ABSTRACT
Enzymes are one of the most important biomolecules which catalyze a variety of reactions. Because of their high specificities and catalytic efficiencies, enzymes are widely used for practical purposes. However, most of the enzymes, except for those isolated from thermophilic organisms, are usually heat labile and rapidly lose their activities at high temperatures. Such instability limits the application of enzymes for practical purposes. Because a thermophilic counterpart of a mesophilic enzyme is not always available, it is important to develop a technique to increase protein stability. Various interactions important for protein stability have been identified by introducing a series of mutations in a given protein and analyzing the effect of the mutations on the protein stability (1-4). They include hydrophobic interactions, hydrogen bonds, electrostatic interactions, and van der Waals interactions. It has been reported that either a hydrophobic interaction created by a buried single methylene or methyl group (5) or an intramolecular hydrogen bond (6) contributes roughly 1.3 kcal/mol to the protein stability. The number of these interactions in each protein molecule is usually beyond 100 in total. Nevertheless, most of the proteins are stabilized only by 10 kcal/mol in the free energy (∆G) on average. These facts indicate that a protein structure is built on a delicate balance of numerous stabilizing and destabilizing interactions within a protein molecule. Therefore it is expected that protein stability can be greatly increased by adding a single stabilizing interaction or removing a single destabilizing interaction. Two strategies have been developed to increase protein stability. One is a computer-assisted design and the other is a directed evolution.
