RESUMEN
The polysaccharide O-carboxymethyl-poly-beta-cyclodextrin was synthesized (molecular mass 13,000 Da, 40% carboxy groups) and attached to the surface of bovine pancreatic trypsin. The resulting neoglycoenzyme retained high proteolytic and esterolytic activity and contained approx. 1.0 mol of polymer/mol of enzyme. The optimum temperature for trypsin activity was increased by 10 degrees C after this transformation. Thermostability of the polymer-enzyme complex was increased by about 14 degrees C over 10 min incubation. The conjugate was also more resistant to thermal inactivation at different temperatures, ranging from 45 to 60 degrees C, demonstrating the influence of supramolecular and polymer-protein electrostatic interactions on trypsin thermostabilization. Additionally, the conjugate was 36-fold more resistant to the action of the anionic surfactant SDS. This modification also protected the enzyme from autolysis at alkaline pH.
Asunto(s)
Carboximetilcelulosa de Sodio/química , Tripsina/química , beta-Ciclodextrinas/química , Animales , Catálisis , Bovinos , Activación Enzimática , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Sustancias Macromoleculares , Peso Molecular , Páncreas/enzimología , Unión Proteica , Desnaturalización Proteica , Electricidad Estática , Temperatura , Tripsina/síntesis químicaRESUMEN
Alpha-oxoglutaric acid was attached to trypsin via reductive alkylation with NaBH4 thereby introducing metal-chelating groups at the protein surface. The thermostability of the modified enzyme was increased by 6.5-13 degrees C and its resistance to autolytic degradation was improved 2- to 4-fold in 5 mM ZnCl2, MnCl2 or MgCl2.