Mrigwani_2022_Biotechnol.Bioeng__

Thermobifida fusca cutinase (TfCut2) is a carboxylesterase (CE) which degrades polyethylene terephthalate (PET) as well as its degradation intermediates [such as oligoethylene terephthalate (OET), or bis-/mono-hydroxyethyl terephthalate (BHET/MHET)] into terephthalic acid (TPA). Comparisons of the surfaces of certain CEs (including TfCut2) were combined with docking and molecular dynamics simulations involving 2HE-(MHET)(3,) a three-terephthalate OET, to support the rational design of 22 variants with potential for improved generation of TPA from PET, comprising 15 single mutants [D12L, E47F, G62A, L90A, L90F, H129W, W155F, deltaV164, A173C, H184A, H184S, F209S, F209I, F249A and F249R], 6 double mutants [H129W/T136S, A173C/A206C, A173C/A210C, G62A/L90F, G62A/F209I, G62A/F249R], and 1 triple mutant [G62A/F209I/F249R]. Of these, 9 displayed no activity, 3 displayed decreased activity, 3 displayed comparable activity, and 7 displayed increased (~1.3 to ~7.2 fold) activity against solid PET, while all variants displayed activity against BHET. Of the variants that displayed increased activity against PET, 4 displayed more activity than G62A, the most-active mutant of TfCut2 known till date. Of these 4, 3 displayed even more activity than LCC (G62A/F209I, G62A/F249R and G62A/F209I/F249R), a CE known to be ~5-fold more active than wild-type TfCut2. These improvements derived from changes in PET binding and not changes in catalytic efficiency. This article is protected by copyright. All rights reserved.