Characterization of levansucrase from Zymomonas mobilis: levan and FOS production by soluble and immobilized enzyme

Trabajo presentado en el XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Madrid (España) del 3 al 6 de septiembre de 2013.Levansucrases (EC 2.4.1.10) catalyse the transfer of the fructosyl moiety from sucrose to different acceptors such as: (1) sucrose, yielding fructooligosaccharides (FOS) that can be further fructosylated forming levan, a polymer with ß(2-6) bonds in the backbone and ß(2-1) branches; (2) water, resulting in sucrose hydrolysis. In this work, a levansucrase from Zymomonas mobilis (LEV-Y, from Amano) was characterized and three isoforms were found and studied. The reaction products using sucrose as substrate were analysed by High-Performance Anion-Exchange Chromatography (HPAEC-PAD). The number of FOS synthesized by the soluble enzyme was significantly higher compared with previous reports [1], including various oligosaccharides that were identified by mass spectrometry and NMR. Production of levan was studied as well. The enzyme was further immobilized by entrapment in calcium alginate gel and the resulting beads were dehydrated to obtain DALGEEs (Dry ALGinate Entrapped Enzymes) [2]. Different strategies were assessed to minimize enzyme loss (lixiviation) through the pores. The first strategy was based on the property of the levansucrase [3] by which the quaternary structure of the protein changes with the pH of the medium: at pH values below 6.0, the protein increases its size by forming microfibrils, therefore making the enzyme more suitable for immobilization by entrapment. The second immobilization approach was also targeted to increase the size of the levansucrase by crosslinking enzyme molecules with a transglutaminase. The effect of immobilization on enzyme behaviour was also analysed.Peer Reviewe