Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

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.

Use of Alcalase in the production of bioactive peptides: A review

“…At 45 °C and pH 9, the modified enzyme could be used for six cycles of 6 h without a detectable decrease in enzyme activity [149]. The same group showed the synergy of different immobilization causes in the Alcalase immobilization on amino-glutaraldehyde: the enzyme was readily immobilized on amino-glutaraldehyde at low ionic strength while it was not immobilized on the amino support, and neither on amino glutaraldehyde at high ionic strength [152]. The immobilization pH value determined the activity versus casein.…”

Parameters necessary to define an immobilized enzyme preparation

“…The case of aminated supports activated with glutaraldehyde is one such example. Here the enzyme may become immobilized via ion exchange, hydrophobic interaction and covalent reaction (which is the slower one) (80,81,82,83,84,85,86,87).…”

Immobilization of Lipase A from Candida antarctica onto Chitosan-Coated Magnetic Nanoparticles

“…Glutaraldehyde is generally used as the activating agent to covalently immobilize enzymes, especially on aminated supports [67], due to its high versatility [68,69]. When it is used, glutaraldehyde reacts with primary amino groups of the enzyme and the support; thus, a heterofunctional support is generated, having both physical and chemical interaction capacities [70,71].…”