Direct binding and characterization of lipase onto magnetic nanoparticles.
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.
Huang
Shih-Hung
SH
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 701, ROC.
Liao
Min-Hung
MH
Chen
Dong-Hwang
DH
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
D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D008049:Lipase