We have reported previously on the kinetics of thermal inactivation at 80–120°C of the extracellular proteinase from Pseudomonas fluorescens 22F (Schokker & van Boekel, 1997, 1999b). During these studies, we noted some inactivation during the heating up and cooling periods, but allowed for this by calculating the residual activity as a fraction of the activity after the heating up period of 2 min followed by cooling to 0°C. However, it may be of interest to evaluate the extent of inactivation during these heating up and cooling periods. If the temperature dependence of the reaction rate behaves according to Eyring's theory, inactivation would, of course, be slower than at the final heating temperature. However, during the heating and cooling of the enzyme solution, the temperature also passes the region in which autoproteolysis occurs (Schokker & van Boekel, 1998a). Prolonged residence time in the critical zone for autoproteolysis may cause increased inactivation, as has been demonstrated in electrophoresis experiments for proteinases from other Ps. fluorescens strains (Barach & Adams, 1977; Richardson, 1981; Diermayr et al. 1987). Consequently, the inactivation during the first few minutes would be dependent on factors influencing both autoproteolytic and thermal inactivation.

In most of our heating experiments (Schokker & van Boekel, 1997, 1999b), inactivation during heating up was relatively rapid compared with inactivation at the final heating temperature, leading to a biphasic inactivation curve. This was also found for proteinases from many other Ps. fluorescens strains. In some studies the inactivation during heating up was not taken into account when analysing the kinetics of thermal inactivation (Patel et al. 1983; Yan et al. 1985; Fairbairn & Law, 1986), which led to misinterpretation of the mechanism or the kinetic values. Others explained the biphasic inactivation curve by autoproteolysis (Barach & Adams, 1977; Richardson, 1981; Stepaniak & Fox, 1983; Kroll & Klostermeyer, 1984; Diermayr et al. 1987), or stabilization by Ca2+ of a small portion of the proteinase to heat inactivation (Stepaniak & Fox, 1983; Azcona et al. 1988).

In this paper we discuss the influence of protein, enzyme purification and Ca2+ activity on inactivation during the heating up and cooling periods. The aim of this study was to determine, using kinetic modelling, whether the inactivation during heating up and cooling periods could be explained by autoproteolysis and thermal inactivation, or whether other mechanisms are involved in the strong initial inactivation.