Research Of Cu/Zn-SOD Extraction Optimization And Preparation Of PAH/PSS Micro Capsule Using Layer-by-Layer Self-assembly Technique

SOD (superoxide dismutase, SOD) widely exists in animals, plants and algae,in this study,we based on the ox blood as experimental materials, in addition,we conducted a study of the best extraction conditions of Cu/Zn-SOD extraction from cow blood, and then we preparation Cu/Zn-SOD coated with polyelectrolyte micro capsule using the LBL technique, finally, we take an experiment of trypsin hydrolysis on the polyelectrolyte micro capsule. The results are as follows.(1)We study the four factors such as concentration of Cu2+and Zn2+、thermal denaturation temperature and denaturation time effect on Cu/Zn-SOD activity and determined the best extraction condition of Cu/Zn-SOD using the L25(4,5)orthogonal experiment based on the four factors,and then we determined the enzyme activity through the method of the pyrogallol autoxidation、the content of protein measured by using coomassie brilliant blue staining、the protein purity through PAGE.it turned out that we can obtain Cu/Zn-SOD with higher activity and specific activity when the concentration of copper and zine ion is6mmol/L and12mmol/L,the thermal denaturation temperature and denaturation time is90℃and35min.eventually,the activity of Cu/Zn-SOD is4.14×103U/mL and the specific activity is6.74×104U/mg.(2) The layer-by-layer self-assembly technique was used to prepare the polyelectrolyte micro capsule containing Cu/Zn-SOD.amorphous type calcium carbonate as a template,Cu/Zn-SOD adsorbed to the calcium carbonate particles using the principle of coprecipitation. assembly [poly (sodium styrene sulfonate), PSS] and [poly (allyamine hydrochloride), PAH] in the surface of calcium carbonate,we acquire the particles that calcium carbonate as the mother nucleus and the PAH/PSS for shell,we dissolve calcium carbonate using ethylenediamine tetraacetic acid(EDTA) and then add G.2M copper ions and zine ions to restore the Cu/Zn-SOD activity,in the end,we get the polyelectrolyte micro capsule containing Cu/Zn-SOD.(3) We conducted trypsin hydrolysis experiment of Cu/Zn-SOD packaged with polyelectrolyte and nature Cu/Zn-SOD,the result is the activity of Cu/Zn-SOD is3.02×103U/mL before parcel,protein content is0.059mg/mL and specific activity is5.14×104 U/mg.after package,Cu/Zn-SOD activity is2.61×103U/mL,protein content is0.047mg/mL reducing0.012mg/mL than they did before and the rate of parcel package is79.41%,the specific activity is5.14×104U/mg.after4hours hydrolyzed by trypisn,the activity of Cu/Zn-SOD packaged with polyelectrolyte is1.99×103U/mL, relative specific activity is75.53%,the activity of nature Cu/Zn-SOD is8.65×102U/mL,relative specific activity is28.71%,it turned out that the Cu/Zn-SOD with polyelectrolyte micro capsule really played a protective effect of Cu/Zn-SOD activity.