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Separation and purification method of haloperoxidase

A technology of halogen peroxide and peroxidase, which is applied in the direction of biochemical equipment and methods, enzymes, enzymes, etc., can solve the problem of less research on physiological functional proteins of red algae, increase the difficulty of enzyme separation and purification, and affect the detection of enzyme activity, etc. problems, to achieve the effect of low cost, easy operation and wide application prospects

Inactive Publication Date: 2009-03-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

Red algae of the genus Gracilaria are rich in polysaccharides (30-70% of the dry weight of algae) and phycobiliproteins (60% of soluble proteins), which affect the detection of enzyme activity and increase the difficulty of enzyme separation and purification. This is red The main reason for the lack of research on algae physiological functional proteins

Method used

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  • Separation and purification method of haloperoxidase
  • Separation and purification method of haloperoxidase
  • Separation and purification method of haloperoxidase

Examples

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Embodiment 1

[0026] Take 200g wet weight of Asparagus algae, grind it into powder with liquid nitrogen; add 200ml Tris-HCl buffer solution (0.02mol / L, pH6.5), soak at 0-5°C for 30h, and filter with gauze. Centrifuge at 4000g for 15min at 4°C, and take the supernatant. Crude extract plus solid (NH 4 ) 2 SO 4 To 15% of the final mass concentration, centrifuge at 7000g for 20min at 4°C. The precipitate was discarded, and the supernatant was used again (N H 4) 2 SO 4 Precipitate (65% of mass concentration), centrifuge at 10000g for 15min at 4°C. The precipitate was collected and dialyzed with 0.02mol / L, pH6.5 Tris-Cl buffer solution for 15 hours to obtain a crude enzyme solution of haloperoxidase; the molecular weight cut-off of the dialysis membrane was 14000. The dialyzed enzyme liquid sample was separated on a DEAE-sepharose column, and 500ml of Tris-HCl buffer containing NaCl concentration varied from 0.05 to 1.0M was eluted with a linear gradient of 0.02mol / L, pH 6.5. Collect at i...

Embodiment 2

[0032] Take 400 grams of Asparagus algae with a wet weight, grind it into powder with liquid nitrogen; add 300ml Tris-HCl buffer solution (0.08mol / L, pH8.5), soak at 6-8°C for 65h, and filter with gauze. Centrifuge at 7000g for 40min at 8°C, and take the supernatant. Crude extract plus solid (NH 4 ) 2 SO 4 To 25% of the final mass concentration, centrifuge at 14000g for 40min at 8°C. The pellet was discarded, and the supernatant was washed again with NH 4 ) 2 SO 4 Precipitate (85% of mass concentration), centrifuge at 15000g for 30min at 8°C. The precipitate was collected and dialyzed with 0.08mol / L, pH8.5 Tris-Cl buffer for 48 hours to obtain a crude enzyme solution of haloperoxidase; the molecular weight cut-off of the dialysis membrane was 14,000. The dialyzed enzyme solution sample was separated on a DEAE-cellulose column, and 500ml of Tris-HCl buffer solution containing NaCl concentration varied from 0.1 to 2.0M with a linear gradient of 0.08mol / L and pH8.5 was use...

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Abstract

The invention relates to a method for separating and purifying a halogen peroxidase. The method comprises the followings steps: using asparagus cultivated in Chinese waters as a raw material, and performing crude extraction, ammonium sulfate fractional precipitation, and ion exchange chromatography to remove abundant structural polysaccharides and phycoerythrin so as to obtain pure halogen peroxidase the molecular weight of which is about 60KD through electrophoresis. The halogen peroxidase is the only enzyme so far widely accepted and related to organic halide biosynthesis, which not only allows a plurality of organic halides to form halogen organic compounds having physiological activity, but also has the functions of catalyzing sulfur oxidation, epoxidation, indole oxidation and other reactions, thus the halogen peroxidase attracts more and more attentions in the aspects of drug synthesis and the synthesis of high added-value compounds. The halogen peroxidase is to be separated and purified from the asparagus cultivated in Chinese waters for the first time.

Description

technical field [0001] The invention relates to the separation and purification of haloperoxidase, that is, the establishment of a process for extracting and purifying haloperoxidase by using Gracilaria lemaneiformis as a raw material. Background technique [0002] Haloperoxidase is ubiquitous in seaweed, especially in red algae, and there are many studies on haloperoxidase of coralline algae (Corallina officinalis) at home and abroad. The growth of coralline algae has a strong seasonality and the amount of collection is small, which has become a bottleneck in the development and utilization of haloperoxidase. Asparagus, a unique species of Gracilaria, has become the third largest cultured large seaweed in China due to its fast adaptability and high growth rate. Red algae of the genus Gracilaria are rich in polysaccharides (30-70% of the dry weight of algae) and phycobiliproteins (60% of soluble proteins), which affect the detection of enzyme activity and increase the diffi...

Claims

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Application Information

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IPC IPC(8): C12N9/08
Inventor 张卫李海燕靳艳虞星炬金美芳吴佩春
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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