title: Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: characterization and application for enzymatic inhibition assays.
abstract: Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe3O4-SiO2) possessed three dimensional core-shell structures with an average diameter of ~20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g(-1). The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The Km and the Vmax values (0.02 mM, 6.40 U·mg(-1) enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg(-1) enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds.
authors.affiliation: Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Yuan-Ting
authors.initials: YT
authors.lastname: Zhu
authors.affiliation: Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China.
authors.forename: Xiao-Yun
authors.initials: XY
authors.lastname: Ren
authors.affiliation: Department of Chemistry and Biochemistry, Jackson State University, 1400 Lynch St., Jackson, MS 39217, USA.
authors.forename: Yi-Ming
authors.initials: YM
authors.lastname: Liu
authors.affiliation: Changzhi Medical College, Changzhi 046000, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China.
authors.forename: Lin-Sen
authors.initials: LS
authors.lastname: Qing
authors.affiliation: Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China. Electronic address: liaoxun@cib.ac.cn.
authors.forename: Xun
authors.initials: X
authors.lastname: Liao
mesh_terms: D000818:Animals; D002264:Carboxylic Acids / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D057075:Enzyme Assays / Q000379:methods*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D058185:Magnetite Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D053761:Nanocomposites / Q000737:chemistry / Q000648:ultrastructure; D010179:Pancreas / Q000201:enzymology*; D059027:Recycling; D017550:Spectroscopy, Fourier Transform Infrared; D034421:Sus scrofa; D013696:Temperature; D013818:Thermogravimetry
chemical_list: D002264:Carboxylic Acids; D004800:Enzymes, Immobilized; D058185:Magnetite Nanoparticles; D008049:Lipase
keywords: Bioreactors; Covalent cross-linking; Enzyme activity; Immobilized enzymes; Lipase; Magnetic nanoparticles