Microfluidic Reactor for Continuous Flow Biotransformations with Immobilized Enzymes: the Example of Lactose Hydrolysis by a Hyperthermophilic β_cont;‐Glycoside Hydrolase

A microfluidic reactor was developed for continuous flow biocatalytic transformations with immobilized enzymes. The microreaction device integrated a microstructured multichannel plate fabricated by microinjection molding from liquid silicon rubber material, with an appropriately interfaced and temperature‐controlled housing. Enzyme immobilization was carried out under flow conditions using the fully assembled microreactor. Covalent protein attachment was utilized, via cross‐linking with glutardialdehyde onto the amino‐silanized microstructured surface of the reaction plate. Scanning electron microscopy (SEM) and continuous flow enzyme kinetic studies were employed to characterize the immobilization of a thermophilic β‐glycoside hydrolase. SEM analysis revealed enzyme binding in aggregated clusters, preferentially around passive mixing elements that were introduced in an alternating left‐and‐right pattern along each microchannel. Using an immobilization protocol optimized with respect to the pH in each step, about 1600 units of activity were bound per m 2 of microstructure. Although retention of activity in the surface‐bound enzyme was only 3–4 %, the Michaelis‐Menten constant for lactose was similar (≈ 70 mM) in free and immobilized hydrolase. The free energy of activation for lactose hydrolysis ( E a ) was decreased from 72 kJ/mol in the free enzyme to 38 kJ/mol in the immobilized enzyme. Covalent attachment of the enzyme onto the microchannel walls via a dendrimeric linker restored the original E a value. The immobilized enzyme microreactor was used for continuous conversion of 100 mM lactose at 80 °C and operated at a stable substrate conversion of 60 % for five days.