Separation and purification method and immobilization method of papain, and product thereof

A papain, separation and purification technology, applied in the direction of immobilized enzymes, biochemical equipment and methods, enzymes, etc., can solve problems such as many steps, large impact on enzyme activity, and increased enzyme ash content

Inactive Publication Date: 2011-11-23
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the existence of many steps in the separation and purification method of papain in the prior art, the cost is high, the enzyme activity is greatly affected, and the ash content in the enzyme will increase, and the method is not suitable for industrial production test scale-up etc. provide a method for separation and purification of papain with good biocompatibility, simple operation steps, mild operating conditions, easy amplification of the process, less product enzyme activity loss, no organic solvent residue, and greatly simplified immobilization steps Papain immobilization method and resulting product

Method used

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  • Separation and purification method and immobilization method of papain, and product thereof
  • Separation and purification method and immobilization method of papain, and product thereof
  • Separation and purification method and immobilization method of papain, and product thereof

Examples

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Embodiment 1

[0069] 1. Dissolve 40g papaya milk in 100ml 50mM cysteine ​​solution (containing 5mM EDTA, pH 6.5) at 4°C, centrifuge (20,000×g, 4°C, 15min) to remove insoluble matter, and measure the supernatant protein concentration (measured by conventional Coomassie brilliant blue staining method), the protein concentration of the supernatant is adjusted to 10 mg / ml with 50 mM cysteine ​​solution, and then the pH of the solution is adjusted to 6.5 (adjusted with 6M NaOH or HCl solution), which is the initial crude enzyme solution; meanwhile, configure 40% polyethylene glycol 2000 solution; configure 40% (w / w) ammonium sulfate solution, and adjust the pH to 6.5;

[0070] At 20°C, the initial crude enzyme solution, polyethylene glycol solution, ammonium sulfate solution and deionized water were mixed and dissolved together, wherein the mass percentage of the initial crude enzyme solution was 40%, and the mass percent of polyethylene glycol 2000 The percentage content is 30%, the mass percen...

Embodiment 2

[0075] At 4°C, 30g of papaya slurry was dissolved in 200ml of 25mM cysteine ​​solution (containing 3mM EDTA, pH 6.0), centrifuged (20,000×g, 4°C, 15min) to remove insoluble matter, and the supernatant protein concentration was measured with 25mM cysteine ​​solution to adjust the protein concentration of the supernatant to 10mg / ml, and then adjust the pH of the solution to 6.0 (adjusted by 6M NaOH or HCl solution), which is the initial crude enzyme solution; configure 40% polyethylene glycol 4000 solution; Simultaneously, configure 40% (w / w) sodium sulfate solution, adjust pH to be 6.0;

[0076]At 20°C, the initial crude enzyme solution, polyethylene glycol solution, sodium sulfate solution and deionized water were mixed and dissolved together, wherein the mass percentage of the initial crude enzyme solution was 35%, and the mass percent of polyethylene glycol 4000 The percentage content is 25%, the mass percentage content of sodium sulfate is 19%, and the mass percentage conte...

Embodiment 3

[0079] At 4°C, 20g of papaya powder was dissolved in 100ml of 30mM cysteine ​​solution (containing 0.5mM EDTA, pH 5.8), centrifuged (20,000×g, 4°C, 15min) to remove insoluble matter, and the supernatant protein concentration was measured. Use 30mM cysteine ​​solution to adjust the protein concentration of the supernatant to 15mg / ml, and then adjust the pH of the solution to 5.8 (adjusted by 6M NaOH or HCl solution), which is the initial crude enzyme solution; meanwhile, configure 40% polyethylene glycol 6000 solution; 40% (w / w) phosphate solution at pH 5.8 from 40% (w / w) K 2 HPO 4 and 40% (w / w) NaH 2 PO 4 Prepared.

[0080] At 20°C, the initial crude enzyme solution, polyethylene glycol solution, phosphate solution and deionized water were mixed and dissolved together, wherein the mass percentage of the initial crude enzyme solution was 30%, and the mass percent of polyethylene glycol 6000 The percentage content is 18%, the mass percentage content of phosphate is 10%, and ...

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Abstract

The invention discloses a separation and purification method and an immobilization method of papain, and a product thereof. The separation and purification method comprises the following steps: 1, obtaining an initial crude enzyme solution through dissolving a raw material containing papain, removing insoluble substances, and adjusting the protein concentration to 10-20mg / ml; 2, mixing the initial crude enzyme solution with polyethylene glycol, an inorganic salt and water, dissolving, and carrying out phase separation after all component are uniformly mixed, wherein the content of the initial crude enzyme solution is 30-40%, the content of polyethylene glycol is 10-30%, the content of the inorganic salt is 10-25%, and the pH value is 5.5 to 6.5; and 3, taking a polyethylene glycol phase obtained after the phase separation, and recovering the papain through an ion exchange method. The immobilization method is that papain is immobilized in the polyethylene glycol phase which is enriched in papain. The separation and purification method allows good biological compatibility, simple operation steps, and mild operation conditions to be realized, processes to be easily amplified, less enzyme activity loss of products and no organic solvent residual to be achieved, and immobilization steps to be substantially simplified.

Description

technical field [0001] The invention relates to a papain separation and purification method, its immobilization method, and the obtained product. Background technique [0002] Papain is an important plant-derived protease with a wide range of applications. It can not only catalyze the hydrolysis of protein, but also has many activities such as amidase activity, esterase activity, transaminase activity, transesterase activity and hydantoinase activity under specific conditions. Papain can hydrolyze antibodies to prepare Fab fragments, synthesize peptides, ester amino acid surfactants, amino acid esters and other compounds, which have been widely used in food, biology, chemical industry, detergent, medicine, cosmetics and other fields. [0003] Papain is extracted from papaya. The product obtained by drying the papaya slurry directly or slightly after refining is the crude product of papain, which generally contains papain (Papain, EC 3.4.22.2), glycine endopeptidase (Glycyl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12N11/00
Inventor 苏二正魏东芝李明亮龚翔宇
Owner EAST CHINA UNIV OF SCI & TECH
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