Substrate-Binding Mode of a Thermophilic PET Hydrolase and Engineering the Enzyme to Enhance the Hydrolytic Efficacy

Abstract

Polyethylene terephthalate (PET) is among the most extensively produced plastics, but huge amounts of PET wastes that have accumulated in the environment have become a serious threat to the ecosystem. Applying PET hydrolytic enzymes to depolymerize PET is an attractive measure to manage PET pollution, and searching for more effective enzymes is a prerequisite to achieve this goal. A thermostable cutinase that originates from the leaf-branch compost termed ICCG is the most effective PET hydrolase reported so far. Here, we illustrated the crystal structure of ICCG in complex with the PET analogue, mono­(2-hydroxyethyl)­terephthalic acid, to reveal the enzyme–substrate interaction network. Furthermore, we applied structure-based engineering to modify ICCG and screened for variants that exhibit higher efficacy than the parental enzyme. As a result, several variants with the measured melting temperature approaching 99 °C and elevated PET hydrolytic activity were obtained. Finally, crystallographic analyses were performed to reveal the structural stabilization effects mediated by the introduced mutations. These results are of importance in the context of understanding the mechanism of action of the thermostable PET hydrolytic enzyme and shall be beneficial to the development of PET biodegradation platforms.

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

“…As a result, LCC ICCG has a more rigid substrate-binding groove, yet it still allows PET binding at elevated temperatures as a result of increased polymer chain mobility. 23 …”

“…In a recent study, LCC ICCG was further engineered to obtain an A59K/V63I/N248P variant with a T m of 98.9 °C. 23 Nonetheless, the T opt of this variant for degrading low-crystallinity PET (lcPET, <15% crystallinity) was 74 °C, only 2 °C higher than that for the precursor LCC ICCG. 5 At reaction temperatures above 70 °C, water-plasticized lcPET rapidly crystallizes to high-crystallinity PET (hcPET), which is much harder to hydrolyze.…”

“… 28 Zeng et al reported the structure of an inactive S165A mutant of LCC ICCG complexed with mono-(2-hydroxyethyl) terephthalate (MHET). 23 Consequently, more structures of PET hydrolases with bound substrate analogues are needed. This would enable more precise computational simulations for elucidating the mechanisms of enzymatic depolymerization and predicting more efficient mutants.…”

“…Calcium ions (Ca 2+ ) can boost the thermostability of many bacterial PET hydrolases (changes in T m (Δ T m ) = 10–16 °C and Δ T opt ≥ 10 °C). ,− Engineering of residues in the Ca 2+ binding sites has proven to be a useful approach for increasing the T m of several PET hydrolases by up to 26 °C. ,,,− Introduction of a disulfide bridge at this site (D238C/S283C) markedly increased the melting point ( T m ) of LCC from 84.7 to 94.5 °C. In a recent study, LCC ICCG was further engineered to obtain an A59K/V63I/N248P variant with a T m of 98.9 °C . Nonetheless, the T opt of this variant for degrading low-crystallinity PET (lcPET, <15% crystallinity) was 74 °C, only 2 °C higher than that for the precursor LCC ICCG .…”

Conversion of polyethylene terephthalate into pure terephthalic acid through synergy between a solid-degrading cutinase and a reaction intermediate-hydrolysing carboxylesterase

“…A green approach to its degradation involves the use of microbederived carboxylesterases (CEs). [1][2][3][4][5][6][7][8][9][10][11] CEs display low substrate specificity (in functions ranging from trans-esterification, or synthesis, of ester bonds to hydrolysis of carboxylic esters, thioesters and amides). 12 Cutinases are CEs that act upon cutin (a waxy substance covering plant leaves).…”