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Introduction: Laccases are blue-multicopper containing enzymes that are known to play a role in the bioconversion of recalcitrant compounds. Their role in free radical polymerization of aromatic compounds for their valorization remains underexplored. In this study, we used a pBAD plasmid containing a previously characterized CotA laccase gene (abbreviated as Bli -Lacc) from Bacillus licheniformis strain ATCC 9945a to express this enzyme and explore its biotransformation/polymerization potential on β-naphthol. Methods: The protein was expressed from TOP10 cells of Escherichia coli after successful transformation of the plasmid. Immobilized metal affinity chromatography (IMAC) was used to generate pure protein. The biocatalytic polymerization reaction was optimized based on temperature, pH and starting enzyme concentration. 1H and 13C solution nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and solid-state NMR (ssNMR) were used to characterize the formed polymer. A one-gram conversion reaction was done to explore applicability of the reaction in a pilot-scale. Results: The polymerization reaction generated a brown precipitate, and its chemical structure was confirmed using 1H and 13C NMR and FTIR. SsNMR revealed the presence of two different orientational hydroxyl functional groups in the polymer in addition to the presence of a very small amount of ether linkages (< 2%). This analysis elucidated that polymerization occurred mainly on the carbons of the aromatic rings, rather than on the carbons attached to the hydroxyl groups, resulting in a condensed ring or polynuclear aromatic structure. The reaction was optimized, and the highest yield was attained under conditions of 37°C, pH 10 and a starting enzyme concentration of 440 nM in 50 mM phosphate buffer. A one-gram conversion yielded 216 mg of polymer as dry mass. The crystal structure of the enzyme was solved at 2.7 Å resolution using X-ray crystallography and presented with a hexagonal space group. The final structure was deposited in the Protein Databank (PDB) with an ID−9BD5. Discussion: This article provides a green/enzymatic pathway for the remediation of phenolics and their valorization into potential useful polymeric materials. The comprehensive analysis of the formed polymer provides insight into its structure and functional moieties present. Based on the yield of the one-gram conversion, this synthetic method proves useful for a pilot-scale production level and opens opportunities to invest in using this polymer for industrial/environmental applications. [ABSTRACT FROM AUTHOR]

Strong polyelectrolytes ( i.e. , macromolecules whose charge density is independent of the medium's pH) are invaluable assets in the soft matter toolbox, as they can readily disperse in aqueous media, complex to oppositely charged species - polymers and small molecules alike - and can be implemented in a plethora of applications, ranging from surface modification to chelating agents and lubricants. However, the direct synthesis of strong polyelectrolytes in a controlled fashion remains a challenging endeavour, and their in-depth characterisation is often limited. Additionally, producing a set of charged macromolecules with the same chain length but varying counterions would open doors towards a fine control of the polymer's chemistry and physical properties. Unfortunately, this either necessitates the direct polymerisation of several monomers with potentially varying reactivities, or a time-consuming ion exchange from a single batch. Herein we explore the facile and efficient production of strong polyanions through the deprotection of a poly(3-isobutoxysulphopropyl methacrylate) using a range of inorganic and organic iodide-containing salts. Owing to the contrasting nature of their counterions, the resulting polyanions exhibit a wide range of glass transition temperatures, which follow a non-monotonic trend with increasing counterion size. While all polymers readily dissolve in water, some can also be dissolved in non-aqueous media as well. This strategy, applied to block copolymers, permits the production of a library of amphiphilic macromolecules with consistent hydrophilic and hydrophobic blocks, yet varying nature of their polyanionic segments. All amphiphiles, regardless of their counterions, readily disperse in aqueous media and form well-defined micelles featuring a hydrophobic core and a charged hydrophilic shell, as evidenced by dynamic light scattering, ζ -potential and transmission electron microscopy. Additionally, a handful of block copolymers are capable of yielding polymer micelles in organic solvents, opening an avenue to the build-up of nanostructured soft matter in non-aqueous media., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)