Development of microreactors with surface-immobilized biocatalysts for continuous transamination

Abstract

The industrial importance of optically pure compounds has thrown a spotlight on ω-transaminases that have shown a high potential for the synthesis of bioactive compounds with a chiral amine moiety. The implementation of biocatalysts in industrial processes relies strongly on fast and cost effective process development, including selection of a biocatalyst form and the strategy for its immobilization. Here, microscale reactors with selected surface-immobilized amine-transaminase (ATA) in various forms are described as platforms for high-throughput process development. Wild type ATA (ATA-wt) from a crude cell extract, as well as Escherichia coli cells intracellularly overexpressing the enzyme, were immobilized on the surfaces of meander microchannels of disposable plastics by means of reactor surface silanization and glutaraldehyde bonding. In addition, a silicon/glass microchannel reactor was used for immobilization of an ATA-wt, genetically engineered to contain a silica-binding module (SBM) at the N-terminus (N-SBM-ATA-wt), leading to immobilization on the non-modified inner microchannel surface. Microreactors with surface-immobilized biocatalysts were coupled with a quenching system and at-line HPLC analytics and evaluated based on continuous biotransformation, yielding acetophenone and l-alanine. E. coli cells and N-SBM-ATA-wt were efficiently immobilized and yielded a volumetric productivity of up to 14.42 g L−1 h−1, while ATA-wt small load resulted in two orders of magnitude lower productivity. The miniaturized reactors further enabled in-operando characterization of biocatalyst stability, crucial for successful transfer to a production scale.