Optimizing Continuous‐Flow Biocatalysis with 3D‐Printing and Inline IR Monitoring

Abstract

Enzymatic biocatalysis typically generates less waste, uses less water, and minimizes energy consumption compared to traditional chemical methods. Efficient, cell‐free biosynthesis relies on the reuse of its valuable biocatalysts. Immobilization of enzymes on solid supports, such as enzyme carrier resins (ECRs), offers a reliable and widely deployed approach to maximize enzyme turnover in cell‐free biosynthesis. We focus on two major bottlenecks associated with optimizing cell‐free biocatalysis. First, we apply our lab’s 3D‐printed labware to screen ECRs in 96‐well mini‐reactors to optimize enzyme immobilization conditions. Second, we introduce inline infrared spectroscopy to monitor bioreactor output and maximize enzyme productivity. Urease provides a model system for examining immobilization conditions and continuous assessment of biocatalyst performance. As required for the high substrate concentrations to improve process efficiency and minimize waste, urease was studied in unusually high concentrations of its substrate—molar concentrations of urea. The optimized reactor processed 3.24 L of 4.00 M urea at an average volumetric productivity of 13 g·L‐1·h‐1 over 18 h and achieved an estimated productivity number of >17.4 kg urea processed per g of immobilized urease Type‐IX. This workflow can be generalized to most biocatalytic processes and could accelerate adoption of cell‐free biosynthesis for greater chemical sustainability.