Abstract
The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better "evolvability" of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (T m) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T m compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T m 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.