Chemoenzymatic peptide synthesis offers a sustainable alternative to traditional solid-phase peptide synthesis for manufacturing therapeutic peptides such as semaglutide. However, cost-efficient industrial implementation requires enzyme immobilization to enable the catalyst recovery and continuous processing. This work presents the first demonstration of a bioinformatics-guided immobilization strategy for a peptiligase variant, successfully applied in a continuous-flow packed-bed reactor for semaglutide fragment ligation. Using computational tools, we predicted optimal immobilization sites and systematically screened covalent and noncovalent methods. EP400/SS-aldehyde emerged as the superior support, maintaining >85% of the immobilized activity after 10 reuse cycles. Translation to continuous flow revealed substrate hydrolysis as a competing side reaction at high enzyme loads. Optimization of the protein loading, to 2.5 mg/g, suppressed this unwanted reaction, achieving 95% conversion with 30 min of residence time, matching solution-phase performance. This establishes a robust framework for immobilizing peptiligases in industrial bioprocesses.

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