Site-specific and random immobilization of thermolysin-like proteases reflected in the thermal inactivation kinetics

Immobilization of proteins usually leads to random orientation of the molec ules on the surface of the carrier material, whereby mechanistic interpreta tions of changes in properties, such as thermal stability, become very diff icult. Recently, we have prepared several mutant enzymes of the thermolysin -like neutral protease from Bacillus stearothermophilus, containing cystein e residues in different positions on the surface of the protein molecule, T hese enzymes allowed site-specific immobilization to Activated Thiol-Sephar ose and showed that stabilization effects strongly depend on the position o f attachment [Mansfeld, Vriend, Van den Burg, Eijsink and Ulbrich-Hofmann ( 1999) Biochemistry 38, 8240-8245], The greatest stabilization was achieved after immobilization of the mutant enzymes S65C and T56C/S65C within the st ructural region (positions 56-69) where unfolding is initiated. In this stu dy thermal inactivation kinetics of these two mutant enzymes, as well as th ose of the pseudo-wildtype enzyme and thermolysin, were compared for differ ent types of immobilization. Besides site-specific immobilization via thiol groups, the enzymes were bound randomly via their amino groups or by mixed -type binding. The basic matrix was Sepharose 4B in all carriers. Whereas t he enzymes bound site-specifically to Activated Thiol-Sepharose showed clea r first-order inactivation kinetics like the soluble enzymes, the other imm obilized enzyme preparations were characterized by distinct biphasic inacti vation kinetics reflecting the heterogeneity of enzyme molecules on the car rier with respect to thermal unfolding. Site-specific binding resulted in s tronger stabilization than the mixed binding type. However, immobilization to a highly functionalized carrier via amino groups increased stability fur ther, suggesting that multiple fixation outside of the unfolding region 56- 65 is able to increase stability of the enzyme molecules additionally.