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

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 mm min⁻¹ at 50 % substrate conversion (50 mm). The use of wall-coated enzyme microreactors in high-performance biocatalytic reaction engineering is supported strongly.