Abstract
The continuous discovery and engineering of polyethylene terephthalate (PET) hydrolases are critical to advancing sustainable plastic recycling. A significant number of PET hydrolases have been identified to date; nonetheless, high-throughput screening and evaluation of enzyme characteristics remain a key bottleneck in protein engineering. This study develops an ultra-high-throughput fluorescence-activated droplet sorting (FADS) system for screening PET hydrolases, based on pH sensing. The pH change caused by the released depolymerization product, terephthalic acid (TPA), is correlated with the fluorescent variation of the pH-sensitive C-SNARF-4F probe. We applied this method to screen mutant libraries of two PET hydrolases, DepoPETase β and a new enzyme, SdPETase (derived from Saccharopolyspora dendranthemae), identified via genome mining. Variants exhibiting 1.21-fold and 2.65-fold higher hydrolytic activities were successfully obtained for DepoPETase β and SdPETase, respectively. The successful integration of the pH-based assay with FADS highlights its versatility and efficiency for ultra-high-throughput screening of PET hydrolases.