(545f) Intensification of Cephalexin Synthesis in Modular Microfluidic Devices Using Electric Field

Cephalexin is an important β-lactam antibiotic that is enzymatically synthetized from a nucleophile (7-aminodeacetoxycephalosporanic acid – ADCA) and an acyl donor (phenylglycine methyl ester – PGME). Cephalexin is thermodynamically unstable and is typically produced in the kinetic regime. Prolonged exposition of cephalexin to the enzyme results in cephalexin decomposition to ADCA. Hence, good control of reaction conditions is necessary to obtain high cephalexin yield. The problems accompanying the cephalexin synthesis can be partially avoided by using microfluidic platforms.

Cephalexin synthesis can be carried out in a homogeneous arrangement with a soluble enzyme with high catalytic activity. Cephalexin extraction from the reaction mixture limits the successive hydrolysis and allows for the enzyme reuse. Aqueous two-phase systems (ATPS) represents an enzyme-friendly alternative to the extraction provided by organic solvents. In this work, we used ATPS consisting of polyethylene glycol (PEG), phosphate salts and water. We have identified such ATPS composition that is characterized by high affinity of the top PEG phase to cephalexin and the reactants and by very low affinity to the enzyme.

In the next step, we have optimized properties of the reaction mixture to attain high cephalexin concentration in the course of the enzyme reaction. We focus on the effects of temperature, the concentrations of ADCA and PGME and their molar ratio. We identified regimes that lead to ADCA conversion to cephalexin higher than 50 % within a few tens of minutes in a batch arrangement. The batch experiments gave us the estimate on the residence time of the reaction mixture in flow-through microfluidic arrangements.

Two microfluidic reactors-separators for the cephalexin synthesis have been developed. One of them relies on the slug flow arrangement of immiscible aqueous phases. The bottom phase contains the enzyme. The reactants are introduced in droplets of the top phase. The reactants are transferred through the interface into the bottom phase where cephalexin is synthetized. Cephalexin is simultaneously transferred back to the top phase where it is stabilized due to the absence of the enzyme. After phase separation, the bottom phase with the enzyme is led back to the start of the process.

The other arrangement, which is currently tested in our lab, contains a long microcapillary that serves as flow-through reactor. Both reactants and the enzyme are introduced in one phase. The output is led to a parallel flow microextractor where cephalexin and unreacted ADCA and PGME are transported to other phase. Enzyme in the original phase is led back to the microcapillary inlet. Moreover, the parallel flow microextractor is equipped by two electrode chambers. We have found that low intensity electric field applied perpendicularly to the interface allows for 100% separation of the product from the enzyme phase. Parametrical studies relevant to both arrangement will be presented.

Selected References:

Cech, J.; Hessel, V.; Pribyl, M., Chem Eng Sci 2017, 169, 97-105.
Cech, J.; Pribyl, M.; Snita, D., Biomicrofluidics 2013, 7 (5), Artn 054103.
Cech, J.; Schrott, W.; Slouka, Z.; Pribyl, M.; Broz, M.; Kuncova, G.; Snita, D., Biochem Eng J 2012, 67, 194-202.
Vobecka, L.; Romanov, A.; Slouka, Z.; Hasal, P.; Pribyl, M., New Biotechnol 2018, 47, 73-79.
Pribyl, M., Sep Purif Technol 2019, 221, 311-318.

Acknowledgement: The authors thank for the support by the grant of the Czech Science Foundation [grant no. 17-09914S].