Thermozymes: Biotechnology and Structure-Function Relationships
Claire VIEILLE, Alexei SAVCHENKO, and J. Gregory ZEIKUS*
Department of Biochemistry, Biochemistry Building, East Lansing, Michigan, 48824-1319, USA
Thermozymes are a hot research topic because they are remarkable tools for developing commercial biotechnologies and for studying protein stability. Thermozymes are enzymes that evolved in thermophilic microbes (grow at >60℃) and hyperthermophilic microbes (grow at > 80℃). Thermozymes are both resistant to irreversible denaturation and are optimally active at very high temperatures (from >60℃ to -120℃). Thermozymes are one class of extremozymes that share the same catalytic mechanisms with their mesophilic counterparts. Thermozymes generally retain their thermal properties when cloned and expressed in mesophilic hosts indicating that their unique thermal properties are genetically encoded. The specific molecular mechanisms that account for enzyme thermophilicity and thermostability vary from enzyme to enzyme. Recent structural comparisons between mesozymes and thermozymes have validated numerous protein-stabilization mechanisms including hydrophobic interactions, packing efficiency, salt bridges, hydrogen bonds, reduction of conformational strain, loop stabilization and resistance to covalent destruction. Our lab has been researching the molecular determinants for thermozyme activity and stability using enzymes from model thermophilic genera (i.e. Thermoanaerobacter and Thermoanaerobacterium) and hyperthermophilic genera (i.e. Pyrococcus and Thermotoga). Our latest findings on the biochemical and molecular features of thermozymes will be presented here and includes: α-amylase and amylopullulanase used in starch processing; glucose isomerase used in sweetener production; alcohol dehydrogenase used in chemical synthesis; and alkaline phosphatase used in diagnostics. The respective genes and recombinant proteins for these thermozymes were characterized in terms of sequence homologies, specific activities, thermophilicity and unfolding kinetics. Site directed and nested deletion mutagenesis was used to understand structure function relationships. All of these thermozymes display higher stability and activity than their counterparts currently used in the biotechnology industry. In general, thermozymes are more rigid than mesozymes and are more resistant to thermal and chemical denaturants.
