Thermostable variants constructed via the structure-guided consensus method also show increased stability in salts solutions and homogeneous aqueous-organic media.
Enzyme instability is a major factor preventing widespread adoption of enzymes for catalysis. Stability at high temperatures and in the presence of high salt concentrations and organic solvents would allow enzymes to be employed for transformations of compounds not readily soluble in low temperature or in purely aqueous systems. Furthermore, many redox enzymes require costly cofactors for function and consequently a robust cofactor regeneration system. In this work, we demonstrate how thermostable variants developed via an amino acid sequence-based consensus method also showed improved stability in solutions with high concentrations of kosmotropic and chaotropic salts and water-miscible organic solvents. This is invaluable to protein engineers since deactivation in salt solutions and organic solvents is not well understood, rendering a priori design of enzyme stability in these media difficult. Variants of glucose 1-dehydrogenase (GDH) were studied in solutions of different salts along the Hofmeister series and in the presence of varying amounts of miscible organic solvent. Only the most stable variants showed little deactivation dependence on salt-type and salt concentration. Kinetic stability, expressed by the deactivation rate constant k(d,obs), did not always correlate with thermodynamic stability of variants, as measured by melting temperature T(m). However, a strong correlation (R(2) > 0.95) between temperature stability and organic solvent stability was found when plotting T(50)(60) versus C(50)(60) values. All GDH variants retained stability in homogeneous aqueous-organic solvents with >80% v/v of organic solvent.
Vazquez-Figueroa
E
E
School of Chemical and Biomolecular Engineering, Parker H. Petit Institute for Bioengineering and Bioscience, Atlanta, GA 30332-0363, USA.
Yeh
V
V
Broering
J M
JM
Chaparro-Riggers
J F
JF
Bommarius
A S
AS
D000818:Animals; D001412:Bacillus subtilis; D016384:Consensus Sequence; D004795:Enzyme Stability / Q000187:drug effects; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry* / Q000235:genetics*; D006358:Hot Temperature; D007700:Kinetics; D009930:Organic Chemicals / Q000737:chemistry; D011489:Protein Denaturation; D012492:Salts / Q000737:chemistry / Q000494:pharmacology*; D012996:Solutions / Q000737:chemistry; D012997:Solvents / Q000737:chemistry / Q000494:pharmacology*; D013696:Temperature
D009930:Organic Chemicals; D012492:Salts; D012996:Solutions; D012997:Solvents; D042843:Glucose 1-Dehydrogenase