Multivalency Effects on the Immobilization of Sucrose Phosphorylase in Flow Microchannels and Their Use in the Development of a High‐Performance Biocatalytic Microreactor

Abstract Microstructured reactors are emerging engineering tools for the development of biocatalytic conversions in continuous flow. A promising layout involves flow microchannels that are wall‐coated with enzyme. As protein immobilization within closed microstructures is challenging, we suggested a confluent design of enzyme and microreactor: fusion to the silica‐binding module Z basic2 is used to engineer enzymes for high‐affinity oriented attachment to the plain wall surface of glass microchannels. In this study of sucrose phosphorylase, we examined the effects of multiple Z basic2 modules in a single enzyme molecule on the activity and adsorption stability of the phosphorylase immobilized in a glass microchannel reactor. Compared to the “monovalent” enzyme, two Z basic2 modules, present in tandem repeat at the N‐terminus, separated at the N‐ and C‐terminus of an enzyme monomer, or arranged N‐terminally in a protein homodimer, boosted the effectiveness of the immobilized phosphorylase by up to twofold. They attenuated (up to 12‐fold) the elution of the wall‐coated enzyme during continuous reactor operation. The divalent phosphorylase was distributed uniformly on the microchannel surface and approximately 70 % activity could still be retained after 690 reactor cycles. Reaction–diffusion regime analysis in terms of the second Damköhler number (Da II ≤0.02) revealed the absence of mass transport limitations on the conversion rate. The synthesis of α‐ d ‐glucose 1‐phosphate occurred with a productivity of ∼14 m m min −1 at 50 % substrate conversion (50 m m ). The use of wall‐coated enzyme microreactors in high‐performance biocatalytic reaction engineering is supported strongly.