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Engineering a Hyperstable Enzyme by Manipulation of Early Steps in the Unfolding Process

2001; Taylor & Francis; Volume: 19; Issue: 5-6 Linguagem: Inglês

10.3109/10242420108992029

1029-2446

Vincent G. H. Eijsink, Gert Vriend, Bertus van den Burg,

Pancreatic function and diabetes

Protein engineering experiments have recently yielded hyperstable variants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). These variants contain mutations suggested by comparison of TLP-ste with its more thermostable counterpart thermolysin, as well as rationally designed mutations. The key to the successful stabilization strategy was the identification of a "weak" region that is involved in early unfolding events ("unfolding region"). Mutations in this region had large effects on stability, whereas mutations in other parts of the protein generally had minor effects. The mutational strategies that were used as well as characteristics of the engineered hyperstable biocatalysts are reviewed below.