On the bioprotective effects of 3-hydroxybutyrate: Thermodynamic study of binary 3HB-water systems

Abstract

Microorganisms must face various inconvenient conditions; therefore, they developed several approaches for protection. Such a strategy also involves the accumulation of compatible solutes, also called osmolytes. It has been proved that the monomer unit 3-hydroxybutyrate (3HB), which is present in sufficient concentration in poly(3-hydroxybutyrate) (PHB)-accumulating cells, serves as a chemical chaperone protecting enzymes against heat and oxidative stress and as a cryoprotectant for enzymes, bacterial cells, and yeast. The stress robustness of the cells is also strongly dependent on the behavior and state of intracellular water, especially during stress exposure. For a better understanding of the protective mechanism and effect of strongly hydrophilic 3HB in solutions at a wide range of temperatures, a binary phase diagram of system sodium 3HB (Na3HB)-water in equilibrium and the state diagrams showing the glass transitions in the system were constructed. To investigate the activity of water in various compositions of the Na3HB/water system, three experimental techniques have been used (dynamic water sorption analysis, water activity measurements, and sorption calorimetry). First, Na3HB proved its hydrophilic nature, which is very comparable with known compatible solutes (trehalose). Results of differential scanning calorimetry demonstrated that Na3HB is also highly effective in depressing the freezing point and generating a large amount of nonfrozen water (1.35 g of water per gram of Na3HB). Therefore, Na3HB represents a very effective cryoprotectant that can be widely used for numerous applications.

Figure 1

(a) Sorption isotherms for water vapor sorption on Na3HB at 25°C determined by Novasina LabMaster (black), DVS (blue), and sorption calorimetry (red) respectively. (b) Enthalpy of hydration of Na3HB as a function of water content as measured by sorption calorimetry at 25°C. (c) Comparison of water vapor sorption isotherms measured by DVS at 25°C (blue) and 40°C (black), respectively.

Figure 2

DSC thermograms of mixtures of Na3HB and water used for construction of equilibrium phase diagram (scanning rates: 10°C/min, scale bars for heat flow are shown). (a) Example of the evaluation of ice melting (Na3HB concentration 30.3 wt %); (b) endotherms at about −27.5°C representing the eutectic points and second endotherms that correspond to ice melting (Na3HB concentrations from 11.3 to 40.5 wt %); (c) endotherms representing the melting of dihydrates and melting of crystalline Na3HB (Na3HB concentrations from 72.4 to 81.6 wt %); (d) magnified endotherms of crystalline Na3HB melting, marked with the red arrow. To see this figure in color, go online.

Figure 3

The phase diagram in the equilibrium of Na3HB/water complex using DSC (×); sorption calorimetry () and DVS (). All lines are drawn as a guide for the eye to follow the respective phase boundaries.

Figure 4

The dependence of the enthalpy of melting of Na3HB dihydrate on concentration.

Figure 5

Glass transitions obtained by DSC data: (left) temperature dependence and independence of glass transition on water content; (right) heat capacity of glass transitions.

Figure 6

State diagram showing glass transitions in Na3HB/water mixtures as determined by DSC. All lines are drawn as a guide for the eye to follow the respective state boundaries. To see this figure in color, go online.

Figure 7

Amount of nonfreezing water per gram of Na3HB obtained by DSC data as a function of water content on enthalpy of melting of water. To see this figure in color, go online.