The multicopper oxidase enzyme laccase holds great potential to be used for biological lignin valorization alongside a biocompatible ionic liquid (IL). However, the IL concentrations required for biomass pretreatment severely inhibit laccase activity. Due to their ability to function in extreme conditions, many thermophilic enzymes have found use in industrial applications. The thermophilic fungal laccase from Myceliophthora thermophila was found to retain high levels of activity in the IL [C 2 C 1 Im][EtSO 4 ], making it a desirable biocatalyst to be used for lignin valorization. In contrast to [C 2 C 1 Im][EtSO 4 ], the biocompatibility of [C 2 C 1 Im][OAC] with the laccase was markedly lower. Severe inhibition of laccase activity was observed in 15% [C 2 C 1 Im][OAc]. In this study, the enzyme surface charges were modified via acetylation, succinylation, cationization, or neutralization. However, these modifications did not show significant improvement in laccase activity or stability in [C 2 C 1 Im][OAc]. Docking simulations show that the IL docks close to the T1 catalytic copper, likely interfering with substrate binding. Although additional docking locations for [OAc] - are observed after making enzyme modifications, it does not appear that these locations play a role in the inhibition of enzyme activity. The results of this study could guide future enzyme engineering efforts by showing that the inhibition mechanism of [C 2 C 1 Im][OAc] toward M. thermophila laccase is likely not dependent upon the IL interacting with the enzyme surface.
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Effects of Ionic Liquids on Laccase from Trametes versicolor
Interactions between ionic liquids and biomolecules are of great interest due to the intrinsic properties of ionic liquids and the flexibility allowed by mixing and matching cations and anions to create unique ionic liquids. A number of ionic liquid–biomolecule studies have focused on interactions with proteins, including industrially relevant enzymes. One of these, laccase from Trametes versicolor, is a naturally derived enzyme used in the breakdown of phenolic compounds in a wide variety of industries, especially useful in breakdown of lignocellulosic materials. Here, a combination of experiments and molecular dynamics (MD) simulations was used to investigate the interactions of ionic liquids with laccase. Enzyme kinetics assays indicated that ionic liquids composed of tetramethylguanidine (TMG) and either serine or threonine caused significant reduction in enzymatic activity, while kinetics was not impacted by TMG-Asp or TMG-Glu ionic liquids. Similarly, intrinsic fluorescence of laccase in the presence of TMG-Ser and TMG-Thr exhibited a shift in spectral properties consistent with structural destabilization, but again TMG-Asp and TMG-Glu had no impact. MD simulations of laccase and ABTS with/without TMG-Ser ionic liquid provided insight into the deactivation mechanism of laccase. The simulations indicated that TMG-Ser disrupts laccase’s electron transfer mechanism.
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- Award ID(s):
- 1904797
- NSF-PAR ID:
- 10332942
- Date Published:
- Journal Name:
- Biophysica
- Volume:
- 1
- Issue:
- 4
- ISSN:
- 2673-4125
- Page Range / eLocation ID:
- 429 to 444
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/biophysica1040031
