Thermally induced inactivation and aggregation of urease: Experiments and population balance modelling Peter Grancic a,c , Viera Illeova b , Milan Polakovic b , Jan Sefcik a,n a Department of Chemical and Process Engineering, University of Strathclyde, 75 Montrose Street, Glasgow G1 1XJ, UK b Institute of Chemical and Environmental Engineering, Slovak Technical University, Radlinskeho 9, 812 37 Bratislava, Slovakia c Chemical Robotics Laboratory, Department of Chemical Engineering, Institute of Chemical Technology, Technicka 5, 166 28 Praha 6, Czech Republic article info Article history: Received 20 December 2010 Received in revised form 8 June 2011 Accepted 19 July 2011 Available online 7 August 2011 Keywords: Enzyme Protein Kinetics Equilibrium Denaturation Unfolding abstract We present a population balance model for enzyme deactivation and aggregation kinetics with a limited number of physically relevant parameters and use this model to analyse the experimental data for thermal inactivation of jack bean urease. The time dependence of the relative enzymatic activity was found to follow the second order kinetics, which was consistent with pre-equilibrated folding/ unfolding of the native enzyme, followed by irreversible cluster–cluster aggregation of the non-native enzyme resulting in gradual and permanent loss of enzymatic activity. Monomer–cluster aggregation scenario was considered but was not consistent with the observed kinetic order of monomer disappearance at longer times. We analysed time evolution of the average hydrodynamic radius obtained from dynamic light scattering measurements and by fitting these data with our model, we were able to estimate the value of the unfolding equilibrium constant with a reasonable accuracy (K c E0.05 at 80 1C). We were also able to make order of magnitude estimates of the maximum number of enzyme molecules in the aggregated clusters (hundreds) as well as the aggregation rate constant of the non-native enzyme. & 2011 Elsevier Ltd. All rights reserved. 1. Introduction Understanding and controlling aggregation of proteins is crucial for their downstream processing, including purification, sterilisation and storage. Control of protein aggregation state is necessary for improvement of stability of therapeutic proteins as well as industrial enzymes. Protein stability is a particularly relevant issue in the biotechnology and pharmaceutical industries for rational development of novel production, separation and sensing procedures in bioprocessing. Robust control of protein stability is particularly difficult because proteins are often only marginally stable and are highly susceptible to thermal, chemical and physical degradation. Non-native aggregation is particularly problematic because it is encountered routinely during purifica- tion, sterilisation, shipping and storage processes. Enzymes are proteins that act as biocatalysts, participating in chemical reactions in living organisms (Voet and Voet, 1994) as well as in many biotechnological processes (Qin and Cabral, 2002). Urease (EC 3.5.1.5, urea amino hydrolase) is an enzyme that catalyses the hydrolysis of urea into carbon dioxide and ammonia. In many living organisms, urease mediates the use of external and internally generated urea as a source of nitrogen (Qin and Cabral, 2002). Known catalytic mechanisms indicate significant similarities on the level of primary structure between various ureases isolated from bacteria, fungi or higher plants (Zerner, 1991; Karplus et al., 1997). Efficiency of nitrogen utilisa- tion and chemical properties of the nickel containing active site positions urease for a variety of applications in clinical and chemical analyses, environmental protection or biochemical reac- tion engineering (Qin and Cabral, 2002). Urease was the first enzyme isolated in a crystalline form providing a proof of the protein nature of enzymes (Sumner, 1926). Native form of jack bean urease exists as a hexameric structure (consisting of six 91 kDa monomers) with the apparent hydrodynamic radius of 7 nm, which may reversibly dissociate to trimers, depending on the solution conditions (Follmer et al., 2004; Sheridan et al., 2002). Activity of an enzyme refers to its ability to catalyse certain chemical reactions under well defined conditions and can be directly controlled through conformational or structural altera- tions of the enzyme (Voet and Voet, 1994). For jack bean urease, the activity is considered to be proportional to the concentration of its native conformation (Omar and Beauregard, 1994, 1995). In industrial applications, enzymes are often exposed to elevated temperatures, which in turn cause loss of their activity. Enzymatic stability refers to the capability of an enzyme to preserve its Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ces Chemical Engineering Science 0009-2509/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ces.2011.07.050 n Corresponding author. Tel.: þ44 141 5482410. E-mail address: jan.sefcik@strath.ac.uk (J. Sefcik). Chemical Engineering Science 70 (2012) 14–21