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Computationally guided improvement of lipase-catalyzed hydrolysis of PET intermediates in acidic environments
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DOI：10.1016/j.psep.2026.109426.png)
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Abstract
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Poly (ethylene terephthalate) (PET) enzymatic depolymerization is hindered by inefficient hydrolysis of mono(2-hydroxyethyl) terephthalate (MHET), a rate-limiting step that restricts terephthalic acid (TPA) yield. In this study, we developed an integrated semi-rational engineering strategy combining alanine scanning, site-saturation mutagenesis, iterative mutagenesis, and coevolution analysis to enhance the catalytic performance of Candida antarctica lipase B (CALB) under acidic conditions. The resulting triple mutant CALBm (I214V/L303N/V260A) exhibited a 2.3-fold increase in catalytic activity toward MHET and achieved 85.51% conversion to TPA. Kinetic analysis demonstrated improved catalytic efficiency through an enhanced kcat/km, while molecular dynamics simulations revealed strengthened active-site hydrogen-bonding networks and reduced structural flexibility, supporting the experimentally observed catalytic improvement. CALBm also displayed superior thermal and acid stability compared with the wild-type enzyme, highlighting its operational robustness. This work provides an effective route for overcoming the pH-dependent bottleneck in PET depolymerization and offers an acid-compatible, industrially relevant biocatalyst for process intensification in sustainable PET recycling.
Keywords:
PET depolymerization
MHET hydrolysis
enzyme engineering
Candida antarctica lipase B (CALB)
semi-rational design
acidic biocatalysis
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