Model‐based characterization of an amino acid racemase from Pseudomonas putida DSM 3263 for application in medium‐constrained continuous processes

Abstract

The amino acid racemase with broad substrate specificity from Pseudomonas putida DSM 3263 was overproduced and characterized with respect to application in an integrated multi-step process (e.g., dynamic kinetic resolution) that--theoretically--would allow for 100% chemical yield and 100% enantiomeric excess. Overexpression of the racemase gene in Escherichia coli delivered cell free extract with easily sufficient activity (20-50 U mg(-1) total protein) for application in an enzyme membrane reactor (EMR) setting. Model-based experimental analysis of a set of enzyme assays clearly indicated that racemization of the model substrates D- or L-methionine could be accurately described by reversible Michaelis-Menten kinetics. The corresponding kinetic parameters were determined from progress curves for the entire suitable set of aqueous-organic mixtures (up to 60% methanol and 40% acetonitrile) that are eligible for an integrated process scheme. The resulting kinetic expression could be successfully applied to describe enzyme membrane reactor performance under a large variety of settings. Model-based calculations suggested that a methanol content of 10% and an acetonitrile content of 20% provide maximum productivity in EMR operations. However product concentrations were decreased in comparison to purely aqueous operation due to decreasing solubility of methionine with increasing organic solvent content. Finally, biocatalyst stability was investigated in different solvent compositions following a model-based approach. Buffer without organic content provided excellent stability at moderate temperatures (20-35 degrees C) while addition of 20% acetonitrile or methanol drastically reduced the half-life of the racemase.