Characterization of an Immobilized Amino Acid Racemase for Potential Application in Enantioselective Chromatographic Resolution Processes

Abstract

Enantioselective resolution processes can be improved by integration of racemization. Applying environmentally friendly enzymatic racemization under mild conditions is in particular attractive. Owing to the variety of enzymes and the progress in enzyme engineering, suitable racemases can be found for many chiral systems. An amino acid racemase (AAR) from P. putida KT2440 is capable of processing a broad spectrum of amino acids at fast conversion rates. The focus of this study is the evaluation of the potential of integrating AAR immobilized on Purolite ECR 8309 to racemize L- or D-methionine (Met) within an enantioselective chromatographic resolution process. Racemization rates were studied for different temperatures, pH values, and fractions of organic co-solvents. The long-term stability of the immobilized enzyme at operating and storage conditions was found to be excellent and recyclability using water with up to 5 vol% ethanol at 20 °C could be demonstrated. Packed as an enzymatic fixed bed reactor, the immobilized AAR can be coupled with different resolution processes; for instance, with chromatography or with preferential crystallization. The performance of coupling it with enantioselective chromatography is estimated quantitatively, exploiting parametrized sub-models. To indicate the large potential of the AAR, racemization rates are finally given for lysine, arginine, serine, glutamine, and asparagine.