Characterization of Bacterial Laccases and Their Application in the Oxidation of Lignin‑Derived Substrates
(2026) KMBM01 20261Applied Microbiology
Biotechnology
Biotechnology (MSc)
- Abstract
- Laccases are multicopper oxidases capable of oxidizing a broad range of phenolic and non-phenolic compounds, making them attractive biocatalysts for lignin valorization and environmental applications. In this work, two bacterial laccases, LacA from Bacillus amyloliquefaciens and MEK from Psychrobacter sp., were recombinantly produced in Escherichia coli BL21 (DE3), purified, and characterized to structure, oligomeric state, thermal stability, and activity toward lignin-derived substrates.
Structural models of LacA and MEK were generated using AlphaFold and compared with the crystallographic laccase CotA (PDB: 1GSK). Both enzymes retained the conserved multicopper oxidase architecture, with T1, T2, and T3 copper sites in highly similar to... (More) - Laccases are multicopper oxidases capable of oxidizing a broad range of phenolic and non-phenolic compounds, making them attractive biocatalysts for lignin valorization and environmental applications. In this work, two bacterial laccases, LacA from Bacillus amyloliquefaciens and MEK from Psychrobacter sp., were recombinantly produced in Escherichia coli BL21 (DE3), purified, and characterized to structure, oligomeric state, thermal stability, and activity toward lignin-derived substrates.
Structural models of LacA and MEK were generated using AlphaFold and compared with the crystallographic laccase CotA (PDB: 1GSK). Both enzymes retained the conserved multicopper oxidase architecture, with T1, T2, and T3 copper sites in highly similar to CotA, indicating preserved catalytic copper centers. Recombinant expression followed by IMAC purification yielded proteins ofapproximately 60 kDa, confirmed by SDS-PAGE. Size-exclusion chromatography to analyse protein native form revealed that LacA was largely present as high-molecular-weight aggregates, with a smaller dimeric fraction), whereas MEK eluted as a monomeric and highly homogeneous species at 61 kDa.
Thermal stability assessed by differential scanning fluorimetry across pH 3–10 showed a single, relatively low melting transition for LacA, with maximal stability around pH 7-8 (Tm: 40 °C). In contrast, MEK displayed two melting points (Tm1 and Tm2), with the higher transition reaching to 61°C at pH 5-6. Laccase activity measured by ABTS oxidation demonstrated that both enzymes are copper-dependent and more active at pH 5 than at pH 7, with MEK showing particularly strong dependence on CuSO₄.
Application studies with lignin-related substrates (vanillic acid, vanillyl alcohol, vanillylamine, lignin from akasia) and terephthalic acid (TPA) showed that both LacA and MEK oxidize phenolic lignin-derived substrates, whereas the non-phenolic TPA was not transformed. HPLC analysis revealed substantial substrate depletion and the formation of new product peaks, and LC‑MS data for vanillyl alcohol suggested the formation of a dehydrodivanillyl alcohol dimer. Together, these results demonstrate that LacA and MEK are functional bacterial laccases with distinct biophysical properties and complementary potential for the oxidative conversion of lignin-derived substrates. (Less) - Popular Abstract
- Lignin is one of nature’s most abundant materials, forming the tough structural walls of plants. Every year, the pulp, paper, and biofuel industries produce massive amounts of lignin as a byproduct, which is usually just burned for energy. But what if we could transform this "waste" into high-value chemicals instead? This thesis explores that exact possibility by characterizing two bacterial enzymes, LacA and MEK, as potential green tools for oxidizing lignin-derived substrates.
Produced and purified using Escherichia coli bacteria, these two enzymes belong to a family called laccases, known for their ability to catalyze reactions using copper. Advanced computer modeling confirmed they possess the necessary structure to function, but... (More) - Lignin is one of nature’s most abundant materials, forming the tough structural walls of plants. Every year, the pulp, paper, and biofuel industries produce massive amounts of lignin as a byproduct, which is usually just burned for energy. But what if we could transform this "waste" into high-value chemicals instead? This thesis explores that exact possibility by characterizing two bacterial enzymes, LacA and MEK, as potential green tools for oxidizing lignin-derived substrates.
