Immobilization of an amino acid racemase for application in crystallization‐based chiral resolutions of asparagine monohydrate

Abstract

Integration of racemization and a resolution process is an attractive way to overcome yield limitations in the production of pure chiral molecules. Preferential crystallization and other crystallization-based techniques usually produce low enantiomeric excess in solution, which is a constraint for coupling with racemization. We developed an enzymatic fixed bed reactor that can potentially overcome these unfavorable conditions and improve the overall yield of preferential crystallization. Enzyme immobilization strategies were investigated on covalentbinding supports. The amino acid racemase immobilized in Purolite ECR 8309F with a load of 35 mg-enzyme/g-support showed highest specific activity (approx. 500 U/g-support) and no loss in activity in reusability tests. Effects of substrate inhibition observed for the free enzyme were overcome after immobilization. A packed bed reactor with the immobilized racemase showed good performance in steady state operation processing low enantiomeric excess inlet. Kinetic parameters from batch reactor experiments can be successfully used for prediction of packed bed reactor performance. Full conversions could be achieved for residence times above 1.1 min. The results suggest the potential of the prepared racemase reactor to be combined with preferential crystallization to improve resolution of asparagine enantiomers.

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

“…This experiment adds additional stability data to the observations made by Carneiro et al 2020 [21], where the immobilized AAR was operating for two days at 40 °C, and Würges et al 2009 [20], where the free AAR lost 90% of its activity at 35 °C within less than seven days. The observed good stability of the AAR encourages the application of the AAR in continuous production processes.…”

“…The immobilized racemase was prepared based on a protocol described by Carneiro et al 2020 [21]. Under varying process conditions, the reaction kinetics, stability, and reproducibility were tested in a small EFBR.…”

“…In addition, the accessibility of the immobilized enzyme in deeper pores might be limited. It had been shown before that the pore size played a role in the overall activity [21]. Furthermore, the moisture content in the carrier resin might fluctuate, which adds uncertainty about the density and dosage of the carrier.…”

“…The AAR, originating from P. putida KT 2440 DSM 6125 [32], was modified to include a His-tag to facilitate its purification [21]…”

“…The performance of the free racemase has already been studied with regard to pH in a phosphate buffer and in the presence of alcohols [18,20]. The investigation of immobilization conditions for the valuable AAR was reported in our previous work [21]. High enzymatic activity and effective recyclability were achieved with the immobilized preparation on Lifetech™ ECR8309 (Purolite, South Wales, UK).…”