The Preparation Of Novel Magnetic Nanomaterials And Application For Bromelain Purification

Bromelain,as a kind of hydrolytic enzyme,has a good ability of decomposing protein and the characteristic bond of amide and ester.It is widely used in food,medical,feed,cosmetics and other fields.However,it is difficult to extract the high purity bromelian because the structure of plant cells is complex containing many organic compounds,such as phenols,pigments and polysaccharide,which could affect the extraction of bromelian.In this sense,the development of effective methods for separation and purification of bromelain is increasingly essential.In this work,three kinds of magnetic nanomaterials were synthesized and used to separate and purify bromelain.The morphology,structure,and magnetic properties were characterized by Fourier transform infrared spectrometry（FT-IR）,Transmission electron microscopy（TEM）,Thermogravimetric analysis（TGA）,X-ray diffraction（XRD）and Vibrating sample magnetometry（VSM）.The details are as follows:（1）Thecore-shelltemperature-responsive Fe₃O₄@P（GMA-co-NIPAM）/IDA/Ni²⁺magnetic microspheres were prepared,and its morphology was characterized.The optimum adsorption conditions such as temperature,pH and time were optimized,and the maximum amount of adsorption was 117 mg/g.The Fe₃O₄@P（GMA-co-NIPAM）/IDA/Ni²⁺and bromelain mixed solution that reached adsorption equilibrium were denatured at 70°C for 10 min,after renaming at room temperature for 1 h,the bromelain enzyme activity recovered to35%,the enzyme activity returned to 40%after 2 hours,the free enzyme activity is only 7%of the original,demonstrating that the material has a certain thermal protection effect on bromelain.（2）The magnetic microsphere Fe₃O₄@SiO₂@P（NIPAM-co-AIM）/Ni²⁺adsorbent material with adjustable polymer shell was designed and synthesized,and it was applied to the separation and purification of bromelain.The adsorbent material with the ratio of NIPAM and AIM monomer of 1:1 was selected as the research object,and the parameters such as temperature,pH,adsorption time between the material and bromelain were optimized.The optimum adsorption capacity is 198.6 mg/g.Besides,the purification rate of bromelain in the crude extract of pineapple peel was 1.68,the extraction recovery was 61.68%,and the purification effect was significant.By SDS-PAGE analysis,it was proved that the purity of the bromelain solution extracted from the crude extract of pineapple peel had reached the electrophoretic purity.UV–vis,FT-IR and circular dichroism（CD）spectra were determined to demonstrate that process of adsorption and elution had no effect on the structure of bromelain and enzyme activity did not change.（3）Thefunctionalcompositesofmagneticgrapheneoxide GO@Fe₃O₄@PEI-Cu²⁺was designed and synthesized,and its properties and morphology were characterized.The optimal adsorption conditions,such as temperature,pH,time and other parameters,were optimized.The optimum adsorption was 357.4 mg/g.After 6 cycles of GO@Fe₃O₄@PEI-Cu²⁺,the adsorption amount of bromelain can still reach 221 mg/g,which proves that the material has excellent reusability.Finally,by SDS-PAGE analysis,it was proved that the purity of the bromelain solution extracted from the crude extract of pineapple peel had reached the electrophoretic purity.UV–vis,FT-IR and circular dichroism（CD）spectra were determined to demonstrate that process of adsorption and elution had no effect on the structure of bromelain and enzyme activity did not change.