76
almost completely conserved. The thermal stability of the enzyme was also conserved or even increased.
As indicated by circular dichroism, the secondary structure of bovine serum
albumin was not affected by the use of a deep eutectic solvent composed of choline
chloride and glycerol either pure or mixed with water compared with phosphatebuffered saline. On the other hand, changes in the near-ultraviolet region suggested
a modification of tertiary structure in the pure deep eutectic solvent (SanchezFernandez et al. 2017). Bovine serum albumin in pure solvents was irreversibly
denatured by heating to 80 °C, but the results in water are not reported. As far as
lysozyme was concerned, its secondary structure of a mixture of α-helix and β-sheet
was not modified in the chosen deep eutectic solvents: choline chloride with glycerol or urea. Small-angle neutron scattering was used to get a better impression of
the protein conformation in solution. This showed that the globular shape of bovine
serum albumin became less symmetrical in pure deep eutectic solvent, indicating
that the solvation of the protein was modified in this solvent. On the other hand,
when a hydrated deep eutectic solvent was used, the results were not different from
pure water. This was interpreted as water forming a shell around the protein, preventing direct interaction with the deep eutectic solvent. In the case of lysozyme,
the protein was found to be only partially folded in the deep eutectic solvent, irrespective of whether it contained glycerol or urea. This might not have been expected
because high concentrations of urea denature proteins.
Another study of lysozyme structure in deep eutectic solvents was carried out by
Esquembre et al. (Esquembre et al. 2013). They used circular dichroism and also
followed the intrinsic fluorescence of tryptophan residues that gives information
about folding and unfolding of the protein. In this study, the deep eutectic solvents
were composed of choline chloride with either glycerol or urea in a 2:1 molar ratio.
Tryptophan fluorescence of lysozyme was slightly blue shifted in solvents compared
with buffer solution, whereas denatured lysozyme shows a red shift. Heating of
lysozyme caused unfolding in all solvents, but monitoring of the mean fluorescence
energy indicated different processes in buffer and in deep eutectic solvent solution:
in the latter, the protein appeared to pass through a number of intermediate states.
These states were examined by means of circular dichroism spectra. Differences in
the near-ultraviolet region were recorded as a function of temperature and revealed
that modifications in the tertiary structure began to occur at a lower temperature for
the deep eutectic solvent containing urea than for the deep eutectic solvent with
glycerol or with buffer. While the thermally induced unfolding of lysozyme was
completely reversible in buffer, it was only partially so in the deep eutectic solvent
with glycerol and irreversible in the urea-containing deep eutectic solvent. The
biological activity of lysozyme was also tested in media containing various
proportions of the choline chloride:glycerol solvent by its capacity to lyse a Grampositive bacterium. The speed of bacterial degradation decreased as the proportion
of the solvent increased. The authors concluded that a detailed characterization of
the system is necessary before deep eutectic solvent can be used as a solvent for
biocatalysis (Esquembre et al. 2013).
C.-H. Nguyen et al.
almost completely conserved. The thermal stability of the enzyme was also conserved or even increased.
As indicated by circular dichroism, the secondary structure of bovine serum
albumin was not affected by the use of a deep eutectic solvent composed of choline
chloride and glycerol either pure or mixed with water compared with phosphatebuffered saline. On the other hand, changes in the near-ultraviolet region suggested
a modification of tertiary structure in the pure deep eutectic solvent (SanchezFernandez et al. 2017). Bovine serum albumin in pure solvents was irreversibly
denatured by heating to 80 °C, but the results in water are not reported. As far as
lysozyme was concerned, its secondary structure of a mixture of α-helix and β-sheet
was not modified in the chosen deep eutectic solvents: choline chloride with glycerol or urea. Small-angle neutron scattering was used to get a better impression of
the protein conformation in solution. This showed that the globular shape of bovine
serum albumin became less symmetrical in pure deep eutectic solvent, indicating
that the solvation of the protein was modified in this solvent. On the other hand,
when a hydrated deep eutectic solvent was used, the results were not different from
pure water. This was interpreted as water forming a shell around the protein, preventing direct interaction with the deep eutectic solvent. In the case of lysozyme,
the protein was found to be only partially folded in the deep eutectic solvent, irrespective of whether it contained glycerol or urea. This might not have been expected
because high concentrations of urea denature proteins.
Another study of lysozyme structure in deep eutectic solvents was carried out by
Esquembre et al. (Esquembre et al. 2013). They used circular dichroism and also
followed the intrinsic fluorescence of tryptophan residues that gives information
about folding and unfolding of the protein. In this study, the deep eutectic solvents
were composed of choline chloride with either glycerol or urea in a 2:1 molar ratio.
Tryptophan fluorescence of lysozyme was slightly blue shifted in solvents compared
with buffer solution, whereas denatured lysozyme shows a red shift. Heating of
lysozyme caused unfolding in all solvents, but monitoring of the mean fluorescence
energy indicated different processes in buffer and in deep eutectic solvent solution:
in the latter, the protein appeared to pass through a number of intermediate states.
These states were examined by means of circular dichroism spectra. Differences in
the near-ultraviolet region were recorded as a function of temperature and revealed
that modifications in the tertiary structure began to occur at a lower temperature for
the deep eutectic solvent containing urea than for the deep eutectic solvent with
glycerol or with buffer. While the thermally induced unfolding of lysozyme was
completely reversible in buffer, it was only partially so in the deep eutectic solvent
with glycerol and irreversible in the urea-containing deep eutectic solvent. The
biological activity of lysozyme was also tested in media containing various
proportions of the choline chloride:glycerol solvent by its capacity to lyse a Grampositive bacterium. The speed of bacterial degradation decreased as the proportion
of the solvent increased. The authors concluded that a detailed characterization of
the system is necessary before deep eutectic solvent can be used as a solvent for
biocatalysis (Esquembre et al. 2013).
C.-H. Nguyen et al.