Stabilization of the Restriction Enzyme EcoRI Dried with Trehalose and Other Selected Glass-Forming Solutes

Abstract

The stabilization of the restriction enzyme EcoRI by its incorporation into aqueous glass-forming carbohydrate or polymer solutions, followed by vacuum-drying to low moisture, has been studied. Glass-forming solutes included trehalose, sucrose, lactose, maltose, raffinose, maltodextrin DE 10, and poly(vinylpyrrolidone) (molecular weight 40,000, PVP). Among the solutes examined, trehalose and sucrose protected the enzyme most effectively during storage at 37 and 45 degrees C. The restriction enzyme dried with trehalose or sucrose maintained its activity without detectable loss for at least 20 days at 37 degrees C and 12 days at 45 degrees C. In contrast, the activity of the enzyme dried with maltodextrin or PVP was reduced during vacuum desiccation and also it decreased remarkably during storage at the same temperatures. Stored (37/45 degrees C) vacuum-dried trehalose and sucrose systems were either a dense paste or a very viscous syrup, and this indicated that they were not glassy. Moreover, no relationship was found between the glass transition temperatures (Tg) of the pure added solute and enzyme protection during storage, since, e.g., sucrose which has significantly lower Tg values protected the enzyme much better than either maltose, lactose, maltodextrin, or PVP. The trisaccharide raffinose offered good protection of enzyme activity, and its role as a novel excipient matrix for labile enzyme stabilization deserves further investigation. The stability of enzyme EcoRI was rapidly lost when the vacuum-dried trehalose and sucrose systems were humidified to 58% relative humidity and stored at 45 degrees C, and this was attributed to disaccharide crystallization.

Correlations between Molecular Mobility and Chemical Stability During Storage of Amorphous Pharmaceuticals

“…Variations in chemical stability that do not seem to be related to global mobility have also been observed for various solid proteins, including restriction enzyme EcoRI dried with sucrose, trehalose, maltodextrin or PVP, 104 lactase and invertase lyophilized with trehalose, maltose, lactose, sucrose, raffinose, maltodextrin, casein or PVP, 22,[105][106][107][108] and glucose-6-phosphate dehydrogenase lyophilized with dextran, sucrose or raffinose. [109][110] It is not easy to explain the lack of correlation between chemical stability and global mobility for proteins because of many complicated factors possibly affecting the chemical stability.…”

Effects of carbohydrate crystallization on stability of dehydrated foods and ingredient formulations

“…During drying, a certain degree of deterioration may occur, and the stabilization of biomaterials by their incorporation into carbohydrate and/ or polymer solutions before drying is a known preservation procedure (Colaço, Sen, Thangavelu, Pinder, & Roser, 1992;Crowe, Crowe, Carpenter, & Winstrom, 1987;Leslie, Israeli, Ligthart, Crowe, & Crowe, 1995;Rossi, Buera, Moreno, & Chirife, 1997). Many sugars are involved in preservation mechanisms of living organisms under extreme conditions.…”

Thermostability and Browning Development of Fungal α-amylase Freeze-dried in Carbohydrate and PVP Matrices

“…Besides the possible effects of the physical characteristics of the matrices discussed above (free volume and plasticization) on α-amylase stabilization, interaction between matrices and enzyme, which could result in specific protective effects, must also be considered. The saccharide raffinose protected enzymes lactase, invertase and restriction enzymes (4,18,23) at the same level as trehalose, in various conditions of temperature and water content. It has been suggested that sugars, like other polyols, are able to make multiple external hydrogen bonds that could replace the essential water molecules involved in the maintenance of the tertiary structure of proteins (1).…”