In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100°C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state (''rigidifying'' mutations such as Gly 3 Ala, Ala 3 Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37°C.Denaturation of proteins at elevated temperatures is usually the result of unfolding, which is followed by an irreversible process, most often aggregation (1). The notion that the unfolding processes involved in irreversible denaturation often have a partial (as opposed to global) character has been confirmed experimentally in several cases (2-4). We have studied the thermal stability and denaturation of a broadspecificity metalloprotease produced by Bacillus stearothermophilus CU21 (ref. 5; called TLP-ste; EC 3.4.24.4) that shares 85% sequence identity with its more stable and better known counterpart thermolysin (ref. 6; Table 1). Thermal denaturation of thermolysin-like proteases (TLPs) also depends on partial unfolding processes that, however, are not followed by aggregation but by autolytic degradation starting at unknown sites in the partially unfolded molecule (7-9). An extensive mutation study in which residues in TLP-ste were replaced by the corresponding amino acid in thermolysin showed that only a few of the 43 substitutions between the two enzymes are important for stability (10). All important substitutions are clustered in the N-terminal domain of the protein, in particular in the 55-69 surface loop (refs. 10-12; Fig. 1). Remarkably, combination of only a few of the stabilizing substitutions identified in the 55-59 region resulted in a TLP-ste variant that was more stable than thermolysin itself (11).Based on the observation that the difference in stability between thermolysin and TLP-ste is mainly determined by mutations in the 55-69 area, we set out to search for several additional stabilizing mutations in this same area. Indeed, several stabilizing mutations involving residues in the 55-69 region have been identified. These include Xaa 3 Pro mutations (13,14), the introduction of a salt bridge (15), and the introduction of a disulfide bridge ...

The thermal inactivation of broad specificity proteases such as thermolysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis. Previous studies have revealed that a surface-located region in the N-terminal domain of the thermolysin-like protease produced by Bacillus stearothermophilus is crucial for thermal stability. In this region a disulfide bridge between residues 8 and 60 was designed by molecular modelling, and the corresponding single and double cysteine mutants were constructed. The disulfide bridge was spontaneously formed in vivo and resulted in a drastic stabilization of the enzyme. This stabilization presents one of the very few examples of successful stabilization of a broad specificity protease by a designed disulfide bond. We propose that the success of the present stabilization strategy is the result of the localization and mutation of an area of the molecule involved in the partial unfolding processes that determine thermal stability.

Thermolysin is a member of a family of homologous proteinases which differ in their resistance to thermally induced unfolding and subsequent autolytic degradation. Site-directed mutagenesis studies of the thermolysin-like proteinase (TLP) from Bacillus stearothermophilus (TLP-ste) show that its reduced resistance to thermally induced autolysis, as compared to thermolysin, is due to only some of the 44 naturally occurring amino-acid differences between them. In fact TLP-ste becomes more resistant than thermolysin by mutation of just a few of these amino-acids. The crucial differences are all localized to a solvent-exposed region in the N-terminal domain of TLP-ste.

The thermostability of neutra1 proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of BaciNus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by proline. The mutations caused large positive (Serd5+Pro, Ala-69-+Pro) or negative (Thr-63+Pro, Tyr-66+Pro) changes in thermostability, which were explained on the basis of molecular modelling of the mutant proteins. The data show that the introduction of prolines at carefully selected positions in the protein can be a powerful method for stabilization.