Although PETase engineering has advanced significantly in recent years, lower throughput methods for protein purification and activity monitoring have hindered the ability to further optimize designed PETase for industrial use. Enzyme engineering at the amino acid level can greatly improve catalytic stability and reaction velocity under process-relevant, often denaturing conditions. Key molecular characteristics of PETase have been identified through extensive biochemical and structural characterization, allowing for more focused and effective use of engineering techniques. New PETase variants have been created using techniques like semi-rational engineering, which entails computationally identifying a library of potentially advantageous mutations, and rational design, which uses evolutionary, structural, and mechanistic information to identify important mutations (Liu et al. 2023).

Increasing PETase activity and melting temperature in comparison to the wild type under chemically specified, buffered conditions has been the aim of these studies. ThermoPETase, which has three point mutations, an 8.81 °C increase in melting temperature, and a 14-fold increase in activity compared to wild-type, and DuraPETase, which has ten individual point mutations and a 300-fold increase in activity and a 31 °C increase in melting temperature compared to wild-type, are examples of successful variants. Although these advancements are noteworthy, the labor-intensive nature of all currently available expression and screening methods for creating these highly active mutants restricts the potential for more engineering toward commercial use. Therefore, there is a need to overcome the technical limitations of the existing state-of-the-art in PETase expression and screening methodology that hinder throughput (Zurier and Goddard 2023).

From an industrial perspective, PETase-driven hydrolysis offers a sustainable alternative to traditional mechanical and thermochemical recycling methods, which are energy-intensive and often result in downcycled products. By enabling the depolymerization of post-consumer PET into monomers, enzymatic recycling permits the closed-loop regeneration of virgin-quality plastics. In pilot-scale experiments, thermostable PETase variants have shown substantial efficacy, achieving up to 90% monomer recovery from bottle-grade PET after 72 h at 40 °C (Kushwaha et al. 2023). These results surpass conventional recycling in terms of energy efficiency and product quality, indicating strong potential for integration into circular economy frameworks.

Moreover, microbial valorization of PETase-derived intermediates is an emerging avenue with both environmental and economic advantages. For example, the metabolic incorporation of TPA into synthetic pathways by engineered Pseudomonas species has enabled the biosynthesis of value-added biopolymers such as polyhydroxyalkanoates (PHAs) (Ferreira et al. 2023). Under bioreactor conditions, such strains have produced 0.5–1 g/L PHA, suggesting a feasible strategy for converting PET waste into biodegradable plastics, thereby adding functional value to waste streams and promoting a sustainable bioeconomy (Kenny et al. 2012).

Despite these promising developments, significant challenges remain that impede the widespread implementation of PETase-based technologies. One major barrier is the cost of enzyme production. Current biotechnological systems, such as recombinant Escherichia coli, yield PETase at a significantly higher cost than chemical catalysts, with recombinant enzyme production costs often reaching several dollars per gram due to complex fermentation and purification processes (da Ferreira et al. 2018; Tufvesson et al. 2011)). In contrast, chemical catalysts, such as metal-based or organic catalysts used for PET depolymerization, are typically produced at costs below $1 per gram due to simpler synthesis and scalability (Tufvesson et al. 2011). This cost discrepancy is primarily attributed to low expression yields, labor-intensive purification steps, and the need for specialized cultivation conditions in biotechnological systems.

Another limitation arises from the intrinsic physicochemical properties of polyethylene terephthalate (PET). Its semi-crystalline structure restricts enzymatic accessibility, necessitating pre-treatment steps to increase amorphous regions, which are more susceptible to enzymatic hydrolysis (Kawai et al. 2020). Techniques such as thermal extrusion (e.g., heating to temperatures above the glass transition point, ~ 70 °C) or chemical amorphization can enhance substrate availability but introduce additional energy inputs and operational complexity, thus reducing the environmental and economic benefits of enzymatic recycling (Tournier et al. 2020). Furthermore, PETase activity is highly sensitive to environmental conditions, particularly pH and temperature. The enzyme exhibits optimal activity at a slightly alkaline pH (around 7.5–8) and temperatures of 30–40 °C, with significant reductions in catalytic efficiency at acidic pH (below 6.5) or elevated temperatures (above 40 °C) (Han et al. 2106). In real-world settings, such as landfills or marine environments, fluctuations in temperature and pH are common, limiting PETase’s practical effectiveness (Kawai et al. 2022). Efforts to engineer more robust and thermotolerant enzyme variants are ongoing, with some progress in improving stability, but these constraints remain unresolved.

In addition to enzymatic limitations, the physical configuration of PET waste presents a significant practical challenge for biodegradation applications. Most experimental studies to date have not used intact PET bottles but rather small PET film pieces, powdered PET, or fibers to facilitate enzyme accessibility and enhance depolymerization rates. For example, Puspitasari et al. (Puspitasari et al. 2021) utilized semi-crystalline PET fibers approximately 20 μm in diameter, while other studies have employed thin PET “coupons” cut from bottles or mechanically ground PET particles to increase surface area (Son et al. 2019). In practical applications, pre-treatment steps such as shredding, milling, or thermal extrusion are currently essential to convert large PET items into smaller, more amorphous fragments that are susceptible to enzymatic attack. At present, the direct biodegradation of full, intact PET bottles remains technically infeasible. Bridging this gap between laboratory conditions and real-world plastic waste streams will require not only continued enzyme engineering but also innovations in substrate preparation, reactor design, and integrated recycling workflows (Austin et al. 2018).

Finally, regulatory and logistical barriers significantly complicate PETase deployment, particularly for environmental applications. The use of genetically modified organisms (GMOs) or the release of active enzymes into open systems requires stringent biosafety and ecological impact assessments to prevent unintended environmental consequences (Xu et al. 2024). Approvals for enzyme release, implementation of containment measures, and long-term monitoring are governed by national and international regulations, such as those under the Cartagena Protocol on Biosafety, which can delay deployment and increase costs (Habib et al. 2024). These regulatory hurdles add complexity to scaling PETase-based technologies for widespread use.

While the development of highly efficient PETase variants offers promising avenues for bioremediation and plastic waste management, the potential environmental release of genetically engineered microorganisms (GEMs) raises important biosafety concerns. Releasing genetically modified organisms (GMOs) into the environment could unintentionally disrupt natural microbial communities or transfer engineered genes to other species. These changes could have unforeseen effects on ecosystems that are not yet fully understood (Wright et al. 2013). To address these concerns, scientists are exploring safety strategies such as building genetic "kill-switches" into the organisms, using strains that cannot survive outside controlled conditions, or focusing on applying only the purified enzymes instead of live bacteria (Brooks and Alper 2021). In addition to these technical solutions, there are also international regulations, such as the Cartagena Protocol on Biosafety, which require thorough risk assessments before any environmental release. As research on PETase continues, balancing environmental benefits with safety considerations will be essential.

In summary, while PETase and its engineered variants offer a promising approach to PET biodegradation and recycling, further innovations are needed to overcome technical, economic, and regulatory challenges. Advances in protein engineering to enhance enzyme stability, process optimization to reduce production costs, and synthetic biology to improve expression systems will be critical to realizing the full potential of PETase within a sustainable plastic lifecycle (Carniel et al. 2024).