Comparative biochemistry of PET hydrolase-carbohydrate-binding module fusion enzymes on a variety of PET substrates.

Enzyme-driven recycling of PET has now become a fully developed industrial process. With the right pre-treatment, PET can be completely depolymerized within workable timeframes. This has been realized due to extensive research conducted over the past decade, resulting in a large set of engineered PET hydrolases. Among various engineering strategies to enhance PET hydrolases, fusion with binding domains has been used to tune affinity and boost activity of the enzymes. While fusion enzymes have demonstrated higher activity in many cases, these results are primarily observed under conditions that would not be economically viable at scale. Furthermore, the wide variation in PET substrates, conditions, and combinations of PET hydrolases and binding domains complicates direct comparisons. Here, we present a self-consistent and thorough analysis of two leading PET hydrolases, LCCICCG and PHL7. Both enzymes were evaluated both without and with a substrate-binding domain across a range of industrially relevant PET substrates. We demonstrate that the presence of a substrate-binding module does not significantly affect the affinity of LCCICCG and PHL7 for PET. However, significant differences exist in how the fusion enzymes act on different PET substrates and solid substrate loading, ranging from a 3-fold increase in activity to a 6-fold decrease. These findings could inform the tailoring of enzyme choice to different industrial scenarios.

Rennison AP ，Prestel A ，Westh P ，Møller MS ... -  《-》

Construction of Fusion Protein with Carbohydrate-Binding Module and Leaf-Branch Compost Cutinase to Enhance the Degradation Efficiency of Polyethylene Terephthalate.

Chen Y ，Zhang S ，Zhai Z ，Zhang S ，Ma J ，Liang X ，Li Q ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Engineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibition.

Recent studies on the enzymatic degradation of synthetic polyesters have shown the potential of polyester hydrolases from thermophilic actinomycetes for modifying or degrading polyethylene terephthalate (PET). TfCut2 from Thermobifida fusca KW3 and LC-cutinase (LCC) isolated from a compost metagenome are remarkably active polyester hydrolases with high sequence and structural similarity. Both enzymes exhibit an exposed active site in a substrate binding groove located at the protein surface. By exchanging selected amino acid residues of TfCut2 involved in substrate binding with those present in LCC, enzyme variants with increased PET hydrolytic activity at 65°C were obtained. The highest activity in hydrolyzing PET films and fibers were detected with the single variant G62A and the double variant G62A/I213S. Both variants caused a weight loss of PET films of more than 42% after 50 h of hydrolysis, corresponding to a 2.7-fold increase compared to the wild type enzyme. Kinetic analysis based on the released PET hydrolysis products confirmed the superior hydrolytic activity of G62A with a fourfold higher hydrolysis rate constant and a 1.5-fold lower substrate binding constant than those of the wild type enzyme. Mono-(2-hydroxyethyl) terephthalate is a strong inhibitor of TfCut2. A determination of the Rosetta binding energy suggested a reduced interaction of G62A with 2PET, a dimer of the PET monomer ethylene terephthalate. Indeed, G62A revealed a 5.5-fold lower binding constant to the inhibitor than the wild type enzyme indicating that its increased PET hydrolysis activity is the result of a relieved product inhibition by mono-(2-hydroxyethyl) terephthalate. Biotechnol. Bioeng. 2016;113: 1658-1665. © 2016 Wiley Periodicals, Inc.

Wei R ，Oeser T ，Schmidt J ，Meier R ，Barth M ，Then J ，Zimmermann W ... -  《-》

Engineered polyethylene terephthalate hydrolases: perspectives and limits.

Polyethylene terephthalate (PET) is a major component of plastic waste. Enzymatic PET hydrolysis is the most ecofriendly recycling technology. The biorecycling of PET waste requires the complete depolymerization of PET to terephthalate and ethylene glycol. The history of enzymatic PET depolymerization has revealed two critical issues for the industrial depolymerization of PET: industrially available PET hydrolases and pretreatment of PET waste to make it susceptible to full enzymatic hydrolysis. As none of the wild-type enzymes can satisfy the requirements for industrialization, various mutational improvements have been performed, through classical technology to state-of-the-art computational/machine-learning technology. Recent engineering studies on PET hydrolases have brought a new insight that flexibility of the substrate-binding groove may improve the efficiency of PET hydrolysis while maintaining sufficient thermostability, although the previous studies focused only on enzymatic thermostability above the glass transition temperature of PET. Industrial biorecycling of PET waste is scheduled to be implemented, using micronized amorphous PET. Next stage must be the development of PET hydrolases that can efficiently degrade crystalline parts of PET and expansion of target PET materials, not only bottles but also textiles, packages, and microplastics. This review discusses the current status of PET hydrolases, their potential applications, and their profespectal goals. KEY POINTS: • PET hydrolases must be thermophilic, but their operation must be below 70 °C • Classical and state-of-the-art engineering approaches are useful for PET hydrolases • Enzyme activity on crystalline PET is most expected for future PET biorecycling.

Kawai F ，Iizuka R ，Kawabata T 《-》

Structural insight into catalytic mechanism of PET hydrolase.

Han X ，Liu W ，Huang JW ，Ma J ，Zheng Y ，Ko TP ，Xu L ，Cheng YS ，Chen CC ，Guo RT ... -  《Nature Communications》