Positive Charge Introduction on the Surface of Thermostabilized PET Hydrolase Facilitates PET Binding and Degradation

Abstract

A thermostable enzyme PET2, found in a metagenome library, has been engineered to improve its hydrolytic activity against polyethylene terephthalate (PET). The PET2 wild-type (WT) showed a melting temperature of 69.0 °C and produced water-soluble reaction products at a rate of 0.40 min–1 (2.4 μM products from 0.1 μM enzyme after 60 min reaction) from an amorphous PET film at 60 °C. Mutations for surface charge modification, backbone stabilization, and formation of additional disulfide bond were introduced into the PET2 WT, and the best mutant (PET2 7M) showed a melting temperature of 75.7 °C and hydrolytic activity of 1.3 min–1 (7.8 μM products from 0.1 μM enzyme after 60 min reaction at 60 °C). X-ray crystal structures of PET2 mutants showed that introduced arginine and lysine residues oriented to the solvent, similar to a PET hydrolase from Ideonella sakaiensis 201-F6. Single-molecule fluorescence imaging revealed that these positively charged surface residues increased binding rate constant of PET2 7M to PET surface 2.7 times, compared with PET2 WT, and resulted in higher activity. Optimal temperature for amorphous PET hydrolysis by PET2 7M (68 °C) was 8 °C higher than that by PET2 WT (60 °C), and hydrolytic activity of PET2 7M at the optimal temperature (2.7 min–1, 16.2 μM products from 0.1 μM enzyme after 60 min reaction) was 6.8 times higher than that of PET2 WT (0.40 min–1). Furthermore, PET2 7M generated reaction products with a constant rate for at least 24 h at 68 °C, indicating long-term thermal stability at the optimal temperature.

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

“… 13 , 16 − 18 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. 5 , 11 , 17 , 19 − 22 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.…”

A cocktail of protein engineering strategies: Breaking the enzyme bottleneck one by one for high UTP production in vitro

“…Disappointingly, all variants were not as active as the R117A variant, despite their improved thermostability compared to the wild type. Nakamura et al (2021) obtained variants with improved thermostability by changing the charge distribution on the surface of the PET enzyme. For Sl UMPK, residue R117 is also located on the surface of the protein, and the R117A variant might affect the surface charge distribution, thereby affecting the thermostability (Figure S7).…”

“…In recent years, with the cross‐application of computer science, computational chemistry, and other disciplines in protein engineering, there is an increasing number of means to address the challenge and a spurt of success stories. For example, reconstructing ancestral sequences by inferring phylogenetic relationships between modern homologs and applying different amino acid substitution models is considered a powerful approach to screen for enhanced thermostability or unique active proteins (Furukawa et al, 2020; Gumulya et al, 2018; Selberg et al, 2021; Spence et al, 2021); iterative conformational dynamics analysis (ICDA) based on molecular dynamics (MD) simulations can continuously improve the reliability of key amino acids used to construct mutant libraries, thereby increasing the likelihood of obtaining target variants, even if the protein models used in the simulations do not achieve the accuracy of real experimental structures (Heo et al, 2019; Li et al, 2021); and remodeling of protein surface charges successfully alters the pH adaptation or thermostability of enzymes (Han et al, 2021; Huang et al, 2021; Nakamura et al, 2021; Wang et al, 2020).…”

Processive Enzymes Kept on a Leash: How Cellulase Activity in Multienzyme Complexes Directs Nanoscale Deconstruction of Cellulose

“… 6 Biological deconstruction relies on cooperative catalysis by multienzyme systems as a common mechanistic principle. The principle is exemplified on a large variety of biomaterials built from hydrolyzable polymer chains (e.g., polysaccharides, 9 − 11 structural proteins, 12 polyesters 11 , 13 , 12 − 15 ). In all cases, the material deconstruction gains efficiency from the spatiotemporally coordinated action of different enzymes with synergetic function in polymer chain depolymerization.…”

“…Biological deconstruction relies on cooperative catalysis by multienzyme systems as a common mechanistic principle. The principle is exemplified on a large variety of biomaterials built from hydrolyzable polymer chains (e.g., polysaccharides, − structural proteins, polyesters ,,− ). In all cases, the material deconstruction gains efficiency from the spatiotemporally coordinated action of different enzymes with synergetic function in polymer chain depolymerization. , Typically, a set of chain end-cleaving and internally chain-cleaving hydrolases act in synergy. , Even in xenobiotic materials such as polyethylene terephthalate, the naturally evolved enzyme system for biological deconstruction of the plastics exploits the synergy between two hydrolases. ,,, …”