Production of Novel Thermostable Esterases from Thermus thermophilus Strain ET-1 in Escherichia coli and Thermus thermophilus HB27 Using the Bifunctional Expression System pTGT-1 and Characterization of the Recombinant Enzymes

The thermophilic bacterium <i>Thermus thermophilus</i> represents a crucial genetic reservoir for exploring thermostable enzymes as valuable biocatalysts for industrial and biotechnology applications. Here, we identify, clone, and characterize Ces1-ET, Est1-ET, and Plp1-ET, three lipolytic enzymes obtained from <i>T. thermophilus</i> strain ET-1 isolated from El Tatio Geothermal Field in Northern Chile. To enable recombinant expression, we constructed the pTGT-1 expression system, a versatile bifunctional shuttle vector compatible with both <i>Escherichia coli</i> and <i>T. thermophilus</i>. The three thermoenzymes Ces1-ET, Est1-ET, and Plp1-ET, were successfully cloned, expressed, and purified using the pTGT-1 system, with a molecular mass of 25 kDa, 36 kDa, and 28 kDa, respectively. The recombinant purified enzymes displayed optimal temperatures at 60 °C, 80 °C, and 70 °C and optimal pH of 7.5, 9.0, and 8.0 for Ces1-ET, Est1-ET, and Plp1-ET, respectively. Functional biochemical assays revealed a broad tolerance to surfactants, detergents, divalent cations, and high salinity, relevant properties for their application in an industrial setting. These thermostable esterases expand the repertoire of thermozymes from <i>Thermus</i> spp., introducing pTGT-1 as an innovative tool for thermophilic protein expression and highlighting <i>T. thermophilus</i> strain ET-1 from El Tatio Geothermal Field as a valuable source of thermostable enzymes for industrial and biotechnology applications.