Coated-wall microreactor for continuous biocatalytic transformations using immobilized enzymes

Microstructured flow reactors are emerging tools for biocatalytic process development. A compelling design is that of the coated‐wall reactor where enzyme is present as a surface layer attached to microchannel walls. However, preparation of a highly active wall biocatalyst remains a problem. Here, a stainless steel microreactor was developed where covalent immobilization of the enzyme in multiple linear flow channels of the reaction plate was supported by a macroporous wash‐coat layer of γ‐aluminum oxide. Using surface functionalization with aminopropyl triethoxysilane followed by activation with glutardialdehyde, the thermophilic β‐glycosidase CelB from Pyrococcus furiosus was bound with retention of half of the specific activity of the free enzyme (800 U/mg), yielding a high catalyst loading of about 500 U/mL. This microreactor was employed for the continuous hydrolysis of lactose (100 mM) at 80°C, providing a space‐time yield of 500 mg glucose/(mL h) at a stable conversion of ≥70%. The immobilized enzyme displayed a half‐life of 15 days under the operational conditions. Due to the absence of hydrophobic solute‐material interactions, which limit the scope of microstructures fabricated from poly(dimethylsiloxane) for biocatalytic applications, the new microreactor was fully compatible with the alternate enzyme substrate 2‐nitro‐phenyl‐β‐D‐galactoside and the 2‐nitro‐phenol product resulting from its hydrolysis catalyzed by CelB.