Produced and purified using Escherichia coli bacteria, these two enzymes belong to a family called laccases, known for their ability to catalyze reactions using copper. Advanced computer modeling confirmed they possess the necessary structure to function, but laboratory tests revealed they behave quite differently. While LacA proved to be somewhat fragile, clumped easily, and lost stability at around 40 °C, MEK emerged as a much tougher candidate, remaining stable up to 60 °C in acidic environments.
When put to the test against various lignin-derived molecules, both enzymes successfully broke down and transformed phenolic compounds. Using high-performance analytical tools, the study tracked these chemical transformations and identified how simpler molecules were linked together into more complex structures. However, neither enzyme could degrade non-phenolic plastics such as TPA.
This research highlights the power of bacterial laccase by proving these biocatalysts can efficiently transform plant-derived molecules. This work paves the way for greener, more sustainable biorefineries that turn industrial waste streams into valuable products. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/student-papers/record/9243093
- author
- Sabbathini, Gabriela Christy LU
- supervisor
- organization
- course
- KMBM01 20261
- year
- 2026
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Bacterial laccases, Multicopper oxidases, lignin-derived substrates, Phenolic compounds, Biocatalysts, Applied microbiology
- language
- English
- id
- 9243093
- date added to LUP
- 2026-06-24 09:27:15
- date last changed
- 2026-06-24 09:27:15
@misc{9243093,
abstract = {{Laccases are multicopper oxidases capable of oxidizing a broad range of phenolic and non-phenolic compounds, making them attractive biocatalysts for lignin valorization and environmental applications. In this work, two bacterial laccases, LacA from Bacillus amyloliquefaciens and MEK from Psychrobacter sp., were recombinantly produced in Escherichia coli BL21 (DE3), purified, and characterized to structure, oligomeric state, thermal stability, and activity toward lignin-derived substrates.
Structural models of LacA and MEK were generated using AlphaFold and compared with the crystallographic laccase CotA (PDB: 1GSK). Both enzymes retained the conserved multicopper oxidase architecture, with T1, T2, and T3 copper sites in highly similar to CotA, indicating preserved catalytic copper centers. Recombinant expression followed by IMAC purification yielded proteins ofapproximately 60 kDa, confirmed by SDS-PAGE. Size-exclusion chromatography to analyse protein native form revealed that LacA was largely present as high-molecular-weight aggregates, with a smaller dimeric fraction), whereas MEK eluted as a monomeric and highly homogeneous species at 61 kDa.
Thermal stability assessed by differential scanning fluorimetry across pH 3–10 showed a single, relatively low melting transition for LacA, with maximal stability around pH 7-8 (Tm: 40 °C). In contrast, MEK displayed two melting points (Tm1 and Tm2), with the higher transition reaching to 61°C at pH 5-6. Laccase activity measured by ABTS oxidation demonstrated that both enzymes are copper-dependent and more active at pH 5 than at pH 7, with MEK showing particularly strong dependence on CuSO₄.
Application studies with lignin-related substrates (vanillic acid, vanillyl alcohol, vanillylamine, lignin from akasia) and terephthalic acid (TPA) showed that both LacA and MEK oxidize phenolic lignin-derived substrates, whereas the non-phenolic TPA was not transformed. HPLC analysis revealed substantial substrate depletion and the formation of new product peaks, and LC‑MS data for vanillyl alcohol suggested the formation of a dehydrodivanillyl alcohol dimer. Together, these results demonstrate that LacA and MEK are functional bacterial laccases with distinct biophysical properties and complementary potential for the oxidative conversion of lignin-derived substrates.}},
author = {{Sabbathini, Gabriela Christy}},
language = {{eng}},
note = {{Student Paper}},
title = {{Characterization of Bacterial Laccases and Their Application in the Oxidation of Lignin‑Derived Substrates}},
year = {{2026}},
}