Preparation method for alkaline phosphatase

A technology for phosphatase and phosphatase activity, which is applied in the field of alkaline phosphatase extraction and preparation, can solve the problems of high cost and difficult labeling, and achieves the effects of low cost, improved comprehensive utilization value and simple operation process.

Active Publication Date: 2012-12-26
西藏天虹科技股份有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In immunology research, AP-labeled antibody has been widely used in enzyme-linked immunofluorescence reaction (ELISA) and Western blot analysis, that is, AP interacts with chromogenic reagent or dephosphorylated substrate that can emit light to reveal the target and detect the enzyme. The existence of the complex, compared with peroxidase, the advantages of AP as a labeling enzyme are high stability and high sensitivity, and the disadvantages are high cost and difficult labeling

Method used

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  • Preparation method for alkaline phosphatase

Examples

Experimental program
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Effect test

Embodiment 1

[0029] (1) Preparation of crude enzyme solution

[0030] 25kg of fresh pig kidney cortex was chopped, added Tris-HCl buffer 75L (50mmol / L, pH7.8), homogenized for 3min (30s×6), stirred at 4°C for 30min, centrifuged at 3000×g for 30min, and the supernatant was It is the crude enzyme solution of alkaline phosphatase.

[0031] (2) acid precipitation

[0032] The resulting crude enzyme solution was adjusted to pH 5.0 with HAc, stirred for 4 h, centrifuged at 8000×g for 30 min, and the precipitate was dissolved in 75 L (50 mmol / L, pH 7.0) Tris-HCl buffer. Add an equal volume of 75 L of n-butanol, stir for 1 h, centrifuge at 1000×g for 30 min, absorb the water layer and dialyze against Tris-HCl buffer (50 mmol / L, pH 7.8), and concentrate to 1 L.

[0033] (3)(NH 4 ) 2 SO 4 salting out

[0034] With 30% saturation (NH 4 ) 2 SO 4 Precipitate, stir for 30min, centrifuge at 6000×g for 30min, and remove the precipitate. Supernatant with 80% (NH 4 ) 2 SO 4 Precipitate, stir fo...

Embodiment 2

[0044] (1) Preparation of crude enzyme solution

[0045] 25kg of fresh pig liver was shredded, added 75L of Tris-HCl buffer solution (50mmol / L, pH7.8), homogenized for 3min (30s×6), stirred at 4°C for 30min, centrifuged at 3000×g for 30min, and the supernatant was Alkaline phosphatase crude enzyme solution.

[0046] (2) acid precipitation

[0047]The resulting crude enzyme solution was adjusted to pH 5.5 with HAc, stirred for 3.5 h, centrifuged at 8000×g for 30 min, and the precipitate was dissolved in 75 L (50 mmol / L, pH 7.0) Tris-HCl buffer. Add an equal volume of 75 L of n-butanol, stir for 1 h, centrifuge at 1000×g for 30 min, absorb the water layer and dialyze against Tris-HCl buffer (50 mmol / L, pH 7.8), and concentrate to 1 L.

[0048] (3)(NH 4 ) 2 SO 4 salting out

[0049] With 30% saturation (NH 4 ) 2 SO 4 Precipitate, stir for 30min, centrifuge at 6000×g for 30min, and remove the precipitate. Supernatant with 80% (NH 4 ) 2 SO 4 Precipitate, stir for 30min...

Embodiment 3

[0059] (1) Preparation of crude enzyme solution

[0060] Wash the small intestine of the pig, take 25kg of its mucous membrane and cut it into pieces, add 75L of Tris-HCl buffer solution (50mmol / L, pH7.8), homogenate for 3min (30s×6), stir at 4°C for 30min, centrifuge at 3000×g for 30min, The supernatant is the alkaline phosphatase crude enzyme solution.

[0061] (2) acid precipitation

[0062] The resulting crude enzyme solution was adjusted to pH 5.0 with HAc, stirred for 3.5 h, centrifuged at 8000×g for 30 min, and the precipitate was dissolved in 75 L (50 mmol / L, pH 7.0) Tris-HCl buffer. Add an equal volume of 75 L of n-butanol, stir for 1 h, centrifuge at 1000×g for 30 min, absorb the water layer and dialyze against Tris-HCl buffer (50 mmol / L, pH 7.8), and concentrate to 1 L.

[0063] (3)(NH 4 ) 2 SO 4 salting out

[0064] With 30% saturation (NH 4 ) 2 SO 4 Precipitate, stir for 30min, centrifuge at 6000×g for 30min, and remove the precipitate. Supernatant with ...

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Abstract

The invention relates to a preparation method for alkaline phosphatase, which mainly comprises the following steps: rubbing and centrifuging the abandoned visceral organs after an animal is slaughtered, thereby obtaining coarse alkaline phosphatase liquid, and then performing the processes of acid deposition, salting-out, column chromatography, dialysis and vacuum freeze drying, thereby finally obtaining the alkaline phosphatase, wherein the specific enzymic activity of the alkaline phosphatase is above 402U/mg. The recovery rate of the alkaline phosphatase prepared according to the preparation method is above 10%; the purifying times is above 1297 times; the whole process has the advantages of short period, typical production equipment, simple operation process, low cost, high efficiency, high product purity, and the like; and the preparation method is suitable for popularization.

Description

1. Technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for extracting and preparing alkaline phosphatase. 2. Background technology [0002] Alkaline phosphatase (alkaline phosphatase, EC3.1.3.1AP) is a non-specific phosphomonoesterase, which can catalyze the hydrolysis reaction of almost all phosphate monoesters to generate inorganic phosphoric acid and corresponding alcohols, phenols, sugars, etc., and can also catalyze Phosphate group transfer reaction, and E. coli AP is also a phosphite-dependent hydrogenase. AP exists in almost all organisms except higher plants, can directly participate in phosphorus metabolism, and plays an important role in the digestion, absorption, secretion and ossification of calcium and phosphorus. [0003] Alkaline phosphatase is mainly used clinically for the diagnosis and differential diagnosis of bone and hepatobiliary system diseases, especially the differential diagnosis of jaundice. For u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16
Inventor 王福华
Owner 西藏天虹科技股份有限责任公司
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