Chemical glycosidation of trypsin with O-carboxymethyl-poly-beta-cyclodextrin: catalytic and stability properties.
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.
Enzyme Technology Group, Center for Biotechnological Studies, University of Matanzas, Autopista a Varadero km 3 1/2, Matanzas, C.P. 44740, Cuba.
Maria L
ML
Villalonga
Gretel
G
Reyes
Alex
A
Fragoso
Roberto
R
Cao
Leyden
L
Fernández
Reynaldo
R
Villalonga
D000818:Animals; D002266:Carboxymethylcellulose Sodium / Q000737:chemistry*; D002384:Catalysis; D002417:Cattle; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D011489:Protein Denaturation; D055672:Static Electricity; D013696:Temperature; D014357:Trypsin / Q000138:chemical synthesis / Q000737:chemistry*; D047392:beta-Cyclodextrins / Q000737:chemistry*
D046911:Macromolecular Substances; D047392:beta-Cyclodextrins; D014357:Trypsin; D002266:Carboxymethylcellulose Sodium