Makryniotis_2025_Int.J.Biol.Macromol_333_148892

The persistent accumulation of plastic waste has intensified the demand for sustainable waste management strategies. Enzymatic degradation has emerged as a promising approach, especially for polymers with hydrolysable bonds like polyethylene terephthalate (PET). PET-degrading enzymes (PETases) catalyze the breakdown of PET into water-soluble intermediates, primarily mono(2-hydroxyethyl) terephthalate (MHET), while further hydrolysis of MHET to terephthalic acid (TPA) is essential for efficient degradation. Structural analysis of IsMHETase, the benchmark MHETase from Ideonella sakaiensis, reveals structural similarities to feruloyl esterases (FAEs) of the tannase family, involved in lignocellulose deconstruction. Building on the structural homology between FoFaeC, a FAE from Fusarium oxysporum, and IsMHETase, specific single-point mutations were designed to enhance FoFaeC's MHETase activity, mimicking IsMHETase active site architecture. FoFaeC-G122S variant exhibited a 1.3- and 4.4-fold increase in specific activity (0.13 Units/mg(enz)) and catalytic efficiency on MHET (26.9 mM(-1) min(-1)), respectively, and 2.0-fold higher activity on PET trimer (110.3 microM(prod)/mg(M(HET)3)). Regarding typical FAE substrates, the variant displayed 2.0- and 1.2-fold higher catalytic efficiency on methyl p-coumarate (MpCA, 6703.1 mM(-1) min(-1)) and methyl caffeate (MCA, 6917.8 mM(-1) min(-1)), respectively. In PET degradation, supplementation of IsPETase with FoFaeC-G122S led to a 17-fold increased MHET hydrolysis, achieving its near-complete conversion to TPA (31.4 micro(TPA)/mg(PET)). Concerning lignocellulose breakdown, FoFaeC-G122S exhibited a 2.0-fold higher ferulic acid release rate (26.5 mg(FA) mg(enz)(-1) mg(DSWB)(-1) h(-1)) from destarched wheat bran compared to the WT, when combined with a GH10 xylanase.