Marina Refaat ¹ Marwa T. ElRakaiby ¹ Mustapha El Hariri El Nokab ² Julien Es Sayed ³ Ahmed Elshewy ^4,5 Khaled O. Sebakhy ^6,7 Nayera Moneib ¹ Tuo Wang ^2* Thomas J. Smith ^8* Mohamed H. Habib ^1,8,9*

• ¹ Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Beni Suef, Egypt
• ² Department of Chemistry, College of Natural Science, Michigan State University, East Lansing, Michigan, United States
• ³ Zernike Institute for Advanced Materials, Faculty of Science and Engineering, University of Groningen, Groningen, Netherlands
• ⁴ Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Giza, Beni Suef, Egypt
• ⁵ Department of Natural and Applied Sciences, College of Arts and Sciences, The American University of Iraq-Baghdad (AUIB), Baghdad, Iraq, Baghdad, Iraq
• ⁶ Department of Materials, Textiles and Chemical Engineering, Centre for Polymer and Material Technologies (CPMT), Ghent University, Ghent, Belgium, Ghent, Belgium
• ⁷ Laboratory for Chemical Technology, Ghent University, Ghent, East Flanders, Belgium
• ⁸ Department of Biochemistry and Molecular Biology, John Sealy School of Medicine, University of Texas Medical Branch at Galveston, Galveston, Texas, United States
• ⁹ Department of Internal Medicine, John Sealy School of Medicine, University of Texas Medical Branch at Galveston, Galveston, Texas, United States

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. 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 polymerization reaction generated a brown precipitate, and its chemical structure was confirmed using 1H and 13C solution nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR). Solid-state NMR (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. This article provides a green/enzymatic pathway for the remediation of phenolics and their valorization into potential useful polymeric materials.