocholine phosphodiesterase used in the present invention is an enzyme derived from Bacillus subtilis GIM1.286, named WGPD-V018. The amino acid sequence of the enzyme is shown in SEQ ID NO:2, and the coding polynucleotide sequence is shown in SEQ ID NO:1.

[0117] Merck's pET24a vector can be used for heterologous expression (see figure 1 ). Adopt respectively the sequence shown as SEQIDNO:3 and SEQIDNO:4 as upstream and downstream primers, carry out PCR reaction with the genome of Bacillus subtilis GIM1.286 as template, obtain the present invention comprising NdeI and XhoI restriction endonuclease sites The full-length nucleotide sequence of the polypeptide is verified by sequencing as shown in SEQ ID NO:1. Then the PCR product was subjected to 1% agarose gel electrophoresis, and the E...

Embodiment 2

[0137] Embodiment 2. The working curve of phosphorus molybdenum blue method detection inorganic phosphorus

[0138] First preparation concentration is the standard inorganic phosphate root solution of 1mg / ml namely is equivalent to the inorganic phosphorus solution (solute is potassium dihydrogen phosphate, solvent is ultrapure water) that concentration is 10.53 μ mol / ml, then dilutes respectively 2 times, 4 times, 8 times, 16 times, 32 times and 64 times to prepare inorganic phosphorus solutions with different concentration gradients.

[0139] Take 40 μl of inorganic phosphorus standard solutions with different concentration gradients, add 100 μl of ammonium molybdate solution, 100 μl of 10 w / v% ascorbic acid aqueous solution and 760 μl of ultrapure water. Then incubate for 20 min in a water bath at 45°C. Finally, the absorbance value was detected at a wavelength of 660 nm. Taking the standard inorganic phosphorus concentration as the ordinate, the absorbance value A 660...

Embodiment 3

[0143] The detection of embodiment 3.GPC

[0144] Precisely weigh the dried GPC standard and dissolve it in 10 ml of ultrapure water to prepare a GPC standard solution. The concentration of this solution is 23.8 mg / ml, which is equivalent to the GPC concentration of 92.53 μmol / ml.

[0145] Then the solution was diluted according to 2 times, 4 times, 8 times, 16 times, 32 times, 64 times, 128 times, 256 times, 512 times, 1024 times and 2048 times.

[0146] Take 80 μl of GPC solutions of various dilutions, add 100 μl of 2× buffer solution, about 1 unit of glycerophosphocholine phosphodiesterase WGPD-V018 prepared in Example 1, and about 1 unit of commercial CIAP enzyme ( NEWENGLAND company, Cat. No. M0290L), supplemented with ultrapure water to a reaction volume of 200 μl. The composition of the above 2× buffer is as follows:

[0147] 200mM Tris HCl, 20mM MgCl 2 , 100mM NaCl, pH8.0

[0148] Place the reaction tube in a 37°C water bath and incubate for 30 min or longer to...

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Abstract

The present invention provides a glycerophosphoryl choline (GPC) degradation or detection enzymologic method, a product and application thereof. The method comprises simultaneous or successive contacting of glycerophosphoryl choline phosphodiesterase and alkaline phosphatase with GPC in a sample for degrading of the GPC; quantification of inorganic phosphorus in a reaction product; and determination of content of the glycerophosphoryl choline in the to-be-tested sample in accordance with the determined inorganic phosphorus content. The method can be used for high sensitivity and high accuracy quick and easy degradation or detection of the GPC, and has broad application prospects.

Description

technical field [0001] The invention belongs to the fields of biotechnology and enzyme engineering. More specifically, the present invention relates to a quick and easy enzymatic method for degrading or detecting the content of glycerophosphocholine and its application. Background technique [0002] Glycerophosphocholine (Glycerophosphocholine, GPC, see the following formula for structure) is a water-soluble small molecular substance that normally exists in the human body. [0003] [0004] GPC is the biosynthetic precursor of the important neurotransmitter acetylcholine (Acetylcholine), which can support the function of the brain and nervous system. In vivo, the most important physiological function of GPC is to cross the blood-brain barrier and provide the necessary choline for the synthesis of acetylcholine and phospholipid (PC). Acetylcholine is an important neurotransmitter in the central nervous system, which helps the brain to complete learning, memory and cognit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48
Inventor 徐正军周美凤许骏杨天奎
Owner 益海嘉里(连云港)生物科技有限公司
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