title: Investigations of reaction kinetics for immobilized enzymes--identification of parameters in the presence of diffusion limitation.
abstract: A method is proposed for identification of kinetic parameters when diffusion of substrates is limiting in reactions catalyzed by immobilized enzymes. This method overcomes conventional sequential procedures, which assume immobilization does not affect the conformation of the enzyme and, thus, consider intrinsic and inherent kinetics to be the same. The coupled equations describing intraparticle mass transport are solved simultaneously using numerical methods and are used for direct estimation of kinetic parameters by fitting modeling results to time-course measurements in a stirred tank reactor. While most traditional procedures were based on Michaelis-Menten kinetics, the method presented here is applicable to more complex kinetic mechanisms involving multiple state variables, such as ping-pong bi-bi. The method is applied to the kinetic resolution of (R/S)-1-methoxy-2-propanol with vinyl acetate catalyzed by Candida antarctica lipase B. A mathematical model is developed consisting of irreversible ping-pong bi-bi kinetics, including competitive inhibition of both enantiomers. The kinetic model, which fits to experimental data over a wide range of both substrates (5-95%) and temperatures (5-56 degrees C), is used for simulations to study typical behavior of immobilized enzyme systems.
authors.affiliation: University of Stuttgart, Institute of Biochemical Engineering (IBVT), Allmandring 31, 70569 Stuttgart, Germany. wrberendsen@gmx.net
authors.forename: Wouter R
authors.initials: WR
authors.lastname: Berendsen
authors.forename: Alexei
authors.initials: A
authors.lastname: Lapin
authors.forename: Matthias
authors.initials: M
authors.lastname: Reuss
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002175:Candida / Q000201:enzymology; D002849:Chromatography, Gas; D004058:Diffusion; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005656:Fungal Proteins; D007700:Kinetics; D008049:Lipase / Q000737:chemistry*; D008956:Models, Chemical; D008962:Models, Theoretical; D012984:Software; D013237:Stereoisomerism; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica