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

• Published in: Journal of biotechnology Vol. 265; pp. 8 - 14
• Main Authors: Flórez Guzman, Glaehter Yhon, Hurtado, Gustavo Buitrago, Ospina, Sonia Amparo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 10.01.2018
• Subjects: Dextran; Dextranase - chemistry; Dextrans - metabolism; Dextransucrase; Electrophoresis; Exopolysaccharides; Fermentation; Glucan; Glucosyltransferase; Glucosyltransferases - chemistry; Glucosyltransferases - isolation & purification; Glucosyltransferases - metabolism; Hydrolysis; Leuconostoc mesenteroides; Leuconostoc mesenteroides - enzymology; Molecular Weight; Sucrose - metabolism; Temperature; Dextran; Glucan; Dextransucrase; Leuconostoc mesenteroides; Exopolysaccharides; Glucosyltransferase
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2017.10.019

•The enzyme dextransucrase of Lm IBUN 91.2.98 synthesizes a polymer type dextran.•The fermentation conditions allowed a high enzymatic productivity.•The DS enzyme interacts with the product allowing its purification by ultrafiltration.•Ds was classified as a glycosyl hydrolase (GH) belonging to family 70. 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 300kDa 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 335.1U/mg, electrophoretic analysis shows absence of subunits, and a molecular weight of 170.1kDa, a Vmax of 28.1U/ml and a Km of 48mM. Optimal conditions of pH, temperature and substrate concentration were 5, 30°C and 584mM sucrose respectively in a ratio of 0.4U/mole of substrate. The produced dextran has a molecular size of 800–1000kDa. 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.