Affiliation: | 1. Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, Madrid, Spain Laboratoire de Biotechnologies Végétales et Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia, Algeria;2. Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, Madrid, Spain Department of Biochemistry, Government College University, Faisalabad, Pakistan;3. Laboratoire de Biotechnologies Végétales et Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia, Algeria;4. Department of Biochemistry, Government College University, Faisalabad, Pakistan;5. Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, Madrid, Spain;6. Departamento de Química, Facultad de Ciencias, Universidad del Tolima, Ibagué, Colombia;7. Laboratoire de Biochimie Appliquée, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia, Algeria |
Abstract: | Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<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 vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. 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 stability 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 three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative 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. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019. |