A FRET-Based Fluorometric Assay for Tracking the Enzymatic Degradation of Poly- l -Lactic Acid

Abstract

Protein engineering has recently emerged as a promising strategy for enhancing the enzymatic recycling of bio-based polymers with the aim of replacing non-degradable petroleum-based plastics with biodegradable ones like poly-l-lactic acid (PLLA). However, a significant challenge exists in the evaluation of the biodegradation efficiency of newly developed enzymes. The precise tracking and continuous rate monitoring of the enzyme-catalyzed hydrolysis remain difficult to achieve with a conventional l-lactate assay kit, which provides only endpoint measurements and is therefore decoupled from the real-time degradation process. To address this issue, we introduce a novel Förster Resonance Energy Transfer (FRET)-based PLLA substrate capable of monitoring real-time PLLA degradation. This was achieved by connecting a highly fluorescent amino carbamoyl coumarin (ACC) dye with a quencher (2,4-dinitrophenyl-l-lysine) via a tetrameric oligo-l-lactate chain. Several fluorescence studies demonstrate that this substrate can effectively detect PLLA-degrading enzyme activity by monitoring increased fluorescence due to enzyme-catalyzed hydrolysis of the oligolactate chain and disruption of the intramolecular FRET system. This substrate can be a potent replacement for the conventional l-lactate assay kit as it allows fast and accurate tracking of the enzyme activity toward PLLA.