Thermostability Engineering of a Class II Pyruvate Aldolase from <i>Escherichia coli</i> by <i>in Vivo</i> Folding Interference

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