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Method for purifying velardon by using metallic affinity membrane

A papain and affinity membrane technology, applied in hydrolase and other directions, can solve the problems of cumbersome process, long separation time, short diffusion path, etc., and achieve the effect of wide source of raw materials, high multiple and high enzymatic activity

Inactive Publication Date: 2011-02-09
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the extraction and purification of papain mainly adopts the precipitation method, but this method is cumbersome, and the product is still mixed with other proteases, the purity is not high, and it cannot meet the needs of the pharmaceutical industry
As people put forward further requirements for purity and safety, chromatographic techniques are applied to the purification of papain, such as ion exchange chromatography and affinity chromatography. Low axial rate limitation, large pressure drop, long separation time, small throughput, cannot use high feed rate
Membrane separation is a separation technology based on the size of molecules. It is characterized by large surface area, short diffusion path, low operating pressure, and large processing capacity. However, traditional membrane separation technology can only separate species with a relative molecular mass difference of more than 10 times. Meet the needs of biochemical separation

Method used

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  • Method for purifying velardon by using metallic affinity membrane
  • Method for purifying velardon by using metallic affinity membrane
  • Method for purifying velardon by using metallic affinity membrane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The activation of the nylon membrane and the modification of the bonded chitosan, the specific steps are as follows:

[0027] (1) The nylon membrane was first hydrolyzed in 1M HCl at 25°C for 24 hours, and then activated with formaldehyde. Ten hydrolyzed nylon membranes were immersed in 20ml of formaldehyde solution (>36.5wt.%), added with 0.2ml of phosphoric acid (85wt.%), reacted at 60°C for 7h, and washed with hot water at 40-50°C for several times.

[0028] (2) The above-mentioned formaldehyde-activated nylon membrane is immersed in 10ml of chitosan solution (dissolved with 1vol.% acetic acid) with a mass fraction of 1.5%, reacted at room temperature for 1h, then transferred to an 80°C oven, took it out after 1h, and used 1vol .% acetic acid and deionized water to wash away unreacted chitosan. The content of chitosan on the nylon film can be tested according to the ninhydrin method.

Embodiment 2

[0030] The modified nylon membrane chelates metal copper ions, and the specific steps are as follows:

[0031] (1) The above modified 10 nylon membranes were immersed in 4.6ml of epichlorohydrin, 46ml of 2M sodium hydroxide and 0.017g of sodium borohydride mixture, reacted at room temperature for 2 hours, and then directly added 23ml of oxygen Chloropropane, 46ml of 2M sodium hydroxide, react at 60°C for 12h, and wash with deionized water several times.

[0032] (2) Immerse the above-mentioned cyclopropane-bound membrane into a mixed solution containing 10ml 8.6M iminodiacetic acid (IDA), 0.14g sodium borohydride and 114.4ml 2M sodium carbonate, react at a constant temperature at 60°C for 12h, and use deionized Wash with water, 5vol.% acetic acid and deionized water several times.

[0033] (3) Immerse the above-mentioned film in 500ml of 100ppm copper sulfate solution and react for 2h to obtain a metal film that chelates copper ions.

Embodiment 3

[0035] Use metal chelate membrane to adsorb papain, and optimize the conditions. The specific steps are as follows:

[0036] (1) Determination of reaction time. Stack 10 modified nylon membranes to form a membrane stack, put them into the membrane bridge, and send 100ml of 1.0mg / ml papain Tris-HCl solution (pH=8.5) with a peristaltic pump at a flow rate of 2.0ml / min. 10min, 20min, 30min, 40min, 50min, 60min, 80min and 100min, carry out adsorption, and measure the amount of papain adsorption. Experimental results such as figure 1 , the optimal response time is selected at 60min.

[0037] (2) Determination of reaction pH. Stack 10 modified nylon membranes to form a membrane stack, put them into the membrane bridge, and send 100ml of 1.0mg / ml papain Tris-HCl solution with different pH values ​​into it with a peristaltic pump, and circulate the reaction for 2 hours at a flow rate of 2.0ml / min , adding papain in Tris-HCl solution with pH values ​​of 5.0, 6.0, 7.0, 8.0, 9.0, 10....

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Abstract

The invention relates to a method for purifying papain with a metal affinity membrane, which comprises the steps that (1) a nylon membrane is activated with formaldehyde and modified with chitosan; (2) the activated and modified nylon membrane reacts with the mixture solution of epichlorohydrin, sodium hydroxide and sodium borohydride; and the membrane is submersed in the mixture solution of sodium carbonate, sodium borohydride and an coupling agent, and in copper sulphate solution, respectively to get the chelating copper ion metal affinity membrane; (3) the metal affinity membrane is foldedup to become a membrane pile, and the membrane pile is arranged in a membrane bridge before the membrane pile is fed into Tris-HCl buffer solution and eluent with a peristaltic pump; subsequently, the membrane pile is fed into papain solution, and is eluted with the eluent, thus getting the target product, namely papain; (4) the enzyme activity and the protein content are tested, and purificationfactor is calculated. The method is quick, simple and convenient, and can be used for separating papain in large quantity, and is applicable for large-scale production. In addition, the activity of the extracted enzyme is excellent.

Description

technical field [0001] The invention relates to the field of protease purification, in particular to a method for purifying papain by using a metal affinity membrane. Background technique [0002] Papain (Papain) referred to as papain, also known as papaya enzyme, enzymatic number EC3.4.22.2, is a pure natural biological enzyme product extracted from the milk of immature papaya fruit and refined by modern bioengineering technology , it is a kind of protease containing thiol (~SH), which has the activity of protease and esterase. Its wide specificity has strong hydrolysis ability to animal and plant proteins, polypeptides, esters, amides, etc., and can catalyze various proteins. It is hydrolyzed into a series of short peptides or amino acids of different lengths, and protein hydrolyzate can also be synthesized into proteinoids. Because of its good thermal stability and natural hygiene and safety, it is widely used in food, leather, textile, daily chemical and pharmaceutical ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/50
Inventor 朱利民何智妍聂华丽
Owner DONGHUA UNIV
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