ABSTRACT
For pressure inactivation studies, today only batch methods are available. For pressure treatments, the enzymic samples should be contained in flexible containers (e.g., microtubes, plastic bags). Because of adiabatic heating, pressure buildup is inevitably associated with temperature increase. At the prevailing high pressure, this temperature increase might provoke a significant reduction of enzyme activity during pressure buildup. The temperature reached during pressure buildup can seriously be reduced by decreasing the rate of compression. To avoid problems arising from the temperature increase during pressure buildup, a possible approach is to exclude the nonisobaric/nonisothermal phase from the inactivation data by starting the time course of the experiment (‘‘zero point’’) after reaching the desired pressure and an additional equilibration period to allow temperature to evolve to its desired value. At that moment, the pressure vessel is decompressed and the activity of the corresponding enzyme sample is considered as the blank. The use of this zero-point approach is merely allowed for firstorder reactions (including the special cases of biphasic and first-order fractional conversion reactions). After pressure-temperature treatment, enzyme solutions are cooled in ice water to stop inactivation and reactivation, and the residual enzymatic activity is measured. To perform kinetic experiments at elevated pressure (up to 1000 MPa), specialized high-pressure equipment, consisting of several individual thermostated pressure vessels, is often used (Fig. 1). Such equipment allows submission of several samples simultaneously to treatments at the same pressure and temperature for preset times.
