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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 ...