Method for purifying lysozyme from milk
A lysozyme, separation and purification technology, applied in the biological field, can solve the problems of long production cycle, high price, limited application scope, etc.
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Embodiment 1
[0133] Primary processing of raw materials
[0134] The purpose of the primary treatment of the raw material is to remove impurities and fats. This embodiment adopts centrifugal method to carry out primary treatment.
[0135] Get 100L of transgenic goat milk (from Shanghai Transgenic Research Center), measure the content of human lysozyme, it is 1g / L. The milk secreted by the transgenic goat is divided into centrifuge bottles, centrifuged at 4° C. at low temperature, 8000-10000 rpm, for 15-30 minutes. After centrifugation, remove the precipitated impurities and the fat in the upper layer, take the skim milk in the middle layer, and filter it with gauze to get the skim whey, a total of 85L, and store it in low temperature storage for later use.
Embodiment 2
[0137] Cationic Resin Treatment and Regeneration
[0138] Resin type: 724 resin.
[0139] 724 resin should be re-used after acid-base treatment, and the used resin can be regenerated and recycled. Methods as below:
[0140] Resin pretreatment:
[0141] (1) Remove impurities with pure water, then stir and soak with 3wt% NaOH solution for 4-6 hours, wash with pure water for several times until the pH value of the effluent is close to 7.5;
[0142] (2) 4wt% HCl solution was stirred and soaked for 4-6 hours, and then washed with pure water for several times until the pH value of the effluent was close to 5.5;
[0143] (3) Stir and soak with 4wt% NaOH solution for 4-6h, wash with pure water several times until the pH value of the effluent is close to 7.5;
[0144] (4) Finally, treat the 724 resin with 0.2 mol / L phosphate buffer solution, pH=6.5, for 1 hour.
[0145] Used Resin Regeneration:
[0146] Stir and soak in 4wt% HCl solution for 4-6 hours, wash with pure water, stir ...
Embodiment 3
[0148] 1. Stirring adsorption of skimmed whey and 724 resin
[0149] Resin consumption: by volume, the resin is weighed at a ratio of 30%, that is, 724 resin:skimmed whey=30:70.
[0150] Add the ready-to-use 724 resin and skimmed whey (85L) into a container or reactor, and stir and adsorb at about 0-4°C for 6 hours.
[0151] 2. Elution of foreign proteins
[0152] After stirring and absorbing, elute with pure water several times until the solution is clear; then use 3 times the resin volume of 0.1mol / L NaCl, 40mmol / L phosphate solution, pH6.5, elution 3 times, each time for 30 minutes , so that impurities are removed.
[0153] 3. Final eluate to elute lysozyme
[0154] The lysozyme adsorbed by the resin was eluted with 5% ammonium sulfate solution (prepared with 40 mmol / L phosphate solution, pH 6.5) equal to the volume of the resin, and eluted 4 times for 40 minutes each time, and the eluate was collected each time. Based on 85L of skimmed whey, a total of 140L of collecte...
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