Preparation of thermostable trypsin-polysaccharide neoglycoenzymes through Ugi multicomponent reaction

A novel synthetic method for preparing enzyme-polysaccharide derivatives is described, based on the use of the Ugi multicomponent reaction. Bovine pancreatic trypsin, the target enzyme, was cross-linked with the anionic polysaccharides O-carboxymethylcellulose (CMC) and sodium alginate in the presence of formaldehyde and t-butyl isocyanide. The protease retained 69-61% and 43-37% of its initial esterolytic and proteolytic activity after cross-linking. The thermostability of the enzyme was enhanced from 49 °C to 57 °C after modification. The resistance to inactivation at 50 °C was 14- and 6-fold increased, and the activation free energy of thermal inactivation at this temperature was 7.2 kJ/mol and 4.9 kJ/mol higher after modification with O-carboxymethylcellulose and sodium alginate, respectively. The enzyme was 15- and 46-fold more resistant to autolytic degradation at pH 9.0 after cross-linking with these polysaccharides.