Immobilization-stabilization of enzymes; variables that control the intensity of the trypsin (amine)-agarose (aldehyde) multipoint attachment

• Published in: Enzyme and microbial technology Vol. 11; no. 6; pp. 353 - 359
• Main Authors: Blanco, Rosa M., Calvete, Juan J., Guisán, JoséM.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 1989; Elsevier Science
• Subjects: agarose; Biological and medical sciences; Biotechnology; enzyme-support multipoint attachment; Fundamental and applied biological sciences. Psychology; Immobilization of enzymes and other molecules; Immobilization of trypsin; Immobilization techniques; Interactions. Associations; Intermolecular phenomena; Methods. Procedures. Technologies; Molecular biophysics; stabilization of trypsin; trypsin; trypsin (amine)-agarose (aldehyde) multiinteraction; stabilization of trypsin; trypsin (amine)-agarose (aldehyde) multiinteraction; enzyme-support multipoint attachment; Immobilization of trypsin; Temperature; Support; Enzyme; Stabilization; Environmental factor; Molecular interaction; Agarose; Colloidal gel; Trypsin; Enzymatic activity; Covalent bond; PH; Mechanism of action; Immobilized enzyme
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/0141-0229(89)90019-7

We have developed a strategy for immobilization-stabilization of trypsin by multipoint covalent attachment to agarose (aldehyde) gels. We have studied the role of four main variables that control the intensity of the trypsin (amine)-agarose (aldehyde) multiinteraction processes: (a) surface density of aldehyde groups in the activated gels, (b) pH of the multiinteraction medium, (c) contact time between insolubilized enzyme and activated support prior to borohydride reduction of the derivatives, and (d) temperature. Different combinations of these four variables have been tested to prepare a number of trypsin-agarose derivatives. All these derivatives preserved 100% of catalytic activity but showed very different stability values. The less stable derivative had exactly the same stability of soluble trypsin in the absence of autolysis phenomena. On the other hand, the three-dimensional structure of the most stable derivative was 5000-fold more stable than the one corresponding to unmodified trypsin. Amino acid analysis of hydrolysates of this very stable derivative reveals that seven lysine residues per trypsin molecule have reacted with the activated support during the process of preparation of the derivative.