Lag Time Spectrophotometric Assay for Studying Transport Limitation in Immobilized Enzymes

Abstract

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.

Bio-Inspired Redox-Adhesive Polydopamine Matrix for Intact Bacteria Biohybrid Photoanodes

“…These include utilizing artificial redox mediating systems based on Osmium, − or quinone-based redox polymers, electrode surface engineering, and synthetic biology. − Quinone-based artificial redox mediating approaches have been reported for a variety of photosynthetic organisms due to their biomimetic properties, as they compete with the natural electron carriers in the micro­organisms for photoinduced electron extraction . At the same time, commercial polymers or bioinspired materials have been utilized to immobilize biocatalyst, either enzymes or whole bacteria, maximizing stability of the biohybrid system . However, the separate synthesis of redox polymers and immobilization matrices complicate the preparation of biohybrid electrochemical systems, limiting their utilization.…”