title: Glyoxyl-disulfide agarose: a tailor-made support for site-directed rigidification of proteins.
abstract: A new strategy has been developed for site-directed immobilization/rigidification of genetically modified enzymes through multipoint covalent attachment on bifunctional disulfide-glyoxyl supports. Here the mechanism is described as a two-step immobilization/rigidification protocol where the enzyme is directly immobilized by thiol-disulfide exchange between the β-thiol of the single genetically introduced cysteine and the few disulfide groups presented on the support surface (3 μmol/g). Afterward, the enzyme is uniquely rigidified by multipoint covalent attachment (MCA) between the lysine residues in the vicinity of the introduced cysteine and the many glyoxyl groups (220 μmol/g) on the support surface. Both site-directed immobilization and rigidification have been possible only on these novel bifunctional supports. In fact, this technology has made possible to elucidate the protein regions where rigidification by MCA promoted higher protein stabilizations. Hence, rigidification of vicinity of position 333 from lipase 2 from Geobacillus thermocatenulatus (BTL2) promoted a stabilization factor of 33 regarding the unipunctual site-directed immobilized derivative. In the same context, rigidification of penicillin G acylase from E. coli (PGA) through position β201 resulted in a stabilization factor of 1069. Remarkably, when PGA was site-directed rigidified through that position, it presented a half-life time of 140 h under 60% (v/v) of dioxane and 4 °C, meaning a derivative eight times more stable than the PGA randomly immobilized on glyoxyl-disulfide agarose. Herein we have opened a new scenario to optimize the stabilization of proteins via multipoint covalent immobilization, which may represent a breakthrough in tailor-made tridimensional rigidification of proteins.
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Madrid, Spain.
authors.forename: Cesar A
authors.initials: CA
authors.lastname: Godoy
authors.forename: Blanca
authors.initials: B
authors.lastname: de las Rivas
authors.forename: Valeria
authors.initials: V
authors.lastname: Grazú
authors.forename: Tamara
authors.initials: T
authors.lastname: Montes
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Guisán
authors.forename: Fernando
authors.initials: F
authors.lastname: López-Gallego
mesh_terms: D004926:Escherichia coli / Q000201:enzymology; D056506:Geobacillus / Q000201:enzymology; D006038:Glyoxylates / Q000737:chemistry*; D008049:Lipase / Q000737:chemistry; D008958:Models, Molecular; D010399:Penicillin Amidase / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry*
chemical_list: D006038:Glyoxylates; D011506:Proteins; C070399:glyoxyl agarose; D012685:Sepharose; D008049:Lipase; D010399:Penicillin Amidase