Stabilization of anti-leukemic enzyme l-asparaginase by immobilization on polysaccharide levan

Biologically active fructose polymer levan from Zymomonas mobilis of different molecular mass (75 and 2000 kDa) was covalently coupled to anti-leukemic enzyme Erwinia carotovora l-asparaginase. The method used for the immobilization of the enzyme involved periodate oxidation of the polysaccharide, followed by reductive alkylation. A gentle periodate oxidation of levan (oxidation degree ≤24%) resulted in the highest residual enzyme activity (≥55%). The Km(app.) of glycoconjugates was higher than the Km of native l-asparaginase. The conjugation of l-asparaginase widened the optimum pH range of the enzyme. The electrophoretic mobility in polyacrylamide gel of glycoconjugates obtained was considerably reduced in comparison with native l-asparaginase. Immobilized l-asparaginase showed significantly higher stability in conditions of increased temperature (40°C and 50°C) and prolonged storage (1 month) in aqueous solutions as compared to the native enzyme. The results are discussed in relation to possible explanations of levan-induced enzyme stabilization, as well as to possible applications of immobilized l-asparaginase research.