Engineering a Stable Protease

Abstract

We have used several approaches to engineer large increases in the stability of the Bacillus serine protease, subtilisin. These include introducing disulfide cross-links, improving electrostatic interactions at calcium ion binding sites, and the use of in vitro random mutagenesis coupled with a phenotypic screen to identify stabilizing mutational events. More than twenty individual stabilizing mutations of subtilisin BPN’ have been identified. Thermodynamic analysis has shown that individually these modifications contribute between 0.3–1.5 Kcal/mol to the free energy of stabilization. We have further found that combining individual stabilizing mutations results in cumulative increases in stability. Calorimetric and crystallographic data demonstrate that increases in the free energy of stabilization are often independent and additive. We therefore have been able to create extremely stable versions of subtilisins in a step by step manner. Thermodynamic stability of subtilisin was also shown to be related to resistance to irreversible inactivation at high temperature and high pH. The most stable versions have half-lives at high pH or high temperature approaching 1000-times longer than the wild type subtilisin BPN’.