A model compounds for the discovery of novel PHA depolymerases – a computational study

Polyhydroxyalkanoates (PHA) represent a sustainable alternative to conventional plastics due to their microbial biological origin, biodegradability, biocompatibility, and structural diversity. The primary challenge in the industrial enzymatic recycling of PHA lies in the relatively slow biodegradation rates, spanning several months to years, depending on environmental conditions and the characteristics of both PHA and the PHA-degrading enzymes. This study involves experimental and computational testing of monomeric and dimeric units of polyhydroxy octanoate (PHO) as suitable substrates for continuous spectrophotometric monitoring of enzymatic reactions. Additionally, we explore the specificity of these novel model substrates for polyhydroxyalkanoate depolymerases (PHOase) in comparison to other esterases (lipase and cutinase). The experimental findings are complemented by computational evaluations. We have modeled the most active enzyme, poly(3-hydroxyoctanoate) depolymerase from Pseudomonas fluorescens, followed by cutinase LCC, which exhibits lower activity on tested substrates, and lipase CalB that doesn't show detectable activity. Our computational insights provide a rationale for these experimental observations.