Stability of lysozyme in aqueous extremolyte solutions during heat shock and accelerated thermal conditions

Abstract

The purpose of this study was to investigate the stability of lysozyme in aqueous solutions in the presence of various extremolytes (betaine, hydroxyectoine, trehalose, ectoine, and firoin) under different stress conditions. The stability of lysozyme was determined by Nile red Fluorescence Spectroscopy and a bioactivity assay. During heat shock (10 min at 70°C), betaine, trehalose, ectoin and firoin protected lysozyme against inactivation while hydroxyectoine, did not have a significant effect. During accelerated thermal conditions (4 weeks at 55°C), firoin also acted as a stabilizer. In contrast, betaine, hydroxyectoine, trehalose and ectoine destabilized lysozyme under this condition. These findings surprisingly indicate that some extremolytes can stabilize a protein under certain stress conditions but destabilize the same protein under other stress conditions. Therefore it is suggested that for the screening extremolytes to be used for protein stabilization, an appropriate storage conditions should also be taken into account.

Figure 1. Molecular structure of the extremolytes betaine, hydroxyectoine, trehalose, ectoine, and mannosylglycerate (firoin).

Figure 2. Proposed mechanism of stabilization of extremolytes, adapted from Arakawa et al. .

In the unfolded state (S_u) proteins undergo the greatest loss of its conformational entropy, which led to the overall shift in equilibrium towards the native state. The stabilization occurs when the entropy difference between two states increases, so that the folded protein (S_f) has fewer tendencies to unfold , .

Figure 3. The effect of extremolytes on the Nile Red fluorescence of the lysozyme-nile red complex after stressing lysozyme at 70°C for 10 minutes (heat shock) in citrate buffer pH 5.0.

Fluorescence spectra recorded on unstressed (solid line) and stressed (dotted line) lysozyme A: without extremolytes, and with extremolyte B: betaine, C: hydroxyectoine, D: trehalose, E: ectoine, and F: firoin.

Figure 4. The effect of lysozyme on M. Lysodeickticus (bioactivity) after stressed at 70°C for 10 minutes (heat shock) and the effect of extremolytes on the bioactivity of lysozyme. * is the level of significance (p<0.05).

Figure 5. The effect of extremolytes on the bioactivity of lysozyme during 4 weeks of storage at 55°C (accelerated thermal condition).

Figure 6. The effect of extremolytes on the unfolding temperature (T _m) of lysozyme.

Unfolding temperatures was measured as transition midpoint analyzed by thermal shift assay using RT-PCR machine of lysozyme on the concentration of 1.0 mg/ml with A: betaine, B: hydroxyectoine, C: trehalose, D: ectoine, and E: firoin.

Figure 7. The effect of firoin on the heat capacity of lysozyme determined by ITC.

Firoin was titrated into lysozyme solution at the temperatures of 10°C (▪), 25°C (□), or 55°C (○).