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Abstract
Starting from a cysteine-free mutant of the thermolysin-like neutral protease from Bacillus stearothermophilus, cysteines were introduced into different positions on the enzyme surface by site-directed mutagenesis. The mutant enzymes were immobilized via the SH-groups to Activated Thiol-Sepharose 4B and their thermostabilities were compared to those of the soluble enzymes. The results showed that the effects of immobilization on stability strongly depend on the site of attachment. Binding of the enzyme via engineered cysteines in the critical unfolding region between residues 56 and 69 led to a considerable increase of thermal stability, whereas the immobilization via a cysteine introduced remote from the unfolding region yielded less stabilization. An extremely strong stabilization was obtained upon binding via T56C where the half-life at 75 °C was increased by the factor of 24.
Published Version
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