77
Harifi-Mood et al. studied the structure and activity of the enzyme catalase from
bovine liver in two deep eutectic solvents: choline chloride:glycerol 1:2 and choline
chloride:urea 1:2 (Harifi-Mood et al. 2017). Fluorescence spectroscopy and circular
dichroism indicated that the secondary structure of the protein was modified in the
deep eutectic solvents, more in the glycerol-containing one than in the urea-based
one. The enzymatic activity was slightly reduced in the deep eutectic solvents compared with phosphate buffer, but the binding affinity of the substrate was increased.
The use of deep eutectic solvents as solvents for the bacterial enzyme laccase was
investigated by Khodaverdian et al. (Khodaverdian et al. 2018). They used natural
deep eutectic solvents based on choline or betaine, the latter leading to better preservation of enzyme activity. In particular, a deep eutectic solvent composed of sorbitol, betaine, and water in equimolar proportions improved the stability of the
enzyme at high temperature compared with aqueous buffer. Once again, the intrinsic fluorescence of tryptophan residues was used to monitor structural changes in
the protein.
The influence of choline-based ionic liquids on the catalytic activity of lipase
from Aspergillus niger was investigated by Nascimento et al. (Nascimento et al.
2019). At low concentrations, all the ionic liquids tested (choline, choline acetate,
choline propanoate, choline butanoate, choline pentanoate, and choline hexanoate)
allowed the activity to be maintained or even enhanced. However, higher concentrations of the anions with longer alkyl chains (pentanoate and hexanoate) inhibited
enzyme activity, suggesting that these formulations could be useful in modulating
lipase activity in vivo.
In living cells, protein folding and refolding is facilitated by other proteins known
as chaperones that play an important role in the response of the cell to stress. The
fact that many of the components of deep eutectic solvents are found in cells and are
related to survival under extreme condition led Niknaddaf et al. to put forward the
hypothesis that these solvents could act as chemical chaperones (Niknaddaf et al.
2018). They conducted experiments to test this using a model of lysozyme aggregation by the reducing agent dithiothreitol. They observed that 20% of choline
chloride:urea solvent in water completely inhibited this aggregation, which is
brought about by the reduction of disulfide bonds in the protein. This might appear
to be contradictory with the results of Esquembre et al. above (Esquembre et al.
2013); however, the mechanism of denaturation was different. They also looked at
refolding by dilution after denaturation with guanidinium chloride. The presence of
the choline chloride:urea solvent in the dilution buffer reduced aggregation compared with simple buffer. Monitoring of the intrinsic fluorescence of tryptophan
confirmed that the solvents promoted correct refolding. The biological activity of
the enzyme, again measured by the ability to lyse bacteria, was also recovered better
when the solvent was present in the renaturation solution (Niknaddaf et al. 2018).
In two articles published in 2018 by Silva et al. another aspect of lysozyme
behavior was considered. In this work, they were attempting to produce nanofibers
from the protein, that is, promoting its denaturation and aggregation. Protein nanofibers have a number of applications in biotechnology, for example, in tissue engineering and biosensors, particularly when combined with polymers. In one study,
2 Deep Eutectic Solvents for Innovative Pharmaceutical Formulations
Harifi-Mood et al. studied the structure and activity of the enzyme catalase from
bovine liver in two deep eutectic solvents: choline chloride:glycerol 1:2 and choline
chloride:urea 1:2 (Harifi-Mood et al. 2017). Fluorescence spectroscopy and circular
dichroism indicated that the secondary structure of the protein was modified in the
deep eutectic solvents, more in the glycerol-containing one than in the urea-based
one. The enzymatic activity was slightly reduced in the deep eutectic solvents compared with phosphate buffer, but the binding affinity of the substrate was increased.
The use of deep eutectic solvents as solvents for the bacterial enzyme laccase was
investigated by Khodaverdian et al. (Khodaverdian et al. 2018). They used natural
deep eutectic solvents based on choline or betaine, the latter leading to better preservation of enzyme activity. In particular, a deep eutectic solvent composed of sorbitol, betaine, and water in equimolar proportions improved the stability of the
enzyme at high temperature compared with aqueous buffer. Once again, the intrinsic fluorescence of tryptophan residues was used to monitor structural changes in
the protein.
The influence of choline-based ionic liquids on the catalytic activity of lipase
from Aspergillus niger was investigated by Nascimento et al. (Nascimento et al.
2019). At low concentrations, all the ionic liquids tested (choline, choline acetate,
choline propanoate, choline butanoate, choline pentanoate, and choline hexanoate)
allowed the activity to be maintained or even enhanced. However, higher concentrations of the anions with longer alkyl chains (pentanoate and hexanoate) inhibited
enzyme activity, suggesting that these formulations could be useful in modulating
lipase activity in vivo.
In living cells, protein folding and refolding is facilitated by other proteins known
as chaperones that play an important role in the response of the cell to stress. The
fact that many of the components of deep eutectic solvents are found in cells and are
related to survival under extreme condition led Niknaddaf et al. to put forward the
hypothesis that these solvents could act as chemical chaperones (Niknaddaf et al.
2018). They conducted experiments to test this using a model of lysozyme aggregation by the reducing agent dithiothreitol. They observed that 20% of choline
chloride:urea solvent in water completely inhibited this aggregation, which is
brought about by the reduction of disulfide bonds in the protein. This might appear
to be contradictory with the results of Esquembre et al. above (Esquembre et al.
2013); however, the mechanism of denaturation was different. They also looked at
refolding by dilution after denaturation with guanidinium chloride. The presence of
the choline chloride:urea solvent in the dilution buffer reduced aggregation compared with simple buffer. Monitoring of the intrinsic fluorescence of tryptophan
confirmed that the solvents promoted correct refolding. The biological activity of
the enzyme, again measured by the ability to lyse bacteria, was also recovered better
when the solvent was present in the renaturation solution (Niknaddaf et al. 2018).
In two articles published in 2018 by Silva et al. another aspect of lysozyme
behavior was considered. In this work, they were attempting to produce nanofibers
from the protein, that is, promoting its denaturation and aggregation. Protein nanofibers have a number of applications in biotechnology, for example, in tissue engineering and biosensors, particularly when combined with polymers. In one study,
2 Deep Eutectic Solvents for Innovative Pharmaceutical Formulations