1. Introduction

The growing usage of enzymes in biodegradation is partially made possible by the evolution of diverse, but complementary, scientific fields. Even though enzymes are natural catalysts, their efficiency, stability, and catalytic turnover is often insufficient to be feasibly applicable [59]^[1]. Thus, diverse enzyme engineering approaches to improve catalytic, stability and productivity properties have been developed. Ultimately, the goal of enzymatic genetic engineering is to modify the amino acid sequences through gene alteration [60]^[2].

Since rational design implies knowledge of protein function and structure, it frequently begins with computer aided design [60]^[2]. The rapid increase of high-resolution protein structures available on the PDB database [63]^[3], and the improvement of structure-prediction tools, such as the recently released AlphaFold [64]^[4], allows for molecular and atomistic perspectives on the structure and function of enzymes. This pipeline combines computational [65]^[5] and experimental [66]^[6] techniques to improve enzymatic thermal stability (through, for example, introduction of additional disulfide bonds), filling protein voids, increase enzymatic turnover and efficiency, or attribute new functions to the enzyme.

At this stage, several PET degrading enzymes have been discovered, and as a lot of work has been done on structural solving and characterization, the emerging trend for the future of the field is the engineering and improvement of these found enzymes. Structure resolution and atomistic understanding of an enzyme is tightly related with the ability to perform rational design, and several of the efforts employed on PET enzymes follow this line of thought, as mutagenesis is used both to confirm amino acid roles, and to improve enzymatic characteristics.

2. Enzymes Involved in PET Degradation

IsPETase presents a highly polarized surface charge, creating a dipole across the macromolecule and resulting in an isoelectric point of 9.6 [75]^[7]. Several PET degrading enzymes present charged surfaces, as evidenced by Figure 5 .

Overall, BhrPETase is a novel enzyme with high PET degrading activity, thermal stability, and a Tm higher than the Tg of PET, features that make it a very promising enzyme for further studies on the biodegradation of PET.

Database query for novel PET degrading enzymes followed a set of criteria inspired by enzymes with well characterized activity on PET, namely the presence of a conserved Ser-His-Asp catalytic triad in the vicinity of an oxyanion hole made up of a Met residue and an aromatic residue, a terminal disulfide bridge essential for enzymatic thermostability, and a conserved DxDxR(Y)xxF(L)C sequence near the first cysteine of the disulfide bridge. Even though no further characterization of these enzymes has been published, the experimentally validated metagenomic methodology search used and the most promising enzymes identified suggest an encouraging path for identifying novel enzymes and microorganisms with relevant PET degrading activity.

PET2 ability to remain active at higher drastic temperatures makes it an extremely attractive enzyme for PET degradation. When 100 μg of enzyme were tested with 14 mg amorphous PET as substrate, 900 μM of TPA were produced after a 24 h period incubation, confirming PET2 demonstrates interesting PET degrading activity and is able to fully degrade it to TPA.

3. Microorganisms with PET Degradation Activity

The evidence that a given organism can degrade PET or other polyester materials and use them as a carbon source frequently leads to the identification of a PET degrading enzyme. However, in many cases it is not possible (or it has not yet been done) to identify or isolate the specific enzyme responsible for PET depolymerization. In this section, some of these microorganisms are described.

Liebminger et al. [251]^[8] screened for microorganisms with PET degradation activity by incubating environmental samples with 3PET as the only carbon source for 10 days. From the active samples, a fungal strain identified as Penicillium citrinum showed the best growth. Although an enzyme was purified, characterization and identification of this presumed polyesterase was not possible. The experiments with PET fabric and 3PET as substrates revealed optimal conditions for enzymatic activity to be 30 °C and pH 8.2. The enzyme successfully altered the surface of the PET fabric and degraded 3PET to TPA, MHET, BHET, and BA. Given the described results, it is to be expected that further biochemical and structural characterization of this novel polyesterase may reveal a new promising PET degrading enzyme.

