Improving plastic degrading enzymes via directed evolution.

Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering. For example, directed evolution has been used to improve the catalytic activity and thermostability of polyethylene terephthalate (PET)-degrading enzymes, although its use for the improvement of other desirable properties, such as solvent tolerance, has been less studied. In this review, we aim to identify some of the knowledge gaps and current challenges, and highlight recent studies related to the directed evolution of plastic-degrading enzymes.

Polyethylene Terephthalate Hydrolases in Human Gut Microbiota and Their Implications for Human Health.

Polyethylene terephthalate (PET), primarily utilized for food and beverage packaging, consistently finds its way into the human gut, thereby exerting adverse effects on human health. PET hydrolases, critical for the degradation of PET, have been predominantly sourced from environmental microbial communities. Given the fact that the human gut harbors a vast and intricate consortium of microorganisms, inquiry into the presence of potential PET hydrolases within the human gut microbiota becomes imperative. In this investigation, we meticulously screened 22,156 homologous sequences that could potentially encode PET hydrolases using the hidden Markov model (HMM) paradigm, drawing from 4984 cultivated genomes of healthy human gut bacteria. Subsequently, we methodically validated the hydrolytic efficacy of five selected candidate PET hydrolases on both PET films and powders composed of micro-plastics (MPs). Notably, our study also unveiled the influence of both diverse PET MP powders and their resultant hydrolysates on the modulation of cytokine expression in macrophages. In summary, our research underscores the ubiquitous prevalence and considerable potential of the human gut microbiota in PET hydrolysis. Furthermore, our study significantly contributes to the holistic evaluation of the potential health hazards posed by PET MPs to human well-being.

Unveiling PET Hydrolase Surface Dynamics through Fluorescence Microscopy.

PET hydrolases are an emerging class of enzymes that are being heavily researched for their use in bioprocessing polyethylene terephthalate (PET). While work has been done in studying the binding of PET oligomers to the active site of these enzymes, the dynamics of PET hydrolases binding to a bulk PET surface is an unexplored area. Here, methods were developed for total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) microscopy to study the adsorption and desorption dynamics of these proteins onto a PET surface. TIRF microscopy was employed to measure both on and off rates of two of the most commonly studied PET hydrolases, PHL7 and LCC, on a PET surface. It was found that these proteins have a much slower off rates on the order of 10-3  s-1 , comparable to non-productive binding in enzymes such as cellulose. In combination with FRAP microscopy, a dynamic model is proposed in which adsorption and desorption dominates over lateral diffusion over the surface. The results of this study could have implications for the future engineering of PET hydrolases, either to target them to a PET surface or to modulate interaction with their substrate.

Significance of poly(ethylene terephthalate) (PET) substrate crystallinity on enzymatic degradation.

Poly(ethylene terephthalate) (PET) is a semi-crystalline plastic polyester material with a global production volume of 83 Mt/year. PET is mainly used in textiles, but also widely used for packaging materials, notably plastic bottles, and is a major contributor to environmental plastic waste accumulation. Now that enzymes have been demonstrated to catalyze PET degradation, new options for sustainable bio-recycling of PET materials via enzymatic catalysis have emerged. The enzymatic degradation rate is strongly influenced by the properties of PET, notably the degree of crystallinity, XC. The higher the XC of the PET material, the slower the enzymatic rate. Crystallization of PET, resulting in increased XC, is induced thermally (via heating) and/or mechanically (via stretching), and the XC of most PET plastic bottles and microplastics exceeds what currently known enzymes can readily degrade. The enzymatic action occurs at the surface of the insoluble PET material and improves when the polyester chain mobility increases. The chain mobility increases drastically when the temperature exceeds the glass transition temperature, Tg, which is ∼40 °C at the surface layer of PET. Since PET crystallization starts at 70 °C, the ideal temperature for enzymatic degradation is just below 70 °C to balance high chain mobility and enzymatic reaction activation without inducing crystal formation. This paper reviews the current understanding on the properties of PET as an enzyme substrate and summarizes the most recent knowledge of how the crystalline and amorphous regions of PET form, and how the XC and the Tg impact the efficiency of enzymatic PET degradation.

