Cold-adapted carboxylesterases from Alcanivoracaceae active with a wide range of synthetic polyesters

Members of the family Alcanivoracaceae are widespread in marine environments, where they play central roles in hydrocarbon degradation and populate plastics-associated microbiomes, with notable enzymatic potential toward ester- and olefin-based polymers. To further investigate their enzymatic potential, we selected 21 candidate enzymes from the α/β-fold hydrolase superfamily, specifically carboxylesterase Family V from genome-sequenced representatives of the genera Alcanivorax, Alloalcanivorax, and Isoalcanivorax. Genes encoding enzymes were cloned and heterologously expressed in E. coli, of which eleven were purified and subjected to substrate specificity analyses including six previously reported and partially characterised carboxylesterases from A. borkumensis SK2, used as benchmarks. All enzymes showed activity against soluble model p-nitrophenyl ester substrates with acyl chain lengths ranging from C2 to C12 and against bis(benzoyloxyethyl) terephthalate (3PET) and polycaprolactone (PCL2). During 3PET hydrolysis, product accumulation followed the order: benzoic acid >  > MHET > terephthalic acid. Five enzymes hydrolysed polycaprolactone (PCL14), poly-DL-lactide (PDLLA), and polybutylene adipate (PBA). All five enzymes displayed temperature optima around or below 50 °C and retained high activity at low temperatures (5-20 °C), consistent with adaptation to marine environments. Enzymes also exhibited moderate solvent tolerance, neutral-to-alkaline pH optima, and low thermostability, with melting temperatures (T m) between 31 and 48 °C. Overall, enzymes from Alcanivoracaceae exhibited promising potential for synthetic polyesters' biodegradation, especially under low-temperature conditions, suggesting potential application for degrading specific polyester-based plastics with lower molecular weight, and their utility in further enzyme engineering for plastic recycling and upcycling. KEY POINTS: • Members of Alcanivoracaceae are a rich resource of polyester-degrading enzymes. • All selected and analysed Family V esterases exhibited high activities and stabilities at low temperatures and solvent tolerance. • Characterised enzymes were active with a broad range of polyesters.