Study On Immobilization Of Enzyme Based On Magnetic Fe₃O₄ Nanoparticles

Magnetic Fe₃O₄nanoparticles have good application prospects in enzyme immobilization due to their superparamagnetic properties,good biocompatibility,large specific surface area,and easy separation from reaction systems.Catalase（CAT）has the ability to convert hydrogen peroxide into water and oxygen,and has the effect of anti-oxidative stress,which can effectively protect organisms and is widely used in food processing,textile and paper making and other fields.β-glucuronidase（β-GUS）mainly acts on the glycosidic bond of the substrate,and can hydrolyze the glucuronic acid bond of glycyrrhizic acid（GL）to generate more pharmacologically active glycyrrhetic acid（GA）,which has potential application in the field medicine.The immobilized enzyme greatly improves the thermal stability and operational stability of the free enzyme,and it is easy to achieve reusability and reduce costs.In this paper,superparamagnetic Fe₃O₄nanoparticles were used to immobilize CAT andβ-GUS.The main work is as follows:（1）The target gene kat E was constructed into the plasmid vector p ET28a-His,and then transferred into E.coli BL21（DE3）which could express and obtain CAT with modified6×His Tag at the ends of amino and carboxyl.The CAT was directly immobilized on magnetic Fe₃O₄nanoparticles via a solvothermal method（Fe₃O₄-CAT）.However,Fe₃O₄-CAT is difficult to separate in the reaction solution.（2）The Fe₃O₄nanoparticles were modified with transition metal ions Ni²⁺（Fe₃O₄@Ni）,and the CAT was immobilized by a metals chelation between Ni²⁺and His-Tagged to form Fe₃O₄@Ni-CAT.Uner the optimization of conditions,the amount of immobilized CAT reached 376.12 mg/g,and the Fe₃O₄@Ni-CAT activity reached 4946 U/g.A three-factor and three-level experiment was designed by the response surface methodology Box-Behnken（BBD）center combination,and the reaction temperature（X1）,solution p H（X2）and substrate concentration（X3）were selected as the influencing factors,and the catalysis of Fe₃O₄@Ni-CAT was investigated.The H₂O₂reaction was optimized,and the optimal reaction conditions were obtained as follows:added Fe₃O₄@Ni-CAT of 0.025 g in the H₂O₂of 14.5 mmol/L solution（p H6.4）at 40℃,the H₂O₂removal rate reached to84.32%,which was consistent with the fitting results 83.63%are basically the same.After repeated use of 10 batches of H₂O₂,the clearance rate still maintained its initial 69.07%,with certain reusability.（3）The Fe₃O₄nanoparticles were modified with hexadecyl quaternary ammonium salt（CTAC）to obtain Fe₃O₄@CTAC nanoparticles,and were used to immobilize on E.coli BL21（DE3）containingβ-GUS to obtain in situ immobilizedβ-GUS（GUS@C@Fe₃O₄）.The optimal immobilization conditions for preparing GUS@C@Fe₃O₄were reaction 2 h,bacterial content（dry weight）6 g/L,temperature 30℃,p H 5.The study of enzyme activity characteristics found that GUS@C@Fe₃O₄exhibited the maximum enzyme activity at40℃and p H 5.And it has better thermal stability and p H stability than free cells.GUS@C@Fe₃O₄catalytic hydrolysis of glycyrrhizic acid to produce glycyrrhetic acid was optimized,and the highest conversion rate of glycyrrhizic acid was 88.28%at 40℃and p H 5 with a reaction time of 2 h.The conversion rate of repeated use of 5 batches remains at 73.03%,which has a certain reusability.