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
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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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