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

TLDR

Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources and is rapidly expanding in production, creating an urgent need for efficient recycling technologies that convert PLA into lactic acid rather than CO₂. Screening 90 purified microbial α/β‑hydrolases identified two uncharacterized enzymes, ABO2449 from *Alcanivorax borkumensis* and RPA1511 from *Rhodopseudomonas palustris*, that hydrolyze emulsified PLA. Both enzymes also degrade polycaprolactone and other polyesters, completely hydrolyze solid PLA into lactic acid monomers, dimers, and oligomers, and structural analysis of RPA1511 reveals a wide‑open α/β‑hydrolase fold with a catalytic triad essential for activity, demonstrating that microbial carboxyl esterases are effective biocatalysts for polyester depolymerization and recycling.

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.