Three-Enzyme Phosphorylase Cascade Immobilized on Solid Support for Biocatalytic Synthesis of Cello−oligosaccharides

Enzyme cascades are promising for multistep biocatalytic synthesis, but their effective use beyond the proof-of-concept stage is challenging. Strategies to recycle the individual enzymes are critical for the applicability of such cascades. Immobilization on solid support is well developed for single enzymes but remains difficult for enzyme ensembles. Here, we show a controlled co-immobilization of three glycoside phosphorylases to establish a highly active and recyclable biocatalyst for the conversion of sucrose and glucose into soluble (short-chain) cello−oligosaccharides. We use protein fusion with the binding module Z_basic2 to enable non-covalent surface tethering of all enzymes according to a uniform principle and in a programmable fashion. We thus achieve loading of the phosphorylases in an activity ratio optimal for the overall conversion and for controlling the cello−oligosaccharide chain length (≤6), hence the solubility, in the reaction. We demonstrate efficient production of ∼12 g/L cello−oligosaccharides with integrated enzyme re-use, retaining ∼85 % of the overall initial activity after five reaction cycles. This study presents a major advance toward the practical use of systems bio-catalysis on solid support.