Previously we have examined the inactivation of unpurified extracellular proteinase from Pseudomonas fluorescens 22F diluted in demineralized water (Schokker & van Boekel, 1998) in the range 40–70°C. It appeared that the inactivation was most probably caused by intermolecular autoproteolysis, which is the hydrolysis of unfolded proteinase molecules by native (not yet unfolded) molecules. It has been reported that purification of proteinases from Pseudomonas spp. enhances the susceptibility of the proteinase to autoproteolysis (Barach et al. 1976; Griffiths et al. 1981; Leinmüller & Christophersen, 1982; Kroll, 1989; Kumura et al. 1991). On the other hand, when the proteinase is heated in milk or when proteins are added to the enzyme solution, the rate of inactivation by autoproteolysis diminishes (Barach et al. 1978; Kroll & Klostermeyer, 1984; Stepaniak et al. 1991). Apparently, proteins stabilize the proteinase against inactivation by autoproteolysis.

Substrate or other ligands stabilize many enzymes against limited proteolysis. Binding of these substances to the enzyme molecule, either to the catalytic centre or to amino acid residues on the enzyme molecule surface, may impose steric difficulties so that the susceptible peptide bonds are protected against proteolysis (Mihalyi, 1978). Such binding may also cause a conformational change of the enzyme molecule, such that susceptible peptide bonds cannot be attacked or that the conformation is stabilized against unfolding (Mihalyi, 1978). In the latter case an increase in the denaturation temperature (Td) would be expected.

In the case of proteinases, addition of substrate to the enzyme solution may protect the enzyme by a third mechanism. Besides autoproteolysis of the proteinase, the added proteins can be digested. An enzyme molecule digesting a protein is not available at the same time for autoproteolysis, so that the substrate may act as a competitive inhibitor against autoproteolysis.

The aim of this study was to determine the mechanism of protection of the proteinase from Ps. fluorescens 22F by sodium caseinate.