title: Direct binding and characterization of lipase onto magnetic nanoparticles.
abstract: Lipase was covalently bound onto Fe(3)O(4) magnetic nanoparticles (12.7 nm) via carbodiimide activation. The Fe(3)O(4) magnetic nanoparticles were prepared by coprecipitating Fe(2+) and Fe(3+) ions in an ammonia solution and treating under hydrothermal conditions. The analyses of transmission electron microscopy (TEM) and X-ray diffraction (XRD) showed that the size and structure of magnetic nanoparticles had no significant changes after enzyme binding. Magnetic measurement revealed the resultant lipase-bound magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu/g (only slightly lower than that of the naked ones (64 emu/g)), a remanent magnetization of 1.0 emu/g, and a coercivity of 7.5 Oe. The analysis of Fourier transform infrared (FTIR) spectroscopy confirmed the binding of lipase onto magnetic nanoparticles. The binding efficiency of lipase was 100% when the weight ratio of lipase bound to Fe(3)O(4) nanoparticles was below 0.033. Compared to the free enzyme, the bound lipase exhibited a 1.41-fold enhanced activity, a 31-fold improved stability, and better tolerance to the variation of solution pH. For the hydrolysis of pNPP by bound lipase at pH 8, the activation energy within 20-35 degrees C was 6.4 kJ/mol, and the maximum specific activity and Michaelis constant at 25 degrees C were 1.07 micromol/min mg and 0.4 mM, respectively. It revealed that the available active sites of lipase and their affinity to substrate increased after being bound onto magnetic nanoparticles.
authors.affiliation: Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 701, ROC.
authors.forename: Shih-Hung
authors.initials: SH
authors.lastname: Huang
authors.forename: Min-Hung
authors.initials: MH
authors.lastname: Liao
authors.forename: Dong-Hwang
authors.initials: DH
authors.lastname: Chen
mesh_terms: D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis / Q000737:chemistry*; D004355:Drug Stability; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry* / Q000648:ultrastructure*; D008280:Magnetics / Q000295:instrumentation*; D008422:Materials Testing / Q000379:methods*; D008863:Microspheres; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods*; D010316:Particle Size; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature
chemical_list: D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D008049:Lipase