Preservation Effect of Vitreous non Reducing Carbohydrates on the Enzymatic Activity, Denaturation Temperature and Retention of Native Structure of Lysozyme
Luz María Martínez,* Marcelo Videa, Francisco Mederos, and Yanel de Moral
Department of Chemistry. Instituto Tecnológico y de Estudios Superiores de Monterrey, Campus Monterrey, Av. Garza Sada 2501 Sur, Monterrey, N.L., 64849, México. (+52) 81 83 58 2000 ext 4511,*luzvidea@itesm.mx
Received January 5, 2011.
Accepted May 6, 2011.
Abstract
In order to develop more efficient methodologies to preserve protein based products at room temperature, a study on the preservation of the enzyme lysozyme in glassy matrices made from three different carbohydrates: trehalose, sucrose and raffinose was carried out. The lysozyme–carbohydrate systems were evaluated structurally by Fourier Transform Infrared (FTIR) spectroscopy, to analyze the intermolecular interactions between carbohydrate and protein. Their thermal stability was characterized using differential thermal analysis (DTA), which allowed the measurement of glass transition temperatures (Tg) of the vitreous sugars and differential scanning calorimetry (DSC), which was used to measured the Tg of sugars in the sugar enzyme systems as well as the thermal denaturation temperature of lysozyme (Td). The structural studies revealed that sucrose is the most effective sugar for the preservation of the native conformation of lysozyme during lyophilization. Nevertheless, analysis of enzymatic activity showed, after storing the enzyme at room temperature for more than five weeks, that the highest activity retention was achieved when preserved in the presence of trehalose and raffinose. Freeze–dried lysozyme in the absence of sugars partially lost its native conformation during the lyophilization and lost 20% of its biological activity when stored at room temperature.
Keywords: Sugar glass, freeze–drying, preservation of proteins, enzymes.
Resumen
Con el propósito de encontrar métodos más eficientes para preservar productos con base a proteínas a temperatura ambiente se estabilizó la enzima lisozima en presencia de tres diferentes carbohidratos: trehalosa, sacarosa y rafinosa. Los sistemas lisozima–carbohidratos fueron evaluados estructuralmente por espectroscopía de infrarrojo por transformada de Fourier (FTIR) para analizar las interacciones intermoleculares carbohidrato–proteína. También se hizo un estudio de estabilidad térmica usando técnicas de calorimetría diferencial de barrido (DSC) y análisis térmico diferencial (DTA) con las que se determinó la temperatura de transición vítrea (Tg) de los azúcares amorfos puros y de los sistemas azúcar enzima, así como la temperatura de desnaturalización térmica de la lisozima (Td). Los estudios estructurales demostraron que la sacarosa es capaz de preservar más efectivamente la conformación nativa de la lisozima durante el secado por liofilización; Sin embargo, el análisis de actividad biológica mostró que la enzima, después de haber sido almacenada a temperatura ambiente por más de 5 semanas, retuvo mayor actividad cuando fue preservada en presencia de trehalose y rafinosa. La lisozima liofilizada en ausencia de sacáridos perdió parcialmente su conformación nativa durante el secado y perdió un 20% de actividad biológica al ser almacenada a temperatura ambiente.
Palabras clave: Azúcares vítreos, liofilización, preservación de proteínas, enzimas.
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]]>Acknowledgements
We acknowledge the research funds provided by the Tecnológico de Monterrey CAT–120, Zambrano–Hellion and CONACyT (106847) for the financial support of this research. We also thank Dr. Jean Nicolas Aebischer (University of Applied Sciences of Western Switzerland) who facilitated his calorimetry instrumentation.
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