Investigating novel bacterial laccases with biotechnologically relevant attributes

Laccases are versatile biocatalysts with applications in a variety of fields ranging from the bioremediation of pollutants to the production of valuable materials from renewable plant resources. At present, fungal laccases have been more extensively applied in biotechnological fields due to their high-redox potential. However, they exhibit poorer activity in alkaline pH and high temperature conditions when compared to their lowredox bacterial counterparts. These discrepancies in oxidative abilities have been correlated to differences in axial ligands coordinating their Type 1 (T1Cu) copper site. Fungal high-redox laccases exclusively display phenylalanine (Phe) axial residues, while low-redox bacterial enzymes harbor a methionine (Met) amino acid at the same position. In this thesis, a novel Phe-axial-ligand-containing bacterial laccase, appropriately designated as LacPhe, was mined through bioinformatics from landfill leachate metagenomes. Despite sharing high sequential similarities with low-redox bacterial enzymes, the predicted folding patterns of LacPhe were more analogous to fungal laccases with high-redox potential. Cloning and expression of this enzyme further confirmed it possessed higher redox potential compared to typical bacterial laccases. Indeed, LacPhe was able to directly oxidize the high-redox reporter substrate violuric acid (E0 = 1.1 V). Its oxidative activity against this substrate was also comparable to the one observed for the model high-redox fungal enzyme from Trametes versicolor (TvL). Moreover, midpoint potential (E1/2) estimations of the redox potentials for LacPhe and TvL obtained through cyclic voltammetry also placed these two enzymes in the same oxidative category. LacPhe recorded an E1/2 value of 780 mV vs. the normal hydrogen electrode (NHE). Meanwhile, a midpoint potential of 775 mV vs. NHE was measured for TvL, approximately 10 mV lower than its redox potential described in the literature. Therefore, the present research reports the occurrence of a bacterial laccase inherently possessing fungal-like attributes in the form of a phenylalanine axial residue and measured high-redox potential. This finding led to the investigation of other novel bacterial enzymes sharing this sequence feature in wider datasets. Accordingly, over 200,000 complete bacterial genomes were collected from the Reference Sequence database (RefSeq) and mined through bioinformatics. More than 500 putative bacterial laccase-like sequences displaying a phenylalanine axial residue emerged from this analysis. The majority of these sequences were either carried by plant-associated bacteria or source organisms inhabiting deep-sea environments. Structural predictions for a selected subset of enzymes revealed that most of them showed similar conformations to fungal highredox potential laccases. In addition, the predicted folding of these laccase-like sequences also closely resembled the one obtained for LacPhe. These results suggest the wider occurrence of possible high-redox bacterial enzymes in nature that could benefit biotechnological applications.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Civil & Environmental Engineering

Grantor dc:publisher

ResearchSpace@Auckland

Year dc:date.issued

2023

Author dc:creator

• Bonnet, Ohinerau

Advisors dc:contributor.advisor

• Zhuang, Wei-Qin
• Yi, Shan
• Fa'aui, Tumanako

Statement dc:rights

• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Licence dc:rights.uri

• https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm
• https://creativecommons.org/licenses/by-nc-sa/3.0/nz/

Handle dc:identifier.uri

https://hdl.handle.net/2292/68628

OAI identifier oai:identifier

oai:researchspace.auckland.ac.nz:2292/68628