Osmolyte effect on enzymatic stability and reaction equilibrium of formate dehydrogenase

• Published in: Physical chemistry chemical physics : PCCP 2022-11, Vol.24 (45), p.2793-27939
• Main Authors: Gajardo-Parra, Nicolás F, Akrofi-Mantey, Harold, Ascani, Moreno, Cea-Klapp, Esteban, Garrido, José Matias, Sadowski, Gabriele, Held, Christoph
• Format: Article
• Language: English
• Subjects: Biocatalysts; Dehydrogenases; Design optimization; Equilibrium; Formate dehydrogenase; Low concentrations; Molecular dynamics; Proteins; Reaction kinetics; Solvation; Stability analysis; Substrates; Thermal stability; Thermodynamic models; Trehalose; Trimethylamine
• ISSN: 1463-9076; 1463-9084
• DOI: 10.1039/d2cp04011e

Osmolytes are well-known biocatalyst stabilisers as they promote the folded state of proteins, and a stabilised biocatalyst might also improve reaction kinetics. In this work, the influence of four osmolytes (betaine, glycerol, trehalose, and trimethylamine N -oxide) on the activity and stability of Candida bondinii formate dehydrogenase cb FDH was studied experimentally and theoretically. Scanning differential fluorimetric studies were performed to assess the thermal stability of cb FDH, while UV detection was used to reveal changes in cb FDH activity and reaction equilibrium at osmolyte concentrations between 0.25 and 1 mol kg −1 . The thermodynamic model ePC-SAFT advanced allowed predicting the effects of osmolyte on the reaction equilibrium by accounting for interactions involving osmolyte, products, substrates, and water. The results show that osmolytes at low concentrations were beneficial for both, thermal stability and cb FDH activity, while keeping the equilibrium yield at high level. Molecular dynamics simulations were used to describe the solvation around the cb FDH surface and the volume exclusion effect, proofing the beneficial effect of the osmolytes on cb FDH activity, especially at low concentrations of trimethylamine N -oxide and betaine. Different mechanisms of stabilisation (dependent on the osmolyte) show the importance of studying solvent-protein dynamics towards the design of optimised biocatalytic processes. The effect of osmolytes was studied on FDH properties: termal stability, initial enzyme activity, long-term stability and reaction equilibrium by experimental methods (UV-VIS and fluorimetrics) supported by theory (PC-SAFT and MD simulations).