Preparation Of Lipase-Inorganic Hybrid Nanoflowers And Its Catalytic Performance

Enzyme-inorganic hybrid nanoflowers are novel immobilized enzyme method developed in recent years,have the characteristics of simple synthesis steps and mild conditions.Moreover,compared with free enzyme,the prepared hybrid nanoflowers immobilized enzyme showed better catalytic performance.Therefore,enzyme-inorganic hybrid nanoflowers is a great promising immobilized enzyme method.In this study,we used lipase from Aspergillus oryzae to prepare lipase-inorganic hybrid nanoflowers(hNF-lipase)with high activity,activated lipase-inorganic hybrid nanoflowers(activated hNF-lipase),and magnetic activated lipase-inorganic hybrid nanoflowers(magnetic activated hNF-lipase),and investigated their catalytic performances.The main research contents are as follows.1.The metal ion had a significant effect on the morphology and activity of hNF-lipase.Compared with monovalent and trivalent metal ions(Ag+,Al3+,Fe3+),divalent metal ions(Mn2+,Zn2+,and Ca2+)and lipase are more easily to form hybrid nanoflowers with regular shape.When monovalent and trivalent metal ions(Ag+,Al3+,Fe3+)were used to synthesize hNF-lipase,activity recovery of hNF-lipase were 4.87%,3.48%,and 0.71%,respectively.However,when divalent metal ions(Mn2+,Zn2+and Ca2+)were used to synthesize hNF-lipase,activity recovery of hNF-lipase were 24.11%,30.20%and 43.05%,respectively.Under optimized preparation conditions(the concentration of phosphate radical of PBS is 4 mM,concentration of lipase is 0.02 mg/mL,concentration of calcium chloride is 12.5 mM and pH of PBS is 8),the activity recovery of hNF-lipase was 34%higher than before optimization,reached 77.4%.In addition,the Tween-80 can significantly activate the activity of hNF-lipase.When tween-80 concentration is at 0.15 mM,the maximum activity recovery of activated hNF-lipase is 172.08%,which is 2.2 times higher than the activity recovery of hNF-lipase.Fourier transform infrared(FT-IR)spectroscopy analysis showed that compared to hNF-lipase,the ?-helix content of activated hNF-lipase was decreased and ?-sheet was increased.This result showed that under the interface activation of Tween-80,the "?-helix lid" of the active center of lipase is opened,causing an active conformation of lipase.Therefore,the activity of activated hNF-lipase increased.Moreover,the optimal catalytic temperature and pH of hNF-lipase and activated hNF-lipase were 40 ? and 4,which were the same as those of free lipase.Compared with hNF-lipase and activated hNF-lipase,the michaelis constant(Km)of free lipase was the lowest,indicating that immobilization decrease the affinity of substrate and enzyme.However,the Vmax/Km of activated hNF-lipase was larger than that of free lipase and hNF-lipase,indicating that activated hNF-lipase exhibited the highest catalytic efficiency.In addition,compare with free lipase,hNF-lipase and activated hNF-lipase exhibited better temperature tolerance,denaturant tolerance and storage stability.Moreover,after reused six times,the residual activity of hNF-lipase and activated hNF-lipase maintained 60.7%and 67.6%,respectively.2.Magnetic activated hNF-lipase was successfully prepared by using direct embedding and covalent cross-linking methods to integrate Fe3O4 magnetic nanoparticles(MNPs)into activated hNF-lipase.The results showed that when concentration of Fe3O4 MNPs was 0.02 mg/mL,the maximum activity recovery of magnetic activated hNF-lipase prepared by direct embedding method was 189.64%.However,the highest activity recovery of magnetic activated hNF-lipase synthesized by covalent cross-linking method was 174%when concentration of glutaraldehyde was 0.1%,time of glutaraldehyde treatment carriers was 30 min and linking time was 1.5 h.Moreover,the optimal catalytic temperature and pH of free lipase and magnetic activated hNF-lipase were 40? and 4.The Km of the magnetic activated hNF-lipase was less than the free lipase,indicating that the magnetic activated hNF-lipase has a stronger affinity to the substrate than the free lipase.However,the Vmax/Km of magnetic activated hNF-lipase was less than that of free lipase,which indicates that the catalytic efficiency of magnetic activated hNF-lipase is lower than that of free lipase.In addition,compare with free lipase,magnetic activated hNF-lipase exhibited better temperature tolerance,methanol resistance and storage stability.Moreover,after repeated use ten times,the residual activity of magnetic activated hNF-lipase and activated hNF-lipase maintained 83.7%and 26%,respectively.