ABSTRACT
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.
Subject(s)
Carboxymethylcellulose Sodium/chemistry , Trypsin/chemistry , beta-Cyclodextrins/chemistry , Animals , Catalysis , Cattle , Enzyme Activation , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Macromolecular Substances , Molecular Weight , Pancreas/enzymology , Protein Binding , Protein Denaturation , Static Electricity , Temperature , Trypsin/chemical synthesisABSTRACT
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.
