Characterization of a novel stable biocatalyst obtained by protein engineering

Protein engineering is a powerful tool for the improvement of the propertie s of biocatalysts, Previously we have applied protein engineering technolog ies to obtain an extremely stable variant of the thermolysin-like protease from Bacillus stearothermophilus [Van den Burg, Vriend, Veltman, Venema and Eijsink (1998) Proc. Natl, Acad. Sci, U.S.A, 95, 2056-2060]. This variant is much more resistant to denaturing conditions (temperature and denaturing agents) than the wildtype enzyme. An extensive enzymic characterization wa s undertaken to explore the suitability of the variant as a biocatalyst at high temperatures. By comparing a range of variants with increasing thermal stabilities we show that the additivity of the mutations is accompanied by an increase in activity at elevated temperatures in accordance with the Ar rhenius law, The results suggest that the constructed protease variants cou ld be suitable alternatives to proteases that are currently used industrial ly, Our studies demonstrate how protein engineering can be exploited to obt ain high-performance biocatalysts.