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

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.

Immobilization Screening and Characterization of an Alcohol Dehydrogenase and its Application to the Multi-Enzymatic Selective Oxidation of 1,-Omega-Diols

“…This divergence between thermal and operational stability might rely on the different inactivation mechanisms triggered during either the thermal shock (in absence of substrates) or the batch oxidation process (in presence of substrate and cofactor). A similar divergence has been reported for a pyruvate aldolase thermostabilized through directed evolution (Bosch et al, 2021).…”

The Unexplored Importance of Fleeting Chiral Intermediates in Enzyme-Catalyzed Reactions

“…Among many possibilities, these include: Oxyanions with four different substituents at the respective C atom, occurring in the mechanism of lipases, esterases and proteases. − Protonated oxyanions with four different substituents at the tetrahedral C atom, formed in the multistep (retro)-aldolase reaction pathway from β-hydroxy ketones and aldehydes, but also in related enzyme catalyzed reactions that involve the formation of enamine and/or Schiff base intermediates covalently linked to catalytic lysine residues. − Oxyanions with four different substituents at the tetrahedral C atom as in racemases and epimerases. − Protonated oxyanions in which two of the four substituents at the respective tetrahedral C atom are formally identical, but are differently stabilized by supramolecular noncovalent H-bonding, as in the mechanisms of fluoroacetate dehalogenase, − a carbonic anhydrase mutant, and most epoxide hydrolases. − …”

“…Protonated oxyanions with four different substituents at the tetrahedral C atom, formed in the multistep (retro)-aldolase reaction pathway from β-hydroxy ketones and aldehydes, but also in related enzyme catalyzed reactions that involve the formation of enamine and/or Schiff base intermediates covalently linked to catalytic lysine residues. − …”

Rational Design of a Thermostable 2′-Deoxyribosyltransferase for Nelarabine Production by Prediction of Disulfide Bond Engineering Sites

“…In this respect, the use of different strategies to get robust biocatalysts is a habitual practice in the pharma industry. These include the immobilization of the enzymes onto different matrixes [ 22 , 23 ], the use of enzymes from extremophilic organisms [ 24 , 25 ], or the tailor-made engineering of more stable enzymes [ 26 , 27 ].…”