Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases

Abstract

Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial α/β-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble α-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 Å resolution and revealed a classical α/β-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.

Beyond oil degradation: enzymatic potential of Alcanivorax to degrade natural and synthetic polyesters

“…PHB, PBS and PCL) (Sekiguchi et al, 2011), although the mechanisms involved were not previously identified. In another study that performed an in vitro screening for PLA esterases, researchers identified that the enzyme ABO2449 encoded by Alcanivorax borkumensis had a strong hydrolytic activity on PLA as well as on a range of other aliphatic polyesters, that is, PHBV, PCL and PES (Hajighasemi et al, 2016). A homologue of the ABO2449 α/β-hydrolase was encoded by Alcanivorax sp.…”

“…A previously characterized esterase from Alcanivorax borkumensis that showed a strong hydrolytic activity on aliphatic polyesters (ABO2449; Hajighasemi et al, 2016) and that had a conserved homologue in our Alcanivorax sp. 24, i.e.…”

“…The hydrolysis of aliphatic polyesters such as poly(caprolactone) (PCL), poly(hydroxybutylate) (PHB) and poly(butylene succinate) (PBS) by some Alcanivorax isolates has also been shown (Sekiguchi et al, 2011;Zadjelovic et al, 2019), although the underlying molecular mechanisms behind the ability of these strains to degrade such polyesters remained unknown. Interestingly, during the screening for polylactic acid (PLA) esterases, researchers identified that the enzyme ABO2449 encoded by Alcanivorax borkumensis had a strong hydrolytic activity on PLA as well as on a range of other aliphatic polyesters, that is, poly(hydroxybutylateco-valerate) (PHBV), PCL and poly(ethylene succinate) (PES) (Hajighasemi et al, 2016).…”

Future manufacturing and remanufacturing of polymeric materials

“…Supercritical depolymerization in water and alcohols has been demonstrated with epoxy and polyethylene. Enzymatic depolymerization of polylactic acid and other polyesters is known . Devulcanization is possible, and was used in both world wars to supplement virgin rubber supplies, but modern vulcanization chemistry has made this increasingly difficult …”

Structural insights into the unique polylactate‐degrading mechanism of Thermobifida alba cutinase

“…Hajighasemi et al . described a putative PLA‐degrading mechanism of a bacterial carboxylesterase, based on structural studies complex with polyethylene glycol (PEG) molecule, which is completely different from cutinase. But the mode of hydrolyzing action of cutinases with PLAs is not fully understood.…”