New model compounds for the efficient colorimetric screening of medium chain length polyhydroxyalkanoate (mcl-PHA) depolymerases reveal mechanism of activity

Plastic pollution presents a significant environmental problem contributing to increased CO₂ emissions and persistently accumulation in ecosystems. Biobased polymers, like polyhydroxyalkanoates (PHAs), offer a part of a solution with their biodegradability and reduced carbon footprint. However, effective end-of-life strategies, such as controlled enzymatic depolymerization, are crucial for sustainability, relying on efficient PHA depolymerases (PHAases). Here we describe the synthesis of two new chromogenic compounds derived from polyhydroxyoctanoate (PHO) and their application in a continuous, quantitative spectrophotometric assay for PHO depolymerase and other medium chain lengths PHAase activity within 10 min. These substrates allow activity measurement at temperatures above 45 °C, simplifying the comparison of PHAases and aiding enzymatic degradation progress. The study also explores enzyme specificity and identifies key amino acids involved in PHO recognition by PfPHOase. The 3-hydroxyoctanoyl moieties of both compounds were found to bind specifically to a groove formed by the amino acids Phe96, Phe125, Ile171, and Val230, which are highly conserved in known mcl-PHA depolymerases.