96 97 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 3D printable plastics that break down pollution Related publication: Zhang,W. H., Day, G. J., Zampetakis, I., Carrabba, M., Zhang, Z., Carter, B. M., Govan, N., Jackson, C., Chen, M., & Perriman, A.W. Three-dimensional printable enzymatically active plastics. ACS Applied Polymer Materials , 3, 6070–6077 (2021). DOI: 10.1021/ acsapm.1c00845 Publication keywords: Nanocomposite; Nanomorphology; Functional bionanomaterials; Enzyme; Nanoconjugate; 3D printing; Melt electrowriting S ome pollutants - such as the organophosphates used in pesticides and chemical weapons - persist in the environment. Self- decontaminatingmaterials could alleviate environmental pollution or form smart personal protective equipment. Incorporating enzymes into solid materials would allow us to take advantage of their fast and efficient reaction kinetics. However, current strategies, such as surface immobilisation of enzymes or the use of biomimetic hydrogels, are not always successful. Researchers have developed amethod tomodify the surface of a native enzyme with amixture of anionic and cationic polymer surfactants. This enhances the thermal resilience of themodified enzymes aswell as enabling their dissolution in organic solvents tomake co-dispersions with plastic polymers. To investigate the compatibility of these constructs with additive manufacturing fabrication techniques, the authors studied the protein structure to ensure the enzyme remained folded and active under the conditions required for material fabrication. The unique B23 beamline facilities offered the power and reliability tomeasure Circular Dichroism (CD) of the samples sandwiched between two fused silicawindows held horizontally to assess the protein secondary structure of the solvent-free enzymes. The beamline also allowed accurate characterisation of the thermal dependent unfolding transition of themelts above 100 °C up to 250 °C, unattainablewithbench-top CD instruments. Temperature-dependent Wide-Angle X-ray (WAXS) studies were also conducted on beamline I22 at Diamond Light Source to investigate the transition of the conjugates from lyophilised powders to annealedmelts. This research demonstrated that the fabricationmethods of organophosphate degrading enzymes through 3D printing, thermal moulding, andmelt electrowriting retaining their active structure were reproducible, the sine qua non condition for any commercial application. Organophosphates (OPs), found in nerve agents and pesticides, pollute the environment and can persist for several years in industrial materials, such as polymer paint coatings or tar roads 1 . Accordingly, the display or incorporation of hydrolytic enzymes into novel materials offers a route to imbuing such materials with self-decontaminating properties to counteract subsequent expose routes. Enzymes are an attractive option, as they often exhibit rapid reaction kinetics, function under green conditions, and can be modified to tailor their activities towards specific toxic compounds. Accordingly, there has been a focused research effort to integrate enzymes into solid materials through surface immobilisation or via the development of biomimetic hydrogels. However, there are limitations that reduce their widespread industrial utility. For example, surface-immobilised enzymes may be denatured, depleted or fouled and thus require a tailored environment to preserve surface activity, whereas enzymes immobilised in hydrogel matrices typically have limited mechanical and environmental stability, and can require specific conditions to retain their gel phase 2 . A key advance would be to successfully incorporate enzymes into more ubiquitous structural materials that have widespread utility. Our strategy involves re-engineering the surface of an enzyme such that it is readily dispersible in a hydrophobic medium; to provide a route towards their integration with suitable feedstock materials (e.g., polymers or plastics) for the fabrication of “smart” solid-state structures. In this scenario, the enzyme exists as an integral component within the material, which could even present new active surfaces through tuneable material degradation profiles, thus overcoming the issues of denaturation or fouling. Previously, we have reported on the electrostatic conjugation of polymer surfactantmolecules to thesurfaceof anenzyme to formanenveloping, dynamic corona 3 . This corona provides an interface with a polarity compatible with the desired dispersion media and also acts as a surrogate hydration shell to support correct protein folding and dynamics. Moreover, a well-designed corona has the potential to even improve the performance of enzymes by increasing the rate of substrate and product transport, by varying the dielectric constant in the vicinity of the active site 4 . In our present study, cationic and anionic polymer surfactants (S + and S − , respectively) were sequentially conjugated to phosphotriesterase from Agrobacterium radiobacter (arPTE). The resulting aqueous protein-polymer surfactant nanoconjugates, [arPTE][S + ][S − ], were lyophilised and thermally annealed to form solvent-free protein“melts”: dense, tacky biofluids composed purely of enzyme and surfactant (Fig. 1). Differential scanning calorimetry (DSC) performed on these biofluids revealed an endothermic melting transition at 33.9 ± 0.1 ° C, which was corroborated by a loss of crystalline features when measured by temperature-dependent Synchrotron RadiationWide-Angle X-ray Scattering (SR–WAXS), confirming a midpoint melting transition temperature of approximately 33.6 ° C. Demonstrating compatibility of these hybrid materials with modern high- resolution fabrication techniques is key for their adoption as a viable and practical material. Accordingly, the folding and thermostability of the arPTE in the melt was assessed by measuring its secondary structure content via Synchrotron Radiation–Circular Dichroism (SR–CD). The low sample volume, its inherent protein concentration and hyper-thermal stable properties