Thermal Inactivation of Penicillin Acylase from Escherichia coli: Rate-Limiting Step and Role of Protein Aggregation

Using kinetic and structural methods - differential scanning calorimetry, dynamic laser light scattering, turbidimetry, and mathematical modeling - a comprehensive study of the process of thermal inactivation of penicillin acylase (PA) from Escherichia coli was conducted. The first step of the process is denaturation of the protein globule. As denatured protein accumulates, start aggregates are formed, and after reaching a critical amount, rapid clustering of aggregates (formation of clusters) occurs, which proceeds in the kinetic regime of diffusion-limited cluster-cluster aggregation. A distinctive feature of the process of PA thermal aggregation is absence of enzyme dissociation into subunits and very prolonged accumulation of start aggregates. The rate-limiting step of the PA thermal inactivation process is denaturation of the protein globule and pronounced tendency of the denatured enzyme to aggregate makes this process irreversible. When searching for the ways to stabilize PA, primary attention should be paid not to prevention of protein aggregation but to preservation its three-dimensional structure.