title: Reversible immobilization of invertase on Sepabeads coated with polyethyleneimine: optimization of the biocatalyst's stability.
abstract: Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE-agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers-octamers at low pH value). The optimal enzyme preparation remained fully active after a 15-day incubation at 50 degrees C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 degrees C higher than that of soluble enzyme.
authors.affiliation: Departmento de Biocatálisis, Instituto de Catalisis, CSIC, Campus Universidad Autónoma, Cantoblanco, Madrid, Spain.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Torres
authors.forename: Cesar
authors.initials: C
authors.lastname: Mateo
authors.forename: Manuel
authors.initials: M
authors.lastname: Fuentes
authors.forename: Jose M
authors.initials: JM
authors.lastname: Palomo
authors.forename: Claudia
authors.initials: C
authors.lastname: Ortiz
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernández-Lafuente
authors.forename: Jose M
authors.initials: JM
authors.lastname: Guisan
authors.forename: Andrea
authors.initials: A
authors.lastname: Tam
authors.forename: Moreno
authors.initials: M
authors.lastname: Daminati
mesh_terms: D019281:Dimerization; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008863:Microspheres; D011094:Polyethyleneimine; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D055672:Static Electricity; D013696:Temperature; D043324:beta-Fructofuranosidase
chemical_list: D004800:Enzymes, Immobilized; D011094:Polyethyleneimine; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase