| Directed evolution, inspired by Darwinian evolution in Nature, is an effective approach for protein design. An industrially-important enzyme, subtilisin E, has been chosen as the research target. Important methodologies for directed evolution have been developed, including optimizing the error-prone polymerase chain reaction (PCR) to allow easy and precise control of the mutation rate, optimizing DNA shuffling for high fidelity recombination, and developing three new in vitro recombination methods: random priming recombination (RPR), defined primer recombination (DPR) and staggered extension process (StEP) recombination.; Using these techniques, subtilisin E isolated from the mesophilic organism Bacillus subtilis has been rapidly converted into its thermophilic counterpart (without compromising its activity). After five generations of directed evolution, the resulting variant 5-3H5 is as stable as its naturally-occurring thermostable homolog, thermitase, isolated from the thermophilic organism Thermoactinomyces vulgaris. The half-lives of thermal inactivation at 83{dollar}spcirc{dollar}C of both 5-3H5 and thermitase are 3.5 min. Their temperature optima are 76{dollar}spcirc{dollar}C, 18{dollar}spcirc{dollar}C higher than that of wild type subtilisin E. In addition, 5-3H5 is more active than wild type subtilisin E over the whole range of temperatures. The mutations responsible for the enhanced thermostability were identified and mapped into the structure of subtilisin E. Our findings strongly support the notion that thermal stability is achieved by the cumulative effect of small improvements at many locations within the protein molecule. Thus, not surprisingly, the pursuit of a 'holy grail' of rules for protein thermostabilization was deemed unsuccessful. However, as demonstrated here, directed evolution is a generally applicable, highly effective approach to increase protein thermostability.; The concepts and techniques developed for directed evolution may also be applied to solving problems associated with molecular evolution in Nature. For example, due to significant sequence divergence, identification of the adaptive mutations, neutral mutations and deleterious mutations in evolutionarily-related proteins is a difficult task. We developed a convenient method to identify functional mutations by gene recombination and sequence analysis of a small sampling of the recombined library exhibiting the evolved behavior. As a demonstration, this approach was used to identify the two thermostable mutations out of ten mutations in a laboratory-evolved thermostable subtilisin E variant. |