required the superior signal-to-noise ratios, narrow pathlengths and large variable temperature range offered through beamline B23 at Diamond Light Source, compared to conventional CD apparatus. Notably, SR–CD revealed a midpoint denaturation temperature ( T m ) of 102.4 ± 2.0 ° C for the [arPTE][S + ][S − ] melt, a significant increase in the thermal stability compared to aqueous native arPTE enzyme ( T m = 71.2 ± 0.6 ° C; Fig. 2). This midpoint protein denaturation temperature indicated that the biofluid could tolerate the high temperatures applied during material fabrication without denaturation of the enzyme and loss of function. The polymer surfactant layer facilitated the dispersion of the nanoconjugate in a range of organic solvents, a necessary step to incorporating the enzyme with material feedstocks . Chloroform was used to produce co-dispersions of [arPTE][S + ][S − ] and polycaprolactone (PCL). PCL was of interest as it is biocompatible, biodegradable, and can be thermally extruded or moulded at ca. 60 ° C, thus was compatible with [arPTE][S + ][S − ]. Removal of the solvent from these co-dispersions gave solid enzymatically active plastic materials. The [arPTE][S + ][S − ]–PCL enzyme plastics could also be directly fabricated using thermal extrusion or moulding of the solid matrix after removal of the solvent. This provided access to 3D printed filaments and thermally moulded monoliths, allowing for the fabrication of larger structures. Significantly, 3D printed [arPTE][S + ][S − ]–PCL rings (1% enzyme w/w) exhibited enzymatic activity, persisting beyond 650 hours. Furthermore, the high resolution 3D fabrication technique Melt Electrowriting (MEW) was used to extrude precise "micro- meter" resolution structures at 70 °C from [arPTE][S + ][S − ]–PCL and analogous superfolder green fluorescent protein co-dispersions, [sfGFP][S + ][S − ]–PCL, such as fabric meshes with ultrafine threads (<5 µm, 0.1% enzyme w/w; Fig. 3). Interestingly, Quantitative Nanomechanics (QNM) mapping of the MEW plastic fibres revealed that the identity of the guest protein (arPTE or sfGFP) impacted upon the morphology and mechanical properties of composite materials.When compared with the neat PCL, both [sfGFP][S + ][S − ]–PCL and [arPTE][S + ][S − ]– PCL exhibited morphologies with increased levels of ordered structure that was indicative of high order molecular orientation and crystallinity, and the overall statistical average modulus of these materials were found to be 26.2 ± 11.6 MPa, 133.7 ± 49.8 MPa and 499.6 ± 182.0 MPa for PCL, [sfGFP][S + ][S − ]–PCL, and [arPTE][S + ][S − ]–PCL, respectively. In conclusion, we showed that functional bionanomaterials comprising enzymes and synthetic polymers provide an attractive opportunity to increase the diversity of chemical environments encountered by protein-based components. Our methodology for re-engineering the native protein surface by the sequential conjugation of anionic and cationic polymer surfactants can yield hyperthermal stable nanoconjugate melts capable of dispersion in a range of dielectric media. These melts could be co- dispersed with polymers, such as PCL, to yield feedstocks compatible with a range of modern fabrication techniques, including 3D printing and melt electrowriting, to fabricate bespoke, enzymatically active solid structures with tuneable degradation profiles and mechanical properties. Specifically, we utilised the phosphotriesterase arPTE to manufacture enzyme– plastics capable of hydrolysing organophosphorus compounds and exhibited catalytic activity persisting across multiple assays and throughout prolonged exposure to an aqueous environment. References: 1. Jang, Y. J. et al. Update 1 of: destruction and detection of chemical warfare agents. Chemical Reviews 115, PR1– PR76 (2015). DOI: 10.1021/acs. chemrev.5b00402 2. Basso, A. et al. Industrial applications of immobilized enzymes–A review. Molecular Catalysis 479, 110607 (2019). DOI : 10.1016/j.mcat.2019.110607 3. Brogan, A. P. S. et al. Enzyme activity in liquid lipase melts as a step towards solvent-free biology at 150 °C. Nature Communications 5, 5058 (2014). DOI: 10.1038/ncomms6058 4. Zhang,W. H. et al. Sequential electrostatic assembly of a polymer surfactant corona Increases activity of the phosphotriesterase arPTE. Bioconjugate Chemistry 30, 2771–2776 (2019). DOI: 10.1021/acs.bioconjchem.9b00664 Funding acknowledgement: This research was funded by the EPSRC (EP/N026586/1) that was awarded in collaboration with the Defence Science and Technology Laboratory (Dstl). Corresponding author: Dr Graham J. Day, University of Bristol,
[email protected] Prof. AdamW. Perriman, University of Bristol,
[email protected] Soft CondensedMatter Group Beamlines B23 and I22 Figure 1: Schematic demonstrating the formation of the solvent-free protein melt. The surface of the enzyme (arPTE) or protein (sfGFP) is modified via the sequential addition of cationic and anionic polymer surfactants and is then lyophilised to create a dry powder. Upon heating, the powder melts form a solvent-free liquid. Crucially, this does not inhibit the biological function, as shown by the retention of sfGFP fluorescence. Figure 2: The absorbance at 230 nm as a function of temperature of the unmodified arPTE in aqueous solution (blue) compared to the [arPTE][S + ][S − ] melt showing that the melt has an enhanced thermostability. Figure 3: Melt electrowriting prints fine PCL threads. (a) active enzyme-PCL fabrics ([arPTE] [S + ][S − ]–PCL); Fabric shown is 3 cm 2 in size; (b) Close examination of the [arPTE][S + ][S − ]–PCL fabric with widefield microscopy shows that it is comprised of tight tangles of enzyme–PCL threads arranged in a discrete repetitive pattern (fibres <5 µm in thickness); (c) Enzymatic activity of the material is retained after the melt electrowriting process, as shown through widefield fluorescence microscopy of the fabric in the presence of Coumaphos, a phosphothioate that is hydrolysed by arPTE into the fluorescent product chlorferon (excitation wavelength 355 nm, emission peak 460 nm) .