Rational engineering of cutinase for enhanced biodegradation of polyvinyl acetate and polyacrylates to control...

In the pulp and paper industry, residual sticky substances accumulate during white water circulation, forming deposits that cause paper defects and increase costs. Cutinase is a promising biocatalyst for mitigating stickies, but its industrial application is limited by inherent catalytic constraints. To address this, a strategy integrating structural alignment, virtual screening, and computer-aided design was employed to remodel the substrate-binding pocket of cutinase from Humicola insolens (HiC). Through iterative recombination, variants C11 and E12 were developed, exhibiting enhanced degradation performance toward polyvinyl acetate (PVAc) and polyethyl acrylate (PEA), respectively. C11 increased acetic acid production from PVAc by 1.73-fold compared to the parental M8 variant, while E12 increased ethanol production from PEA by 3.17-fold. Notably, C11 and E12 displayed half-life (t_1/2) of 205.9 h and 51.8 h at 70 °C, corresponding to over 20000-fold and 5000-fold improvements over the wild-type HiC (t_1/2 = 0.01 h at 70 °C), while retaining more than 88% activity at pH 11.0. Molecular dynamics (MD) simulations revealed that the enhanced catalytic activity originated from optimized enzyme-substrate conformations, characterized by shortened nucleophilic attack distances and converged oxyanion hole distributions, stabilizing substrates in catalytically favorable near-attack conformations. Collectively, these engineered cutinase variants represent promising biocatalysts for industrial stickies control, offering an efficient and environmentally friendly strategy.