Costa et al. [252]^[9] showed Yarrowia lipolytica , a widely used yeast, had PET degrading activity. Y. lipolytica was cultured with amorphous PET, PET oligomer 3, post-consumer PET (with high crystallinity), and PET monomers TPA, monoethylene glycol (MEG), and BHET. Highest activity was registered for the amorphous PET samples, with MHET as the main product and TPA as a residual released product. This suggests MHET has an inhibitory effect on the yeast, hindering complete degradation of PET to TPA. Although the yeast was able to use the monomers as a carbon source in the absence of other substrates, the accumulation of PET degradation products seemed to be toxic for yeast growth, and consumption rate was higher for PET oligomers than for the monomers. The described results show that Yarrowia lipolytica is able to express PET degrading enzymes, as evidenced by the release of MHET in large quantities, and further isolation and expression of these enzymes is of high importance for future PET degrading yeast studies.

Arguing that using individual purified enzymes to break down PET might not be the most promising strategy, Roberts et al. [253]^[10] showed that a bacterial consortia containing Bacillus and Pseudomonas species able to use PET as a sole carbon source resulted in more effective PET degradation and surface modification activity. The consortia were identified in a screening of soil samples for lipase activity, and several consortia were built and further analyzed after initial lipase activity was confirmed. PET hydrolytic activity was demonstrated by the depolymerization of amorphous PET to BHET, TPA, and EG by the bacterial consortia. Individual strains identified within the consortia were tested as well, but activity and product release were always slower and lower than in the assays with the full consortia. It was therefore concluded that the bacterial strains in the consortia acted on PET in a synergistic manner, and complete degradation was only possible in the presence of all bacterial strains and respective secreted enzymes. Identification of individual secreted enzymes was not possible, which the authors attributed to biofilm formation within the consortia, and natural tendency of the secreted enzymes to bind the PET sample, hindering isolation.

4. Potential PET Hydrolases with Activity to Be Confirmed

Ao Cut, a cutinase from Aspergillus oryzae , has been shown to degrade PCL and theorized to have a similar effect on PET [254,255]^[11][12]. The two three-dimensional structures available for Ao Cut (PDB: 3GBS and 3QPD) reveal an α/β-serine hydrolase fold, with a central β sheet flanked by α helixes on either side. The catalytic triad (Ser126, Asp181, His194) and oxyanion hole (Ser48 and Gln127) are similar to the reported residues in typical enzymes with PET degrading activity. The two major structural differences that differentiate Ao Cut are Fs Cut the additional disulfide bridge (Cys63–Cys76, in addition to the two well conserved Cys37–Cys115 and Cys177–Cys184) and a longer and deeper groove near the active site. These features could explain the higher thermal stability of Ao Cut, and also higher activity on PCL [255]^[12]. Efforts by Shirke et al. [254]^[11] to increase thermal stability via rational mutation design have been successful in increasing T m by 6 °C, but with a negative effect on PCL degrading activity. It is widely suggested that Ao Cut will have a hydrolytic effect on PET polymer, but only specific experimental activity assays can confirm this assumption.

Huang et al. [257]^[13] reported an esterase from Thermobifida fusca named Tf AXE, with high similarity with Tf HCut and other highly active PET degrading enzymes. Even though specific activity against PET or other polyester polymers has not yet been described for this enzyme, it is consensually regarded as a likely PETase-like enzyme and considered of major interest in state-of-the-art PET bioremediation studies.

Among the p -nitrophenyl acyl esters, Ta Est119 revealed higher enzymatic activity towards p NPB substrate, resulting in a k cat of 4.48 ± 0.21 s −1 [57]^[14], and a specific activity of 2.30 ± 0.02 U/mg in the absence of Ca 2+ and 8.29 ± 0.03 U/mg in the presence of 300 mg of Ca 2 + [210]^[15].

To implement Ta Est119 in the plastic-degrading industry for catalyses of PET substrates, enzymatic activity and thermostability need to be enhanced. For a better understanding of the enzymatic activity, studies of Ta Est119 complexed with PET substrates should be developed.