Lag Time Spectrophotometric Assay for Studying Transport Limitation in Immobilized Enzymes

Enzymes are promising catalysts for bioprocessing. For instance, the enzymatic capture of CO₂ using carbonic anhydrase (CA) is a carbon capture approach that allows obtaining bicarbonate (HCO₃ ^-) with no high-energy input required. However, application in a commercially viable biotechnology requires sufficient enzymatic lifetime. Although enzyme stabilization can be achieved by different immobilization techniques, most of them are not commercially viable because of transport limitations induced by the immobilization method. Therefore, it is necessary to develop assays for evaluating the role of immobilization on transport limitations. Herein, we describe the development of a fast and reproducible assay for screening immobilized CA by means of absorbance measurement using a computer-controlled microplate reader in stop-flow format. The automated assay allowed minimizing the required volume for analysis to 120 μL. We validated the assay by determining lag times and activities for three immobilization techniques (modified Nafion, hydrogels, and enzyme precipitates), of which linear polyethyleneimine hydrogel showed outstanding performance for CA immobilization.