We present an experimental study dealing with the transfer of the enzyme synthesis of cephalexin from a batch arrangement to a continuous-flow microfluidic system with integrated reaction product separation and enzyme recovery. Cephalexin is synthesized by penicillin acylase in a kinetic regime that is characterized by the appearance of a concentration maximum during the enzyme reaction. We determine proper reaction conditions providing a high cephalexin yield and relatively short reaction time by performing a set of batch experiments. Once the residence/reaction time was found, the reaction process is transferred into a continuous mode. We employ an aqueous two-phase system (ATPS) forming two-phase slug flow in a microfluidic capillary as the reaction-separation environment. Such a flow arrangement guarantees a uniform residence time of the reaction mixture in the reaction microcapillary, and it provides in situ extraction of cephalexin. ATPS also offers an easy way for enzyme recycling and addition of fresh reactants as a benefit. We optimized the composition of the ATPS based on phosphates, polyethylene glycol and water so that cephalexin showed a high affinity to one phase and a soluble enzyme to the other phase. Due to the low level of utilization of a soluble catalyst in the continuous flow arrangement, we recycled the reaction phase containing the dissolved enzyme. The recycling loop also contained microdialysis unit for removal of phenylglycine that tends to cause system clogging. We show that the final design of the microfluidic system with recycle can operate continuously for at least 5 h. Our integrated microfluidic platform represents a general solution for enzymatic reactions performed with simultaneous reaction product separation and enzyme recycle.

• University of Chemistry and Technology, Prague

• API

1. API
2. aqueous two-phase system (ATPS)
3. cephalexin
4. continuous flow
5. in situ extraction
6. microfluidic system
7. Research Article