Immobilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F on Eupergit C supports.
Dextransucrase from Leuconostoc mesenteroides B-512F was immobilized on epoxy-activated acrylic polymers with different textural properties (Eupergit C and Eupergit C 250L). Prior to immobilization, dextransucrase was treated with dextranase to remove the dextran layer covering the enzyme surface, thus increasing the accessibility of its reactive groups to the epoxide centers of the support. Elimination of 99% of the initial carbohydrate content was determined by the anthrone method. To prevent enzyme inactivation, the immobilization was carried out at pH 5.4, at which the coupling to the support took place through the carboxylic groups of the enzyme. The effects of the amount (mg) of dextransucrase added per gram of support (from 0.2:1 to 30:1), temperature and contact time were studied. Maximum activity recovery of 22% was achieved using Eupergit C 250L. Using this macroporous support, the maximum specific activity (710 U/g biocatalyst) was significantly higher than that obtained with the less porous Eupergit C (226 U/g biocatalyst). The dextransucrase immobilized on Eupergit C 250L showed similar optimal temperature (30 degrees C) and pH (5-6) compared with the native enzyme. In contrast, a notable stabilization effect at 30 degrees C was observed as a consequence of immobilization. After a fast partial inactivation, the dextransucrase immobilized on Eupergit C 250L maintained more than 40% of the initial activity over the following 2 days. The features of this immobilized system are very attractive for its application in batch and fixed-bed bioreactors.
Departamento de Biocatálisis and Departamento de Ingeniería de Procesos Catalíticos, Instituto de Catálisis, CSIC, Cantoblanco, 28049 Madrid, Spain.
Aránzazu
A
Gómez de Segura
Miguel
M
Alcalde
Malcolm
M
Yates
M Luisa
ML
Rojas-Cervantes
Nieves
N
López-Cortés
Antonio
A
Ballesteros
Francisco J
FJ
Plou
D019149:Bioreactors; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005964:Glucosyltransferases / Q000737:chemistry*; D007934:Leuconostoc / Q000201:enzymology*; D008567:Membranes, Artificial; D011108:Polymers / Q000737:chemistry*; D016062:Porosity; D011485:Protein Binding; D013499:Surface Properties; D013696:Temperature; D014462:Ultrafiltration / Q000295:instrumentation / Q000379:methods*
D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D011108:Polymers; C065843:Eupergit C; D005964:Glucosyltransferases; C020025:dextransucrase