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. 2014 Apr 4:5:150.
doi: 10.3389/fmicb.2014.00150. eCollection 2014.

Glass-forming property of hydroxyectoine is the cause of its superior function as a desiccation protectant

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Glass-forming property of hydroxyectoine is the cause of its superior function as a desiccation protectant

Christoph Tanne et al. Front Microbiol. .

Abstract

We were able to demonstrate that hydroxyectoine, in contrast to ectoine, is a good glass-forming compound. Fourier transform infrared and spin label electron spin resonance studies of dry ectoine and hydroxyectoine have shown that the superior glass-forming properties of hydroxyectoine result from stronger intermolecular H-bonds with the OH group of hydroxyectoine. Spin probe experiments have also shown that better molecular immobilization in dry hydroxyectoine provides better redox stability of the molecules embedded in this dry matrix. With a glass transition temperature of 87°C (vs. 47°C for ectoine) hydroxyectoine displays remarkable desiccation protection properties, on a par with sucrose and trehalose. This explains its accumulation in response to increased salinity and elevated temperature by halophiles such as Halomonas elongata and its successful application in ``anhydrobiotic engineering'' of both enzymes and whole cells.

Keywords: ESR; FTIR; desiccation; enzyme stabilization; glass transition temperature; hydroxyectoine.

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Figures

FIGURE 1
FIGURE 1
Chemical structure of ectoine (A) and hydroxyectoine (B).
FIGURE 2
FIGURE 2
Influence of temperature and salinity on the relative proportion of hydroxyectoine in H. elongata. Error bars represents lowest and highest value of duplicate measurements for both salinities at 30°C and for 15% NaCl at 45°C; all others represent standard deviations of three replicates (in the case of 15% NaCl, upshock) or at least four replicates.
FIGURE 3
FIGURE 3
Growth of H. elongata in minimal medium MM63 with 15% sodium chloride. The experiment was performed with two parallel culture, one at constant 30°C (black dots), the other with a rapid temperature upshock at OD 2 (arrow) from 30 to 50°C (white dots). The inset shows the relative proportions of ectoines in bacterial cells at the early stationary growth phase (point of harvest).
FIGURE 4
FIGURE 4
Survival rates of unstressed H. elongata cells (initial cell number), undried control cells and cells which survived harsh drying for 3 h at 45 °C and 10 mbar (error bars show standard deviations of triplicates).
FIGURE 5
FIGURE 5
Relative enzyme activities of LDH after prolonged air drying at 60°C for 2, 4, and 6 h. Activities of unstabilized enzyme (control) and of enzyme protected by sucrose, trehalose, ectoine, and hydroxyectoine, respectively, are shown (error bars show standard deviations of at least four to a maximum of six replicates).
FIGURE 6
FIGURE 6
Light microscopic photographs of solute matrices which were casted from 2 M solutions of trehalose (A), sucrose (B), ectoine (C), and hydroxyectoine (D) by air drying of 10 μL at 60°C for 2 h.
FIGURE 7
FIGURE 7
The shape of ESR spectra of Tempone in dry hydroxyectoine at 220, 360, and 365 K. Inset – the molecular structure of perdeuterated Tempone. The way of calculation of 2Amax is indicated.
FIGURE 8
FIGURE 8
Temperature dependence of the distance between outermost extremes 2Amax (as in Figure 7) and the relative spectral integrated intensity (normalized to the intensity at 220 K) of the ESR spectra of Tempone in dry hydroxyectoine. Inset – first integral of ESR spectrum (absorption) at 220 K. The integrated intensity is the area (black) under the absorption peak (gray).
FIGURE 9
FIGURE 9
(A) Tempone spectra from dry ectoine and hydroxyectoine at 220 K adjusted for spectral intensity and peak position; (B) a singlet obtained by subtraction of Tempone spectrum in hydroxyectoine from Tempone spectrum in ectoine as in (A); dashed red line is a Tempone spectrum in ectoine. (C) Tempone spectra in dry ectoine at different temperatures. Arrows indicate the position of the central line H0 and narrow low-field line H+1. (D) Tempone spectra in dry ectoine at 300 and 340 K (red line), adjusted for spectral intensity and line position; (E) the difference between spectra in (D).
FIGURE 10
FIGURE 10
Temperature dependence of H+1/H0 and normalized integrated intensity of Tempone spectra in dry ectoine.
FIGURE 11
FIGURE 11
(A) FTIR spectra of dry amorphous ectoine (red line) and hydroxyectoine at room temperature. The intensity of the spectra are normalized to the height of the peak at 1600 cm-1; (B) enlarged finger print region of FTIR spectrum.
FIGURE 12
FIGURE 12
Temperature dependence of the frequency vibration at 1382–1392 cm–1 in dry ectoine and hydroxyectoine (A) and at 1082–1092 cm–1 in dry hydroxyectoine (B).

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