Thermostability

Thermostable enzymes such as Taq polymerase and Pfu DNA polymerase are used in polymerase chain reactions (PCR) where temperatures of 94 °C or over are used to melt apart DNA strands in the denaturation step of PCR.[10] This resistance to high temperature allows for DNA polymerase to elongate DNA with a desired sequence of interest with the presence of dNTP's.

Knowledge of an enzyme's resistance to high temperatures is especially beneficial in protein purification. In the procedure of heat denaturation, one can subject a mixture of proteins to high temperatures, which will result in the denaturation of proteins that are not thermostable, and the isolation of the protein that is thermodynamically stable. One notable example of this is found in the purification of alkaline phosphatase from the hyperthermophile Pyrococcus abyssi. This enzyme is known for being heat stable at temperatures greater than 95 °C, and therefore can be partially purified by heating when heterologously expressed in E. coli.[11] The increase in temperature causes the E. coli proteins to precipitate, while the P. abyssi alkaline phosphatase remains stably in solution.

Another important group of thermostable enzymes are glycoside hydrolases. These enzymes are responsible of the degradation of the major fraction of biomass, the polysaccharides present in starch and lignocellulose. Thus, glycoside hydrolases are gaining great interest in biorefining applications in the future bioeconomy.[12] Some examples are the production of monosaccharides for food applications as well as use as carbon source for microbial conversion in fuels (ethanol) and chemical intermediates, production of oligosaccharides for prebiotic applications and production of surfactants alkyl glycoside type. All of these processes often involve thermal treatments to facilitate the polysaccharide hydrolysis, hence give thermostable variants of glycoside hydrolases an important role in this context.

Protein engineering can be used to enhance the thermostability of proteins. A number of site-directed and random mutagenesis techniques,[13][14] in addition to directed evolution,[15] have been used to increase the thermostability of target proteins. Comparative methods have been used to increase the stability of mesophilic proteins based on comparison to thermophilic homologs.[16][17][18][19] Additionally, analysis of the protein unfolding by molecular dynamics can be used to understand the process of unfolding and then design stabilizing mutations.[20] Rational protein engineering for increasing protein thermostability includes mutations which truncate loops, increase salt bridges[21] or hydrogen bonds, introduced disulfide bonds.[22] In addition, ligand binding can increase the stability of the protein, particularly when purified.[23] There are various different forces that allow for the thermostability of a particular protein. These forces include hydrophobic interactions, electrostatic interactions, and the presence of disulfide bonds. The overall amount of hydrophobicity present in a particular protein is responsible for its thermostability. Another type of force that is responsible for thermostability of a protein is the electrostatic interactions between molecules. These interactions include salt bridges and hydrogen bonds. Salt bridges are unaffected by high temperatures, therefore, are necessary for protein and enzyme stability. A third force used to increase thermostability in proteins and enzymes is the presence of disulfide bonds. They present covalent cross-linkages between the polypeptide chains. These bonds are the strongest because they’re covalent bonds, making them stronger than intermolecular forces.[24]