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Title: Biochemical characterization of multicopper oxidase enzymes from lignin degrading bacteria
Author: Granja Trávez, Rommel Santiago
ISNI:       0000 0004 7961 1495
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2018
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Lignin is an abundant biopolymer found in nature. Because of its carbon content and aromatic nature, lignin is promising renewable feedstock for biotechnological applications. However, the resistance of lignin to chemical and microbial breakdown have limited its utilization. Some enzymes have been involved in lignin depolymerization, among them, the multicopper oxidase subgroup of laccase has shown oxidase activity towards lignin. In addition, since laccase activity only requires oxygen and the only by-product generated is water, these enzymes have been regarded as perfect green catalysts, offering a range of potential applications beyond lignin transformation. Multicopper oxidases (MCOs) have been found encoded in the genome of some bacterial lignin degraders, which might show laccase activity and maybe play a role in lignin degradation. In this study, three MCOs (Pp-CopA, Pf-CopA and Oc-CueO), have been expressed and reconstituted as recombinant laccase-like multicopper oxidases (LMCOs), and they have been kinetically characterized. Pp-CopA, Pf-CopA and Oc-CueO showed activity towards common laccases substrates, such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS); syringaldazine (SGZ); guiacol; 2,6-dimethoxyphenol (DMP); and 2,4-dichlorophenol (DCP). These enzymes showed further activity towards the lignin model compounds: guaiacylglycerol-beta-guaiacyl (GGE) and 2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl (DDVA), generating oxidized dimeric products; and they were active with Ca-lignosulfonate (Borregaard), generating the low molecular weight product vanillic acid. The steady state kinetic parameters have been elucidated for these enzymes with ABTS and SGZ, and their pH profile showed an optimal pH value of ≈4.5 for ABTS and ≈7.5 for SGZ. The crystal structure of Oc-CueO has been determined at 1.1 Å resolution, showing a four-coordinate mononuclear type I copper centre with Met500 as an axial ligand, whereas the trinuclear type 2/3 copper cluster was modelled into the active site. Site-directed mutagenesis was carried out on amino acid residues found in the solvent tunnels, indicating the importance for residues Asp102, Gly103, Arg221, Arg223 and Asp462 for catalytic activity. The host organism of Pp-CopA, Pseudomonas putida KT2440 has been subjected to a double deletion of copA-I and copA-II genes, which led to a diminished growth capability of this strain on different small aromatic compounds, related with lignin degradation, only if copper was present in the media. This study identifies new bacterial multicopper oxidase enzymes with laccase activity and activity for lignin oxidation.
Supervisor: Not available Sponsor: Secretaría de Educación Superior, Ciencia, Tecnología e Innovación, Ecuador
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QK Botany