Method for purifying velardon with cobalt ion metal chelate affinity film

A technology of papain and metal chelation, applied in the direction of hydrolytic enzymes, etc., can solve the problems of low purity, long separation time, and high feeding speed, etc., achieve purification multiples and high enzyme activity, and facilitate extraction and purification. Raw materials source convenience effect

Inactive Publication Date: 2009-04-08
DONGHUA UNIV
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  • 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 with cobalt ion metal chelate affinity film
  • Method for purifying velardon with cobalt ion metal chelate affinity film
  • Method for purifying velardon with cobalt ion metal chelate affinity film

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The activation of the nylon membrane and the modification of the bonded chitosan, the specific steps are as follows:

[0030] (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.

[0031] (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

[0033] The modified nylon membrane chelates metal cobalt ions, and the specific steps are as follows:

[0034] (1) 100 sheets (about 10 grams) of nylon membranes after the above-mentioned modification were immersed in 4.6ml epichlorohydrin, 46ml 2mol / L sodium hydroxide and 5mmol sodium borohydride mixed solution, reacted at room temperature for 2 hours, and then directly applied to Add 23ml of epichlorohydrin and 46ml of 2mol / L sodium hydroxide to the mixed solution, react at 60°C for 12h, and wash with deionized water several times.

[0035] (2) Immerse 10 films (approximately 1 gram) of the above-mentioned combined cyclopropane into a mixed solution containing 86mmol of iminodiacetic acid (IDA), 8mmol of sodium borohydride and 114.4ml of 2mol / L sodium carbonate, and shake at a constant temperature of 60°C After reacting for 12 hours, wash with deionized water, 5vol.% acetic acid and deionized water several times.

[0036] (3) The above-mentioned film is immersed in 500ml of...

Embodiment 3

[0038] Use metal chelate membrane to adsorb papain, and optimize the conditions. The specific steps are as follows:

[0039] (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) 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 reaction time is chosen at 30min.

[0040] (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 The pH values ​​of the Tris-HCl solution added with papain were 5.0, 6.0, 7.0, 8.0, 9.0, ...

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Abstract

The invention relates to a method for purifying the papain by using a chelating affinity membrane of a cobalt ion metal, which comprises the following steps: (1) an activated nylon membrane is modified by the bonding of the chitosan; (2) the modified nylon membrane reacts with the mixed solution of epichlorohydrin, sodium hydroxide and sodium borohydride, then the membrane is immerged respectively into the mixed solution of sodium carbonate, sodium borohydride and coupling agent, and cobalt chloride solution to obtain the metal affinity membrane of the chelating cobalt ion; (3) the metal affinity membrane is piled up to a membrane stack, put onto a membrane bridge, transferred into Tris-HCl buffer and eluate by a peristaltic pump, then into the papain solution, is eluted by eluting agents, and then the papain is produced; and (4) the activity of enzyme and the protein content are measured, and the multiple of purification is calculated. The method has the advantages of rapidness, high efficiency, simpleness and big separation capacity. The extracted enzyme is good in activity and applicable to commercial production.

Description

technical field [0001] The invention relates to the field of protease purification, in particular to a method for purifying papain by using a cobalt ion metal chelate 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 a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50
Inventor 朱利民何智妍聂华丽周毓婷胡映清
Owner DONGHUA UNIV
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