Fritzsche S (2025)
Publication Type: Thesis
Publication year: 2025
In recent years, enzymatic depolymerisation of plastics such as PET has emerged as one promising solution to the problem of environmental pollution. It not only allows existing plastics to be recycled, but also addresses the depletion of fossil resources by reusing the recovered monomers. As with other biocatalytic processes, there are still challenges in implementing this biocatalytic approach on an industrial scale with high economic efficiency compared to existing chemical and physical recycling processes. In this context, this thesis has investigated the main factors influencing the efficiency of enzymatic PET depolymerisation. Different strategies have been applied to characterise the substrate and process conditions, to optimise the biocatalyst properties and to develop approaches for its production and reuse.
The LCC variant ICCG initially selected for this thesis was further optimised by protein engineering, resulting in the ICCGDAQI variant with four combined single site mutations. In addition to the thermostabilisation of ICCG introduced by Tournier et al. (2020), the ICCGDAQI variant improved the product release rate for various post-industrial PET fibre and post-consumer PET bottle substrates by up to 27%, which has already been confirmed for other substrates in further studies (Norton-Baker et al., 2024). The LCC-based variants used in this work are at this time still an excellent choice in terms of catalytic activity and thermostability compared to other biocatalysts, although several promising PET degrading enzymes have been identified or significantly improved by protein engineering in recent years (Sonnendecker et al., 2022; Qiu et al., 2024; Zheng et al., 2024). However, there is still a considerable demand and room for both the identification of novel wildtype enzymes and further protein engineering in the field of PET degrading enzymes. While many approaches in recent years have been aimed at optimising thermostability, with the requirement of hydrolysis temperatures of over 70 °C being discussed, isolation and optimisation of enzymes with high PET degradation rates at moderate temperatures of 30 to 40 °C could be promising (Pirillo et al., 2023). This could reduce process energy costs and pave the way for combined systems with simultaneous recombinant protein production and PET degradation. Screening of metagenomic libraries is particularly useful for achieving such goals (Datta et al., 2020). In the past, these screenings have yielded groundbreaking results such as LCC and PHL7 when examining compost materials (Sulaiman et al., 2012; Sonnendecker et al., 2022). Screening metagenomic libraries from marine or glacial environments for relevant target genes could lead to the identification of promising PET degrading enzymes (Liu et al., 2022a; Guo et al., 2023; Qi et al., 2023). A recent analysis of a global dataset with metagenomes of glaciers in the Alps, Tibet, Arctis and Antarctis identified more than 400 putative PET hydrolase sequences (Qi et al., 2023). Further breakthroughs in protein engineering are also expected in the future through new genetic methods, robotic assisted high-throughput screening and computational design based on artificial intelligence, some of which are already being applied (Cui et al., 2021; Li et al., 2022; Norton-Baker et al., 2024; Zheng et al., 2024).
This thesis has investigated different substrate properties and hydrolysis conditions and their influence on the efficiency of enzymatic PET depolymerisation. Specifically, XC and specific surface area were found to substantially influence the product release rates. Lower XC and higher specific surface area were correlated with higher product release rates. In addition to the pure PET materials already examined in this and other work (Thomsen et al., 2022), it is necessary to characterise and investigate the hydrolysis of polymer blends and mixed materials, such as natural fibre-polyester textile materials (Kaabel et al., 2023), for future applications. Product yield and profile can be influenced by hydrolysis conditions such as substrate pretreatment, pH, temperature and enzyme-substrate ratio, as demonstrated in this and other studies (Świderek et al., 2023; Heinks et al., 2024). Ultimately, however, a more refined compromise between product yield and environmental and economic impact may be required for industrial application.
This work could show that both E. coli and C. glutamicum are suitable organisms for the production of extracellular recombinant cutinase ICCGDAQI. Although the secretion of cutinase in C. glutamicum can be influenced by different signal peptides, the cutinase release obtained with E. coli, due to membrane damage caused by the catalytically active cutinase, was superior. With an extracellular recombinant cutinase production of up to 137 U mL-1, cutinase release in E. coli proved to be superior also to other known organisms such as B. subtilis and K. phaffii. Further optimisation of cultivation conditions and feed strategy is expected to further increase extracellular activity. Other organisms, especially filamentous fungi, could also be promising hosts for recombinant cutinase production. As noted above, mixtures of polyester/plant fibres such as cotton are more common in the textile industry than pure materials. This suggests a process in which all components can be usefully metabolised. In particular, cellulase-producing fungi such as Aspergillus niger or T. reesei could utilise cotton as carbon source for growth, with the recombinantly produced cutinase hydrolysing the remaining polyester fibres. The use of the plant part of blended fabrics has already been demonstrated for A. niger, with the PET fibres recovered in high purity (Hu et al., 2018). Together with other studies showing the simultaneous hydrolysis of PET and cotton by HiC and various cellulases (Kaabel et al., 2023), the described approach represents a realistic and promising way to further optimise biocatalyst production in an economically and ecologically meaningful way.
Although using immobilised enzymes to convert solid substrates has often been described as inefficient, in this work several possible strategies for immobilising the cutinase ICCGDAQI were identified. Under the depolymerisation conditions chosen for this thesis, particle-based and carrier-free methods were found to be inefficient due to limitations in achieving high catalytic activity, accessibility and stability. In contrast, the use of responsive polymers, in particular the pH-responsive polymer Kollicoat®, proved very promising for the repeatable complete degradation of solid PET substrates and for optimal integration into the process flow for recovery of both the enzyme and the target products. Therefore, immobilising the cutinase not only allows a cost-effective reuse of the biocatalyst, but also a higher purity of the target product without protein impurities. For further improvement of this approach, a more detailed characterisation of this immobilisation method in terms of loading, thermal and mechanical stability and transferability to other substrates, enzymes and reaction environments is required.
Overall, this thesis could point out the substantial potential of optimising the enzymatic depolymerisation of PET as a sustainable solution to the problem of plastic waste and depletion of fossil ressources. With growing interest from research groups worldwide and the establishment of a demonstration plant of this technology by CARBIOS (Biopôle Clermont-Limagne, France) supported by major industry players such as L'Occitane, L'Oréal, Nestlé Waters, Patagonia, PepsiCo and Puma, the transition from lab-scale research to industrial application seems achievable (Arnal et al., 2023; CARBIOS, 2025). Developments such as the first fibre-to-fibre shirt in October 2024 (CARBIOS, 2024) show that this technology has the potential to catalyse a circular economy for PET, particularly in major sectors such as food packaging and textiles, offering a promising path to a more sustainable and environmentally friendly future.
APA:
Fritzsche, S. (2025). Enzymatic Recovery of Polyester Monomers: Protein Engineering, Recombinant Production and Immobilisation of Cutinases (Dissertation).
MLA:
Fritzsche, Stefanie. Enzymatic Recovery of Polyester Monomers: Protein Engineering, Recombinant Production and Immobilisation of Cutinases. Dissertation, 2025.
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