Effects of Random Mutagenesis and In Vivo Selection on the Specificity and Stability of a Thermozyme

Abstract

Factors that give enzymes stability, activity, and substrate recognition result from the combination of few weak molecular interactions, which can be difficult to study through rational protein engineering approaches. We used irrational random mutagenesis and in vivo selection to test if a β-glycosidase from the thermoacidophile Saccharolobus solfataricus (Ssβ-gly) could complement an Escherichia coli strain unable to grow on lactose. The triple mutant of Ssβ-gly (S26L, P171L, and A235V) was more active than the wild type at 85 °C, inactivated at this temperature almost 300-fold quicker, and showed a 2-fold higher kcat on galactosides. The three mutations, which were far from the active site, were analyzed to test their effect at the structural level. Improved activity on galactosides was induced by the mutations. The S26L and P171L mutations destabilized the enzyme through the removal of a hydrogen bond and increased flexibility of the peptide backbone, respectively. However, the flexibility added by S26L mutation improved the activity at T > 60 °C. This study shows that random mutagenesis and biological selection allowed the identification of residues that are critical in determining thermal activity, stability, and substrate recognition.

Biomass-degrading glycoside hydrolases of archaeal origin

“…In addition to rational approaches, also random mutagenesis studies involving suitable in vivo selection mechanisms were conducted with hyperthermozymes. One study focusing on the β-glycosidase from Saccharolobus solfataricus showed that mutations far from the active site may have crucial impact on the enzyme's activity and stability profiles [132]. While a mutant with three random mutations showed a twofold enhanced specific activity towards galactosides at 85 °C, the higher flexibility of the enzyme variant that enabled this increase in substrate turnover also led to an almost 300-fold reduced thermal stability.…”

Editorial Catalysts: Special Issue on Novel Enzyme and Whole-Cell Biocatalysts

“…The authors studied the well-defined outer ring of the substrate groove of a non-specific nuclease from Pseudomonas syringae and defined it as a potential target for modulation of the enzymatic performance. Perugino et al [15] demonstrated that random mutagenesis and biological selection allowed the identification of residues that are critical in determining thermal activity, stability and substrate recognition of a β-glycosidase from the thermoacidophile Saccharolobus solfataricus. Cyclodextrin transferase's product specificity was changed finally by Sonnendecker et al [16] by semi-rational mutagenesis, obtaining larger cyclodextrin's rings of up to 12 units.…”