Rational mutagenesis of <i>Thermobifida fusca</i> cutinase to modulate the enzymatic degradation of polyethylene terephthalate
Arpita Mrigwani, Madhav Pitaliya, Harman Kaur, Bharathraj Kasilingam, Bhishem Thakur, Purnananda Guptasarma
Abstract
Abstract Thermobifida fusca cutinase ( Tf Cut2 ) 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 Tf Cut2) 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, ΔV164, A173C, H184A, H184S, F209S, F209I, F249A, and F249R), 6 double mutants [H129W/T136S, A173C/A206C, A173C/A210C, G62A/L90F, G62A/F209I, and G62A/F249R], and 1 triple mutant [G62A/F209I/F249R]. Of these, nine displayed no activity, three displayed decreased activity, three displayed comparable activity, and seven 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, four displayed more activity than G62A, the most‐active mutant of Tf Cut2 known till date. Of these four, three 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 Tf Cut2. These improvements derived from changes in PET binding and not changes in catalytic efficiency.