IN SITU DETERMINATION OF THE STABILITY AND PERFORMANCE OF SOLID-SUPPORTED ENZYME CATALYSTS FOR THE RING-OPENING POLYMERIZATION OF ε-CAPROLACTONE
Sara Orski, Santanu Kundu, Richard Gross, and Kathryn Beers
The enzyme Candida antarctica Lipase B (CAL B), immobilized on a solid support, catalyzes the ring-opening polymerization of ε-caprolactone to make biodegradable polyesters. The immobilization of CAL B occurs through physisorption on a cross-linked poly(methyl methacrylate) (PMMA) bead, where weak hydrophobic interactions between the enzyme and the surface can permit catalyst desorption. Enzyme leaching decreases the concentration of remaining enzymes for polymerization, as well as contaminates and degrades the polycaprolactone (PCL) product. Furthermore, the enzyme surface can induce adsorption of other species which hinder activity of the physisorbed catalyst. Previous studies have determined that water concentration within the polymerization controls the equilibrium between active enzyme-polymer chains that propagate, and free enzyme, which can degrade polyester sites along the polymer. Determination of optimal reaction conditions at the polymer/enzyme interface can afford better control of polymerization from solid-supported catalysts and improve catalyst retention throughout the reaction. Evaluation of surface coverage, efficiency, and stability of CAL B at the surface, however, presents a significant measurement challenge.
A quartz crystal microbalance (QCM) was used to monitor in situ adsorption and desorption at the solid-support/enzyme interface, while deconvoluting background events, such as polymer swelling or non-specific binding. A PMMA thin film (50 nm) on a QCM crystal was fabricated to mimic the surface of the catalyst bead. The film is covalently immobilized through UV irradiation of benzophenone, present in both a surface monolayer and as a crosslinking agent (10 mol%) incorporated into the film. The hydrophobicity of the surface is representative of a native PMMA resin with a static water contact angle of 80°. Enzyme stability and PCL binding to the CAL B surface was monitored as the trace water content of the reaction incrementally increased. This will be used to determine the water content threshold for enzyme desorption and the conditions under which hydrolysis of the enzyme-activated polymer chains occur.