Sunday, April 29, 2007

Screening of methods and supports to immobilize and stabilize Cyclomaltodextrin Glucanotransferase from Thermoanaerobacter

Ana Elisa Amud1, Gercio Rodrigo Presa da Silva1, Cleide M. F. Soares2, Paulo Waldir Tardioli1, Flávio Faria de Moraes1, and Gisella M. Zanin1. (1) Chemical Engineering Department, State University of Maringa, Av. Colombo, 5790, BL E-46, Maringa - PR, 87013-190, Brazil, (2) Instituto de Tecnologia e Pesquisa, Universidade Tiradentes, Av. Murilo Dantas, 300, Farolandia, Aracaju - SE, 49032-490, Brazil

Thermoanaerobacter CGTase was immobilized on different supports using covalent multi-point attachment, physical adsorption and sol-gel encapsulation, aiming at an immobilized enzyme with high activity recovery. The enzyme covalently attached into glyoxyl-silica (silica activated with aldehyde groups), at 25oC, pH 10 and 5h of reaction, showed an inefficient activity recovery. The process yielded an immobilized enzyme with a very low activity recovery, approximately 1.43%. The hydrophobic adsorption of the enzyme on Octadecyl-Sepabeads (epoxy acrylic resin covered by octadecyl groups) at very low ionic strength, 25oC and pH 7.0, yielded also an immobilized enzyme with low activity recovery, approximately 3.83%, although the immobilization yield was satisfactory, approximately 76%. In addition, the immobilized enzyme could easily leak from the support at an ionic strength normally used in the ciclyzation reactions; the support lost approximately 25% of the immobilized enzyme in 5 hours. CGTase encapsulated in a sol-gel matrix yielded also an immobilized enzyme with low activity recovery which was 7.94%, approximately 4 times smaller than that obtained using glyoxyl-agarose as support. The soluble enzyme had a maximum cyclization activity around 80-85oC, whereas the sol-gel encapsulated enzyme had a maximum cyclization activity around 60oC, at pH 6.0. In our continued search for better methods, in a next work we will try to immobilize CGTase on glyoxyl-agarose in the presence of some additives which might preserve the enzyme activity during the immobilization process.