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PublicationBiotechnology Progress
Year2019
Volume35
Pagese2768
International

COOPERATIVITY OF COVALENT ATTACHMENT AND ION EXCHANGE ON ALCALASE IMMOBILIZATION USING GLUTARALDEHYDE CHEMISTRY: ENZYME STABILIZATION AND IMPROVED PROTEOLYTIC ACTIVITY

Authors:Roberto Fernandez-Lafuente
Groups of research:Optimization of biocatalysts and bioprocesses
Sabrina Ait Braham1,2, +, Fouzia  Hussain1,3,+, Roberto Morellon-Sterling2, Shagufta Kamal3, Jakub F. Kornecki1, Oveimar Barbosa4, DjamelEdine Kati5, Roberto Fernandez-Lafuente1*
1Departamento de Biocatálisis. ICP-CSIC, Campus UAM-CSIC Madrid, Spain.
 2Laboratoire de BiotechnologiesVégétales et Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Algeria
3Department of Biochemistry, Government College University, Faisalabad 38000, Pakistan.
4Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué 546,  Colombia.
5 Laboratoire de Biochimie Appliquée. Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Algeria



Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)- agarose beads at different pH values  (less than 20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7 and 9, and in all cases the activity versus Boc‐Ala‐ONp decreased to around 50%. However, the activity versus casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200% and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7 or 9.  The highest stability was always observed when the enzyme was immobilized at pH 9 and the worst one occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the 3 preparations when the inactivation was performed at pH 5.
Thus, this is a practical example on how the co-operative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation.

Keywords:enzyme modulation, ion exchange, protease immobilization, enzyme orientation, glutaraldehyde in enzyme immobilization, synergic immobilization
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