Stabilization of enzymes by immobilization is frequently accompanied by typical changes in inactivation kinetics. Due to previous studies, the surface position of the protein molecule being connected with the carrier was postulated to be deciding for the stabilization success. Stabilization is strongest if that structural region is fixed where unfolding is initiated, whereas it is low if this region is free [1]. Mutants of a thermolysin-like protease from Bacillus stearothermophilus (TLP-ste) allowed to probe this hypothesis. Local unfolding processes in the N-terminal domain leading to rapid autoproteolytic degradation were previously suggested to determine the thermal stability of this protease. Recent studies using site-specific immobilization of the protease via genetically introduced cysteines have shown that protein stabilization is most effective when the enzyme is attached to the carrier at that sensitive region [2].

In this study, the mutant enzymes were used to compare the effect of different modes of immobilization on the inactivation kinetics. The engineered proteases T56C/S65C and S65C were immobilized site-specifically via the cysteines as well as nonspecifically via their amino groups and compared with respect to thermal inactivation. For site-specific immobilization, TLP-ste mutant enzymes were bound to Activated Thiol-Sepharose. For nonspecific immobilization NHS-Activated Sepharose was used. Both carrier materials are derived from the same chemical matrix structure.

All enzymes bound site-specifically to Activated Thiol-Sepharose via defined thiol groups in the enzyme molecule showed a clear first-order inactivation kinetics due to the homogeneity of the enzyme-carrier bond. In contrast, the same enzymes bound nonspecifically via their amino groups were characterized by a distinct biphasic inactivation kinetics. The results are interpreted in terms of the importance of local superfacial regions for protein stability.

[1] Ulbrich-Hofmann, R., Golbik, R., Damerau, W. in Stability and Stabilization of Enzymes (van den Tweel, W.J.J., Harder, A., Buitelaar, R., eds.), Elsevier, London 9, 497-504, 1993.

[2] Mansfeld, J., Vriend, G., Van den Burg, B., Eijsink, V. G. H., Ulbrich-Hofmann, R., Biochemistry, 38, 8240-8245, 1999.