Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda. Activation, inactivation and the kinetic properties of the enzyme

Abstract Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria chorda . The enzyme was activated 1.3-fold in the presence of Ca 2+ and Cl − ions. The ions also stabilized the enzyme increasing the temperature of its maximum activity from 45°C to 50°C. GLase was inactivated by chemical modification with carbodiimide and a carboxyl group of the enzyme was shown essential to the lyase activity. A tryptophanyl residue(s) was also shown to be important for the activity and was probably involved in substrate binding. K m values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1 mM for maltooligosaccharides of degree of polymerization (dp) 4–7, and the k 0 values for the oligosaccharides were similar (42–53 s −1 ). The analysis of these kinetic parameters showed that the enzyme has four subsites to accommodate oligosaccharides. The subsite map of GLase was unique, since subsite 1 and subsite 2 have large positive and small negative affinities, respectively. The subsite map of this type has not been found in other enzymes with exo-action on α-1,4-glucan. The K m and k 0 values for the polysaccharides were lower (0.03 mM) and higher (60–100 s −1 ), respectively, suggesting the presence of another affinity site specific to the polysaccharides.