Acinetobacter haemolyticus lipase KV1 mutants (Var18 and Var181) with enhanced polyethylene terephthalate biodegradation activity over the wild-type enzyme

Polyethylene terephthalate (PET) is a recalcitrant polymer whose increasing production poses environmental challenges, prompting the search for diverse polyester-hydrolyzing enzymes. While PETases and cutinases degrade PET efficiently, lipases are more robust but less effective. This study explores whether rational engineering can enhance the modest PET-degrading capability of a bacterial lipase. Computational modeling and molecular dynamics simulations were employed to design and evaluate mutant variants (Var18 and Var181) of Acinetobacter haemolyticus lipase KV1. The commercially synthesized mutants exhibited optimal activity at 40 °C and pH 8.0, maintained stability across pH 7.0–11.0, and retained > 50% residual activity after 1 h of incubation. PET films treated with Var18 and Var181 showed weight losses of 2.80 ± 0.02% and 2.31 ± 0.02% after 96 h at 40 °C, modest but reproducible improvements relative to the wild-type (0.57 ± 0.01%), though lower than those reported for benchmark PET hydrolases such as PETase or LCC. Thus, the weight loss data represent surface-level erosion rather than definitive proof of bulk depolymerization. Scanning electron micrographs confirmed that the lipase-treated PET showed notable surface morphological changes on the surface of the degraded PET films. The experimental outcome aligned with docking prediction, indicating higher binding affinities for Var18_PET (-6.0 kcal/mol) and Var181_PET (-6.0 kcal/mol) compared to wild-type LipKV1_PET (-2.5 kcal/mol). Although the achieved weight loss (2–3%) is lower than that of benchmark PET hydrolases, the active-site modifications identified here, increased hydrophobicity, and an open binding groove provided a structural foundation for further rounds of mutagenesis to improve lipase-based PET biodegradation.

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