Immobilization of Candida antarctica lipase B on resins and flat films: Effects of physical properties and surface chemistry of matrices.

摘要

Methyl methacrylate resins with identical average pore diameter (250 A) and surface area (500 m2/g) but with varied particle size (35 to 560-710 mum) and polystyrene resins with varied particle size (35 to 350-600 mum) and pore diameter (300 to 1000A´) were employed to study how the immobilization resin particles of varying size and pore diameter influences Candida antarctica Lipase B (CALB) loading, fraction of active sites and catalytic properties for polyester synthesis. CALB was also covalently immobilized onto epoxy-activated macroporous poly(methyl methacrylate) Amberzym beads (235 mum particle size, 220 A pore size) and nanoparticles (nanoPSG, diameter 68 nm) with a poly(glycidyl methacrylate) outer region. The majority of CALB is covalently linked to resins confirmed by changing the immobilization time and by IR microspectroscopy images, The activity of CALB assessed by epsilon-CL ring-opening polymerizations increased in the order of Amberzym-CALB, Novozym 435 and nanoPSG-CALB. Amberzyme-CALB showed excellent stability when reused over three reaction cycles for epsilon-CL ring-opening polymerization and polycondensations reactions. For the former, over three reaction cycles, %-epsilon-CL conversion versus time plots appear identical. Furthermore, a series of epoxy activated extremely flat polymer films composed of poly(GMA/BMA/HEMA) were prepared to study the performance of CALB binding to surfaces varied in wettabi1ity. The occurrence of single CALB molecules and aggregates of CALB at surfaces was determined by AFM imaging and measurements of volume. The change of absolute numbers of protein monomers and multimers on flat films were quantitatively determined, which was used for analysis of CALB specific activity. A hypothesis was formulated to explain how protein conformation and/or orientation influences CALB activity due to the protein-surface and protein-protein interactions by changing film wettability and enzyme loading.