Heike Slusarczyk

Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide

In two cycles of an error‐prone PCR process, variants of formate dehydrogenase from Candida boidinii were created which revealed an up to 4.4‐fold (440%) higher residual activity after entrapment in polyacrylamide gels than the wild‐type enzyme. These were identified in an assay using single precursor molecules of polyacrylamide instead of the complete gel for selection. The stabilization resulted from an exchange of distinct lysine, glutamic acid, and cysteine residues remote from the active site, which did not affect the kinetics of the catalyzed reaction. Thermal stability increased at the exchange of lysine and glutamic acid, but decreased due the exchange of cysteine. Overall, the variants reveal very suitable properties for application in a technical synthetic process, enabling use of entrapment in polyacrylamide as an economic and versatile immobilization method.

Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption

Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is recombinantly expressed as an intracellular protein in Escherichia coli. In order to establish an efficient and reliable purification protocol, an expanded bed adsorption (EBA) process was developed, starting from the crude bacterial homogenate. EBA process design was performed with the goal of finding operating conditions which, on one hand, allow efficient adsorption of the target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solution. A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH with high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml). Additionally, a simplified modelling approach, involving small packed beds for generation of process parameters, was employed for defining the operating conditions during sample application. In combination with extended elution studies, a process was set up, which could be scaled up to 7.5 l of adsorbent volume yielding a total amount of 100,000 U of 94% pure FDH per run. On this scale, 19 l of a benzonase-treated E. coli homogenate of 15% wet-weight (pH 7.5, 9 mS/cm conductivity) were loaded to the pseudo-affinity adsorbent (0.25 m sed. bed height, 5 x 10(-4) m/s fluid velocity). After a series of two wash steps, a particle-free eluate pool was obtained with 85% yield of FDH. This excellently demonstrates the suitability of expanded bed adsorption for efficient isolation of proteins by combining solid-liquid separation with adsorptive purification in a single unit operation.

Cloning and stabilization of NAD-dependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis

The gene of the NAD-dependent formate dehydrogenase from the yeast Candida boidinii was cloned by polymerase chain reaction and expressed in E. coli The recombinant enzyme was stabilized by site-directed mutagenesis. Two cysteine residues probably located at the surface of the protein were exchanged against various aliphatic amino acids and the mutant enzymes were tested for stability against oxidation by air in the presence of catalytic amounts of Cu(II). All mutants were significantly more stable against oxidative stress than the wildtype enzyme. Additionally, the mutant enzymes were characterized with respect to their temperature optima, kinetic constants and activation energy of the enzymatic reaction. The results of these studies indicated that the catalytic properties of the mutant enzymes have not been altered by the mutagenesis compared to the wildtype enzyme.