Mechanism-Based Design of Efficient PET Hydrolases

Abstract

Polyethylene terephthalate (PET) is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages and food, contributing considerably to the global solid waste stream and environmental plastic pollution. While enzymatic PET recycling and upcycling have recently emerged as viable disposal methods for a circular plastic economy, only a handful of benchmark enzymes have been thoroughly described and subjected to protein engineering for improved properties over the last 16 years. By analyzing the specific material properties of PET and the reaction mechanisms in the context of interfacial biocatalysis, this Perspective identifies several limitations in current enzymatic PET degradation approaches. Unbalanced enzyme-substrate interactions, limited thermostability, and low catalytic efficiency at elevated reaction temperatures, and inhibition caused by oligomeric degradation intermediates still hamper industrial applications that require high catalytic efficiency. To overcome these limitations, successful protein engineering studies using innovative experimental and computational approaches have been published extensively in recent years in this thriving research field and are summarized and discussed in detail here. The acquired knowledge and experience will be applied in the near future to address plastic waste contributed by other mass-produced polymer types (e.g., polyamides and polyurethanes) that should also be properly disposed by biotechnological approaches.

Figure 1

Selected milestones of a 16-year-long history of identifying and engineering PET hydrolases. Both optimal reaction temperatures (in varying colors) for PET degradation and normalized maximal conversion rates (diamonds corresponding to logarithmic values on the vertical axis) calculated based on various publications are denoted, regardless of the material properties of applied PET substrates. The successes in raising the degradation performance using certain benchmark enzymes by both protein and process engineering are indicated by arrows with broken lines. TfH: hydrolase from T. fusca DSM43793;HiC: Humicola insolens cutinase; LCC: leaf-branch compost cutinase;^,IsPETase: I. sakaiensis PET hydrolase;^,TfCut2: T. fusca KW3 cutinase; LCC^ICCG: LCC variant with indicated quadruple substitutions; DuraPETase, ThermoPETase, etc.: various thermostabilized IsPETase variants.⁻

Figure 2

Frequently reported mutation hot spots illustrated on the superposed crystal structures of known bacterial PET hydrolases. Backbones shown in the cartoon are derived from the structural superposition with selected homologous enzymes. The catalytic triad (S160, D206, and H237) as well as two aromatic residues (W159 and W185) are involved in the interaction with the monomer analogue 1-(2-hydroxyethyl)-4-methyl terephthalate (HEMT) (A, B) based on the IsPETase structure (PDB ID: 5XH3; the numbering of residues is modified consistently with other structures solved later for easy comprehension). (A) One frequently reported mutation hotspot equivalent to S209 (B) in IsPETase can adopt various residues which might influence the widths of the binding pocket:^, F found in 4CG1, 4EB0, and 7OSB and also conserved in many other PET hydrolases; S found in the IsPETase structure 5XH3; I found in an LCC mutant 6THT; L found in another PET hydrolase 7CUV. (C) One of the putative Ca²⁺ binding sites revealed by cocrystallized structures such as 4WFJ, 5LUL, and 5ZNO can be replaced by a disulfide bridge (6THT, 7CTS, and 7CTR) to thermostabilize several PET hydrolases.

Figure 3

Interfacial biocatalytic hydrolysis of PET and its reaction mechanism. The states of a PET hydrolase are schematically illustrated in the upper right panel. In the lower panel, individual steps of the hydrolysis reaction are schematically shown in line with their activation free-energy barriers in kcal·mol^–1 summarized based on different studies.^, The reaction is initiated by a nucleophilic attack by a catalytic serine resulting in a tetrahedral intermediate stabilized by a catalytic histidine, an aspartic acid, and the oxyanion hole, followed by breakdown of the tetrahedral intermediate 1 into an acyl–enzyme intermediate and release of an alcohol. The aspartate–histidine pair activates the water for attack on the acyl–enzyme intermediate carbonyl, resulting in the formation of the second tetrahedral intermediate. The deacylation of this tetrahedral intermediate releases the carboxylic acid product. The rate-limiting step is regarded as the initial nucleophilic attack and highlighted in red with two free-energy activation barriers denoted. The top number is the Boltzmann-weighted average from 20 QM/MM MD simulations, and the bottom number comes from adiabatic mapping studies.

Figure 4

Potential PET hydrolase screening methods arranged in order of increasing potential throughput. (A) Fluorogenic substrates like fluorescein dilaurate can be trapped in polyester films or particles^, and can be released and hydrolyzed upon polymer hydrolysis, generating a fluorescence signal. (B) Fluorimetric method based on the reaction of terephthalic acid with hydroxyl radicals to form the fluorophore 2-hydroxyterephthalic acid.⁻ Tens of thousands of clones can be screened using microtiter plate-based assays (A, B). (C) Agar plate assay based on the hydrolysis of polyester (PET) nanoparticles.^,, Clear zones (halos) form around clones expressing active polyester-hydrolyzing enzymes, allowing simple visual identification. Millions of clones can easily be screened using this method. (D) Recently reported ultra-high-throughput droplet-based assay for PETase activity. The use of the fluorogenic surrogate substrate fluorescein dibenzoate indicates a low selectivity, since many other esterases would also be identified using this assay. Tens of millions of clones could be analyzed using this method. Combinations of the turbidimetric assay (C) and droplet-based methods (D) seem promising. (E) Ultra-high-throughput assay based on a terephthalic acid biosensor. Cells could be entrapped in hydrogel beads containing reporter cells that express GFP in response to terephthalic acid formed by clones expressing active PET-hydrolyzing enzymes. Because fluorescence-activated cell sorting (FACS) can be used to sort the beads, the throughput of this method is potentially in the hundreds of millions. (F) Envisaged growth selection approach based on the conversion of terephthalic acid to protocatechuic acid, which could be catabolized by engineered strains of E. coli or other model organisms.^, The throughput of this method would be limited only by library size and transformation efficiencies, making it one of the most attractive methods.