New dextransucrase purification process of the enzyme produced by Leuconostoc mesenteroides IBUN 91.2.98 based on binding product and dextranase hydrolysis

This paper examines a new dextransucrase (DS) purification process of the extracellular enzyme (EC 2.4.1.5) produced by Leuconostoc mesenteroides IBUN 91.2.98. The enzyme was purified using a methodology which combines the immobilization of the enzyme in the produced biopolymer dextran, followed by a concentration step by ultrafiltration, using a membrane with a pore size of 300 kDa and subsequent hydrolysis of dextran by action of a dextranase and finally enzyme purification by anion exchange chromatography. The obtained enzyme has a purification factor of 118 and a yield of 26% from the initial extract. The purified dextransucrase has a specific activity of 335.1 U/mg, electrophoretic analysis shows absence of subunits, and a molecular weight of 170.1 kDa, a Vmax of 28.1 U/ml and a Km of 48 mM. Optimal conditions of pH, temperature and substrate concentration were 5, 30 °C and 584 mM sucrose respectively in a ratio of 0.4 U/mole of substrate. The produced dextran has a molecular size of 800-1000 kDa. Both the hydrolytic and transference activity are inhibited by Fe+3 (96.5%) and Al+3 (99.1%), whereas Mg+2 and K produce activation of 36.7% and 27.2%, respectively.