Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase

Abstract

Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substrate-binding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 °C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 °C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic recycling processes.

Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes

“…The value and significance of this method has been demonstrated by several studies that successfully generated improved variants of PET degrading enzymes using MD simulations 14,58–60 . These studies not only confirm a mode of interaction comparable to the one we observe, with a strong proportion of aromatic interactions and simultaneous participation of polar interactions, but also illustrate for example the diverse binding of the substrate by means of several binding modes 16 …”

“…To date, there is some crystallographic data on the substrate interaction of PET degrading enzymes, however they rely on shorter substrate analogues instead of the PET polymer 16,55–57 . In order to circumvent this limitation, MD simulations can be employed, as done here.…”

“…Over the last years, protein engineering has been applied to increase activity and thermostability of these enzymes, enabling future recycling applications. Recently a number of exciting articles have been published reporting improved catalytic activities of either IS, PET2, or LCC 12‐19 . A variety of strategies targeting sequence‐based knowledge, substrate‐binding or the catalytic mechanism have been used including machine‐learning or directed evolution that resulted in enhanced degradation capabilities thereby emphasizing the high potential of engineered PET‐hydrolases.…”

“…Recently a number of exciting articles have been published reporting improved catalytic activities of either IS, PET2, or LCC. 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 A variety of strategies targeting sequence‐based knowledge, substrate‐binding or the catalytic mechanism have been used including machine‐learning or directed evolution that resulted in enhanced degradation capabilities thereby emphasizing the high potential of engineered PET‐hydrolases. In fact, Tournier et al already presented a proof of concept for enzymatic PET recycling employing engineered variants of LCC.…”

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

“…The positive correlation between the buffer concentration and the enzymatic performance of a PET hydrolase has also been reported (Pfaff et al 2022).…”