ENGINEERING AN ENZYME TO RESIST BOILING

In recent years, many efforts have been made to isolate enzymes from e xtremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show h ow a moderately stable enzyme (a thermolysin-like protease from Bacill us stearothermophilus, TLP-ste) can be made hyperstable by a limited n umber of mutations. The mutational strategy included replacing residue s in TLP-ste by residues found at equivalent positions in naturally oc curring, more thermostable variants, as well as rationally designed mu tations. Thus, an extremely stable 8-fold mutant enzyme was obtained t hat was able to function at 100 degrees C and in the presence of denat uring agents. This 8-fold mutant contained a relatively large number o f mutations,whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state (''rigidifying' ' mutations such as Gly --> Ala, Ala --> 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 degrees C.