Engineering the catalytic activity of an Antarctic PET-degrading enzyme by loop exchange.

Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.

Trends in in-silico guided engineering of efficient polyethylene terephthalate (PET) hydrolyzing enzymes to enable bio-recycling and upcycling of PET.

Polyethylene terephthalate (PET) is the largest produced polyester globally, and less than 30% of all the PET produced globally (∼6 billion pounds annually) is currently recycled into lower-quality products. The major drawbacks in current recycling methods (mechanical and chemical), have inspired the exploration of potentially efficient and sustainable PET depolymerization using biological approaches. Researchers have discovered efficient PET hydrolyzing enzymes in the plastisphere and have demonstrated the selective degradation of PET to original monomers thus enabling biological recycling or upcycling. However, several significant hurdles such as the less efficiency of the hydrolytic reaction, low thermostability of the enzymes, and the inability of the enzyme to depolymerize crystalline PET must be addressed in order to establish techno-economically feasible commercial-scale biological PET recycling or upcycling processes. Researchers leverage a synthetic biology-based design; build, test, and learn (DBTL) methodology to develop commercially applicable efficient PET hydrolyzing enzymes through 1) high-throughput metagenomic and proteomic approaches to discover new PET hydrolyzing enzymes with superior properties: and, 2) enzyme engineering approaches to modify and optimize PET hydrolyzing properties. Recently, in-silico platforms including molecular mechanics and machine learning concepts are emerging as innovative tools for the development of more efficient and effective PET recycling through the exploration of novel mutations in PET hydrolyzing enzymes. In-silico-guided PET hydrolyzing enzyme engineering with DBTL cycles enables the rapid development of efficient variants of enzymes over tedious conventional enzyme engineering methods such as random or directed evolution. This review highlights the potential of in-silico-guided PET degrading enzyme engineering to create more efficient variants, including Ideonella sakaiensis PETase (IsPETase) and leaf-branch compost cutinases (LCC). Furthermore, future research prospects are discussed to enable a sustainable circular economy through the bioconversion of PET to original or high-value platform chemicals.

Reaction Pathways for the Enzymatic Degradation of Poly(Ethylene Terephthalate): What Characterizes an Efficient PET-Hydrolase?

Bioprocessing of polyester waste has emerged as a promising tool in the quest for a cyclic plastic economy. One key step is the enzymatic breakdown of the polymer, and this entails a complicated pathway with substrates, intermediates, and products of variable size and solubility. We have elucidated this pathway for poly(ethylene terephthalate) (PET) and four enzymes. Specifically, we combined different kinetic measurements and a novel stochastic model and found that the ability to hydrolyze internal bonds in the polymer (endo-lytic activity) was a key parameter for overall enzyme performance. Endo-lytic activity promoted the release of soluble PET fragments with two or three aromatic rings, which, in turn, were broken down with remarkable efficiency (kcat /KM values of about 105  M-1 s-1 ) in the aqueous bulk. This meant that approximatly 70 % of the final, monoaromatic products were formed via soluble di- or tri-aromatic intermediates.

An Overview into Polyethylene Terephthalate (PET) Hydrolases and Efforts in Tailoring Enzymes for Improved Plastic Degradation.

Plastic or microplastic pollution is a global threat affecting ecosystems, with the current generation reaching as much as 400 metric tons per/year. Soil ecosystems comprising agricultural lands act as microplastics sinks, though the impact could be unexpectedly more far-reaching. This is troubling as most plastic forms, such as polyethylene terephthalate (PET), formed from polymerized terephthalic acid (TPA) and ethylene glycol (EG) monomers, are non-biodegradable environmental pollutants. The current approach to use mechanical, thermal, and chemical-based treatments to reduce PET waste remains cost-prohibitive and could potentially produce toxic secondary pollutants. Thus, better remediation methods must be developed to deal with plastic pollutants in marine and terrestrial environments. Enzymatic treatments could be a plausible avenue to overcome plastic pollutants, given the near-ambient conditions under which enzymes function without the need for chemicals. The discovery of several PET hydrolases, along with further modification of the enzymes, has considerably aided efforts to improve their ability to degrade the ester bond of PET. Hence, this review emphasizes PET-degrading microbial hydrolases and their contribution to alleviating environmental microplastics. Information on the molecular and degradation mechanisms of PET is also highlighted in this review, which might be useful in the future rational engineering of PET-hydrolyzing enzymes.

