Development of a novel dehydrogenase and a stable cofactor regeneration system

Enzyme catalysis is becoming increasingly important for the synthesis of chiral pharmaceuticals and fine chemicals. However, most enzymes have been optimized by nature to perform at specific conditions (i.e., pH, temperature, and medium), generally distinct from the desired process conditions. Advances in molecular biology and protein engineering have allowed researchers to tailor enzymes to meet ever-increasing demands.;The first goal of this work focused on the development of an amine dehydrogenase (AmDH) from a leucine dehydrogenase using site-directed mutagenesis. We aimed at reductively aminating a prochiral ketone to a chiral amine by using leucine dehydrogenase (LeuDH) as a starting template. This initial work was divided into two stages. The first focused mutagenesis to a specific residue (K68) that we know is key to developing the target functionality. Subsequently, mutagenesis focused on residues known to be in close proximity to a key region of the substrate (M65 and K68). This approach allowed for reduced library size while at the same time increased chances of generating alternate substrate specificity. An NAD+-dependent high throughput assay was optimized and will be discussed. The best variants showed specific activity in mU/mg range towards deaminating the target substrate.;The second goal of this work was the development of a thermostable glucose dehydrogenase (GDH) starting with the wild-type gene from Bacillus subtilis. Due to the high cost of nicotinamide cofactors an efficient regeneration system is vital to the development of a process utilizing redox enzymes (including an AmDH). GDH is able to carry out the regeneration of both NADH and NADPH cofactors using glucose as a substrate. We applied the structure-guided consensus method to identify 24 mutations that were introduced using overlap extension. 11 of the tested variants had increased thermal stability, and when combined a GDH variant with a half-life ∼3.5 days at 65°C was generated---a ∼106 increase in stability when compared to the wild-type.;The final goal of this work was the characterization of GDH in homogeneous organic-aqueous solvent systems and salt solutions. Organic media and salts are extensively used in organic synthesis to increase substrate stability and solubility as well as enzyme stability. Engineered GDH variants showed increased stability in all salts and organic solvents tested. Thermal stability had a positive correlation with organic solvent and salt stability. This allowed the demonstration that consensus-based methods can be used towards engineering enzyme stability in uncommon media. This is of significant value since protein deactivation in salts and organic solvents is not well understood, making a priori design of protein stability in these environments difficult.;Lastly, future works for further improving LeuDH and GDH and potential applications are discussed.