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Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity
Author(s) -
Ait Braham Sabrina,
Hussain Fouzia,
MorellonSterling Roberto,
Kamal Shagufta,
Kornecki Jakub F.,
Barbosa Oveimar,
Kati Djamel Edine,
FernandezLafuente Roberto
Publication year - 2018
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.2768
Subject(s) - glutaraldehyde , chemistry , immobilized enzyme , agarose , ionic strength , enzyme , chromatography , covalent bond , enzyme assay , casein , cooperativity , ion exchange , biochemistry , organic chemistry , ion , aqueous solution
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.