Polylactic acid (PLA) is attracting significant interest as a sustainable alternative to conventional plastics. However, its biodegradation rates vary across environments, and its integration into existing recycling infrastructure necessitates the development of complex end-of-life management strategies. Enzymatic depolymerization offers a promising pathway for closed-loop recycling and upcycling of waste plastics by recovering monomeric building blocks. Yet, few enzymes have been identified that exhibit PLA depolymerization efficiency comparable to those known for PET degradation. Here, we report the computational design of an esterase, RPA1511, from Rhodopseudomonas palustris, which exhibits hydrolytic activity against solid PLA but lacks thermal stability. Using a variety of computational enzyme stability design tools, a focused library was constructed for experimental validation. Further accumulation of beneficial mutations resulted in a five-point variant, R5, which showed an 8 °C increase in melting temperature (Tm) and a substantial 11.5-fold increase in relative enzyme activity at optimal temperatures. This variant achieved efficient PLA degradation, converting 85.38% of PDLLA powder into lactate monomers within 72 h at 65 °C, with a 3.3-fold enhancement compared to wild-type RPA1511. Molecular dynamics simulations showed that the V202 W mutation induced structural changes in the substrate binding pocket and potentially formed more productive complexes, while the remaining four mutations improved the variant's thermal stability. This combined approach through computational design yielded an efficient and thermostable PLA depolymerase, potentially facilitating PLA bio-recycling processes.
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