Stabilization of fungal beta-galactosidase in trehalose-salt supercooled media

SANTAGAPITA, PATRICIO R.; BUERA, M. PILAR

Varadero, Cuba

Conferencia; International Conference of Enzyme Technology- RELATENZ 2005; 2005

Red Latinoamericana de Tecnología Enzimática

One of the limitations to the use of enzymes is their reduced shelf-life in aqueous sytems. Enzymes may be stabilized through immobilization by drying in saccharide matrices (1), but a second excipient is generally required to improve sugar protective effects. The non-reducing disaccharide trehalose has a special protector effect on biomolecules, which is not only explained by the capacity of the sugar to form glassy systems, but also by the intermolecular interaction between the biomolecules and sugar trought hidrogen bonds. Inorganic and organic salts have a universal presence in biological systems and they are often added to therapeutics and diagnostic formulations. The effect of electrolytes on the thermophysical properties of sugar systems is of special interest due to their mayor influence on water structure an their possible interactions with biomolecules (2). Salts affect the kinetics of very important changes in sugar systems such as crystallization, hydrolysis and Maillard reactions. Many enzymes such as invertase, b-galactosidase, phosphofructokinase (3) were protected by trehalose, but in any of these studies the physical and chemical stability was globally analyzed. The purpose of the present work was to analyze fungal b-galactosidase stability in supercooled systems of trehalose containing electrolytes. The kinetics of sugar crystallization, hydrolysis and Maillard reaction were also related to enzyme stability. The matrices consisted of freeze-dried solutions containing 20% of trehalose, 0.7 U.E./g matrix of b-galactosidase of Aspergillus oryzae (E.C. 3.2.1.23) and salts (potassium and magnesium chlorides, acetates or citrates at 5:1 sugar:salt molar ratio). The dried matrices were re-humidified for one weak at water activities (aw) of 0.22 and 0.43 (over saturated solutions of CH3COOK and K2CO3, respectively). The thermal treatment was performed at 70°C in a forced-air oven. Thermal transitions, remaining enzyme activity, water content, sugar hydrolysis and the progress of Maillard reaction were analyzed as a function of time. Optical microscopy with polarized light was also employed. The maintenance of native structure is critical for enzyme activity. The results showed that the trehalose matrix protected the dehydrated enzyme but for an effective protection of the enzyme, sugar crystallization should be avoided. The glass transition temperature (Tg) did not change significantly by the presence of salts. Trehalose hydrolysis was almost negligible in all systems, but Maillard reaction was favoured in the presence of organic anions, especially acetate. Except KCl, electrolytes had an inhibitory effect on sugar crystallization kinetics, being citrate the most inhibiting anion. However, none of the salts offered better protection than pure trehalose at aw 0.43. At aw 0.22, potassium citrate provided the best protective action on the enzyme. In this system, less water was retained than in the rest of the salt containing systems, crystallization was inhibited, and the concomitant Maillard reaction showed intermediate values. The kinetic constants for Maillard reaction and for the consumption of glucose (from trehalose hydrolysis) were correlated, but they did not correlate with enzyme activity retention, which was inversely dependent on the ability of the salts to retain water. The key factors to stabilize the enzyme in supercooled trehalose matrices were the presence of potassium citrate and the capability of salts to inhibit sugar crystallization, being   water retention a negative factor in salt systems. The results may contribute to the selection of excipients in the formulation of preservation media which are valuable for many technological applications.