title: Immobilization-stabilization of alpha-chymotrypsin by covalent attachment to aldehyde-agarose gels.
abstract: We have developed a strategy for immobilization-stabilization of alpha-chymotrypsin by multipoint covalent attachment of the enzyme, through its amino groups, to agarosealdehyde gels. We have studied the role of the main variables that control the intensity of these enzyme-support multi-interaction processes (surface density of aldehyde groups in the activated gel, contact time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives, etc.). In this way, we have prepared a number of very different chymotrypsinagarose derivatives. Our best derivatives, with the most intense multipoint attachment, were more stable than one-point attached derivatives and were more than 60,000-fold more stable than soluble enzyme in the absence of autolysis phenomena. In spite of the dramatic stabilization, the catalytic activity of these derivatives is little changed (they only lose 35% of intrinsic activity after this intense enzyme-support multi-interaction process). In addition, we have also demonstrated the very high capacity of 6% aldehyde-agarose gels to immobilize pure chymotrypsin (40 mg enzyme/mL catalyst). Furthermore, we have been able to establish a clear correlation between enzyme-support multipoint covalent attachment, stabilization against very different denaturing agents (heat, urea, organic cosolvents), and insensitivity of those immobilized chymotrypsin molecules to some activating agents.
authors.affiliation: Instituto de Catalisis, Unidad de Biocatalisis C.S.I.C., Campus de la Universidad Autonoma Cantoblanco, 28049-Madrid, Spain.
authors.forename: J M
authors.initials: JM
authors.lastname: Guisán
authors.forename: A
authors.initials: A
authors.lastname: Bastida
authors.forename: C
authors.initials: C
authors.lastname: Cuesta
authors.forename: R
authors.initials: R
authors.lastname: Fernandez-Lufuente
authors.forename: C M
authors.initials: CM
authors.lastname: Rosell