Mechanism and kinetics of inactivation at 40–70°C of the extracellular proteinase from Pseudomonas fluorescens 22F

• Published in: Journal of dairy research 1998-05, Vol.65 (2), p.261-272
• Main Authors: SCHOKKER, ERIX P., VAN BOEKEL, MARTINUS A. J. S.
• Format: Article
• Language: English
• Subjects: Bacteriology; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Metabolism. Enzymes; Microbiology
• ISSN: 0022-0299; 1469-7629
• DOI: 10.1017/S0022029997002719

HPLC size exclusion chromatography experiments showed that during inactivation at 40–70°C of the extracellular proteinase from Pseudomonas fluorescens 22F small molecular mass fragments were formed, indicating that autoproteolysis was at least one of the major causes of inactivation. The formation of small molecular mass fragments and the reaction order indicated that intermolecular autoproteolysis was more likely than intramolecular autodigestion. This was confirmed by computer simulations. The rate constants and the activation enthalpy (ΔH[Dagger]) and entropy (ΔS[Dagger]) for the reactions of the intermolecular autoproteolysis model were derived from computer simulations. ΔH[Dagger] and ΔS[Dagger] of the unfolding reaction were 504 kJ mol−1 and 1252 J mol K−1 respectively. ΔH[Dagger] and ΔS[Dagger] of the refolding reaction were strongly temperature dependent. The estimates for the enthalpy (ΔH0) and entropy (ΔS0) difference between the folded and unfolded state as derived from the reaction rate constants of unfolding and refolding were subject to large deviations, owing to accumulation of errors in the estimation of the kinetic characteristics.