Rapid depolymerization of poly(ethylene terephthalate) thin films by a dual-enzyme system and its impact on material properties.

Enzymatic hydrolysis holds great promise for plastic waste recycling and upcycling. The interfacial catalysis mode, and the variability of polymer specimen properties under different degradation conditions, add to the complexity and difficulty of understanding polymer cleavage and engineering better biocatalysts. We present a systemic approach to studying the enzyme-catalyzed surface erosion of poly(ethylene terephthalate) (PET) while monitoring/controlling operating conditions in real time with simultaneous detection of mass loss and changes in viscoelastic behavior. PET nanofilms placed on water showed a porous morphology and a thickness-dependent glass transition temperature (Tg) between 40°C and 44°C, which is >20°C lower than the Tg of bulk amorphous PET. Hydrolysis by a dual-enzyme system containing thermostabilized variants of Ideonella sakaiensis PETase and MHETase resulted in a maximum depolymerization of 70% in 1 h at 50°C. We demonstrate that increased accessible surface area, amorphization, and Tg reduction speed up PET degradation while simultaneously lowering the threshold for degradation-induced crystallization.

Microbial Polyethylene Terephthalate Hydrolases: Current and Future Perspectives.

Plastic has rapidly transformed our world, with many aspects of human life now relying on a variety of plastic materials. Biological plastic degradation, which employs microorganisms and their degradative enzymes, has emerged as one way to address the unforeseen consequences of the waste streams that have resulted from mass plastic production. The focus of this review is microbial hydrolase enzymes which have been found to act on polyethylene terephthalate (PET) plastic. The best characterized examples are discussed together with the use of genomic and protein engineering technologies to obtain PET hydrolase enzymes for different applications. In addition, the obstacles which are currently limiting the development of efficient PET bioprocessing are presented. By continuing to study the possible mechanisms and the structural elements of key enzymes involved in microbial PET hydrolysis, and by assessing the ability of PET hydrolase enzymes to work under practical conditions, this research will help inform large-scale waste management operations. Finally, the contribution of microbial PET hydrolases in creating a potential circular PET economy will be explored. This review combines the current knowledge on enzymatic PET processing with proposed strategies for optimization and use, to help clarify the next steps in addressing pollution by PET and other plastics.

Enzymatic Remediation of Polyethylene Terephthalate (PET)-Based Polymers for Effective Management of Plastic Wastes: An Overview.

Globally, plastic-based pollution is now recognized as one of the serious threats to the environment. Among different plastics, polyethylene terephthalate (PET) occupies a pivotal place, its excess presence as a waste is a major environmental concern. Mechanical, thermal, and chemical-based treatments are generally used to manage PET pollution. However, these methods are usually expensive or generate secondary pollutants. Hence, there is a need for a cost-effective and environment-friendly method for efficient management of PET-based plastic wastes. Considering this, enzymatic treatment or recycling is one of the important methods to curb PET pollution. In this regard, PET hydrolases have been explored for the treatment of PET wastes. These enzymes act on PET and end its breakdown into monomeric units and subsequently results in loss of weight. However, various factors, specifically PET crystallinity, temperature, and pH, are known to affect this enzymatic process. For effective hydrolysis of PET, high temperature is required, which facilitates easy accessibility of substrate (PET) to enzymes. However, to function at this high temperature, there is a requirement of thermostable enzymes. The thermostability could be enhanced using glycosylation, immobilization, and enzyme engineering. Furthermore, the use of surfactants, additives such as Ca2+, Mg2+, and hydrophobins (cysteine-rich proteins), has also been reported to enhance the enzymatic PET hydrolysis through facilitating easy accessibility of PET polymers. The present review encompasses a brief overview of the use of enzymes toward the management of PET wastes. Various methods affecting the treatment process and different constraints arising thereof are also systematically highlighted in the review.