Many oxidoreductases need nicotinamide cofactors for their reactions. The big obstacle of using these syntheses in industry is the high cost of these nicotinamide cofactors. The work here is about finding novel NADH oxidases from Lactococcus lactis and applying in a cofactor regeneration system with carbonyl reductase or alcohol dehydrogenase. NADH oxidases are useful biocatalysts for regenerating nicotinamide cofactors of biological redox reactions. The annotated alkyl hydroperoxide reductase (AhpR) and the H2O-forming enzyme (nox-2) genes from Lactococcus lactis (L. lactis, L.lac-Nox2) were cloned and proteins were expressed and characterized. They were compared with the H2O-former from Lactobacillus sanfranciscensis (L. sanfranciscensis, L.san-Nox2). AhpR is composed of H2O2-forming NADH oxidase (nox-1) and peroxidase and the net reaction of AhpR is the same as nox-2 when oxygen is the substrate. Both nox-1 and nox-2 are flavoproteins and turnover-limited. In the absence of exogenously added thiols, both nox-1 and nox-1/peroxidase are considerably more stable against overoxidation than nox-2. L.san-Nox2 was crystallized and was found to have ADP ligand, but according to the HPLC results, no ADP ligand was found in the L. lac-Nox-2. Enzyme membrane reactor was used for the application of oxidative reaction of cyclohexanol to cyclohexanone, with isolated enzymes horse liver alcohol dehydrogenase and L.lac-Nox2.

Emissions of CO$_2$ largely contribute to global warming. Carbon dioxide can be captured and used to produce value-added chemicals. This thesis focuses on bioelectrocatalysis as a green and sustainable approach. Our aim was to perform conversion of CO$_2$ to methanol via a multi-enzymatic cascade. However, for reactions involving oxidoreductases, ß-NAD is required as a cofactor. Its use in stoichiometric amounts is unprofitable. We address the issue by employing electrochemical regeneration of the cofactor. For the cascade, we expressed and purified formate dehydrogenase, formaldehyde dehydrogenase and alcohol dehydrogenase. Enzymes activity was tested and found to be low for two enzymes. A reliable method to detect methanol via headspace gas chromatography with a flame ionization detector was developed. We tested the cascade with employed in situ electrochemical cofactor regeneration. After two and a half hours of the reaction 4 µmol methanol were detected. Further research is needed to optimize the setup.

No description available.

No description available.

Nicotinamide cofactor-dependent oxidoreductases have become a valuable tool for the synthesis of high value chiral compounds. The feasibility of biocatalytic processes involving these enzymes stands and falls with the efficiency of the regeneration of cofactors. In this study, we describe a novel NADPH regeneration method based on the natural thioredoxin electron delivery system. Thioredoxin 1 (Trx1) and thioredoxin reductase (TR) from Thermus thermophilus were characterized for the dithiol-dependent reduction of NADP+, revealing good catalytic activities and a particularly remarkable thermostability. The TR/Trx1 system was then coupled with two representative NADPH-dependent oxidoreductases, alcohol dehydrogenase and cyclohexanone monooxygenase. Reaction conditions for both systems were optimized for reaction yield and selectivity. The results demonstrate the feasibility of the TR/Trx1-system for its application as NADPH regeneration system.

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. 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℃ was generated--a ~10⁶increase 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. 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.

No description available.