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The handling of plastic waste and the associated ubiquitous occurrence of microplastic poses one of the biggest challenges of our time. Recent investigations of plastic degrading enzymes have opened new prospects for biological microplastic decomposition as well as recycling applications. For polyethylene terephthalate, in particular, several natural and engineered enzymes are known to have such promising properties. From a previous study that identified new PETase candidates by homology search, we chose the candidate PET6 from the globally distributed, halophilic organism Vibrio gazogenes for further investigation. By mapping the occurrence of Vibrios containing PET6 homologs we demonstrated their ubiquitous prevalence in the pangenome of several Vibrio strains. The biochemical characterization of PET6 showed that PET6 has a comparatively lower activity than other enzymes but also revealed a superior turnover at very high salt concentrations. The crystal structure of PET6 provides structural insights into this adaptation to saline environments. By grafting only a few beneficial mutations from other PET degrading enzymes onto PET6, we increased the activity up to three-fold, demonstrating the evolutionary potential of the enzyme. MD simulations of the variant helped rationalize the mutational effects of those mutants and elucidate the interaction of the enzyme with a PET substrate. With tremendous amounts of plastic waste in the Ocean and the prevalence of Vibrio gazogenes in marine biofilms and estuarine marshes, our findings suggest that Vibrio and the PET6 enzyme are worthy subjects to study the PET degradation in marine environments.

Accumulation of plastic and subsequent microplastic is a major environmental challenge. With the discovery of potent polyethylene terephthalate (PET)-degrading enzymes, a new perspective arose for environmental decomposition as well as technical recycling. To explore the enormous diversity of potential PET-degrading enzymes in nature and also to conveniently employ techniques like protein engineering and directed evolution, a fast and reliable assay platform is needed. In this study we present our versatile solution applying a PET coating on standard lab consumables such as polymerase chain reaction tubes, 96- and 384-well microtiter plates, yielding an adjustable crystallinity of the PET. Combining the reaction vessels with either ultra-high performance liquid chromatography (UHPLC) or fluorometric readout and additional enzyme quantification offers a range of advantages. Thereby, the platform can easily be adapted to diverse needs from detailed analysis with high precision to high-throughput (HT) applications including crude lysate analysis.

Polyethylene terephthalate (PET) is an important synthetic polymer accumulating in nature 2 and recent studies have identified microorganisms capable of degrading PET. While the majority of 3 known PET hydrolases originate from the Actinobacteria and Proteobacteria, here we describe the 4 first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific 5 Hidden-Markov-Model (HMM)-based search algorithm we identified two promiscuous and cold6 active esterases derived from Aequorivita sp. (PET27) and Chryseobacterium jeonii (PET30) acting 7 on PET foil and powder. Notably, one of the enzymes (PET30) was able to hydrolyze PET at 8 temperatures between 4° - 30°C with a similar turnover rate compared to the well-known Ideonella 9 sakaiensis enzyme (IsPETase). 10 PET27 and PET30 homologues were detected in metagenomes encompassing a wide range 11 of different global climate zones. Additional transcript abundance mapping of marine samples imply 12 that these promiscuous enzymes and source organisms may play a role in the long-term 13 degradation of microplastic particles and fibers.

Polyethylene terephthalate (PET) is an important synthetic polymer accumulating in nature and recent studies have identified microorganisms capable of degrading PET. While the majority of known PET hydrolases originate from the Actinobacteria and Proteobacteria, here we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model (HMM)-based search algorithm we identified two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Chryseobacterium jeonii (PET30) acting on PET foil and powder. Notably, one of the enzymes (PET30) was able to hydrolyze PET at temperatures between 4° - 30°C with a similar turnover rate compared to the well-known Ideonella sakaiensis enzyme (IsPETase). PET27 and PET30 homologues were detected in metagenomes encompassing a wide range of different global climate zones. Additional transcript abundance mapping of marine samples imply that these enzymes and source organisms play a significant role in the long-term degradation of microplastic particles and fibers.

With macroscopic litter and its degradation into secondary microplastic as a major source of environmental pollution, one key challenge is understanding the pathways from macro- to microplastic by abiotic and biotic environmental impact. So far, little is known about the impact of biota on material properties. This study focuses on recycled, bottle-grade poly(ethylene terephthalate) (r-PET) and the degrading enzyme PETase from Ideonella sakaiensis. Compact tension (CT) specimens were incubated in an enzymatic solution and thermally and mechanically characterized. A time-dependent study up to 96 h revealed the formation of steadily growing colloidal structures. After 96 h incubation, high amounts of BHET dimer were found in a near-surface layer, affecting crack propagation and leading to faster material failure. The results of this pilot study show that enzymatic activity accelerates embrittlement and favors fragmentation. We conclude that PET-degrading enzymes must be viewed as a potentially relevant acceleration factor in macroplastic degradation.

MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore. Here, we present a near-native, site-specific in situ labeling strategy for MST experiments that enables reliable measurements in cell lysates and that has distinct advantages over routine covalent labeling techniques. To this end, we exploited the high-affinity interaction of tris-NTA with oligohistidine-tags, which are popular for purification, immobilization or detection of recombinant proteins. We used various DYE-tris-NTA conjugates to successfully label His-tagged proteins that were either purified or a component of cell lysate. The RED-tris-NTA was identified as the optimal dye conjugate with a high affinity towards oligohistidine-tags, a high fluorescence signal and an optimal signal-to-noise ratio in MST binding experiments. Owing to its emission in the red region of the spectrum, it also enables reliable measurements in complex biological matrices such as cell lysates allowing a more physiologically realistic assessment and eliminating the need for protein purification. ispartof: Scientific Reports vol:8 issue:1 pages:4977- ispartof: location:England status: published

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of β- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 A structure of Epstein-Barr virus (EBV) BFRF1–BFLF2, as well as an increased resolution 1.48 A structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approximately 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these β- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.