Immobilization of enzymes using non‐ionic colloidal liquid aphrons (CLAs): Activity kinetics, conformation, and energetics

Abstract

This study seeks to examine the ability of non-ionic/non-polar Colloidial Liquid Aphrons (CLAs) to preserve enzyme functionality upon immobilization and release. CLAs consisting of micron-sized oil droplets surrounded by a thin aqueous layer stabilized by a mixture of surfactants, were formulated by direct addition (pre-manufacture addition) using 1% Tween 80/mineral oil and 1% Tween 20 and the enzymes lipase, aprotinin and α-chymotrypsin. The results of activity assays for both lipase and α-chymotrypsin showed that kinetic activity increased upon immobilization by factors of 7 and 5.5, respectively, while aprotinin retained approximately 85% of its native activity. The conformation of the enzymes released through desorption showed no significant alterations compared to their native state. Changes in pH and temperature showed that optimum conditions did not change after immobilization, while analysis of activation energy for the immobilized enzyme showed an increase in activity at higher temperatures. Furthermore, the effect of bound water within the aphron structure allowed for some degree of enzyme hydration, and this hydration was needed for an active conformation with results showing a decrease in ΔH* for the immobilized system compared to its native counterpart.

Catalytically Active Protein Coatings: Toward Enzymatic Cascade Reactions at the Intercolloidal Level

“…In light of various biomedical and biotechnological applications of enzymes, the demand for their stability, catalytic activity under nonoptimal conditions, recovery, and reusability is great and urgent. − An inevitable approach to meeting these demands is their immobilization on solid supports. Parameters such as the geometry of the support (e.g., flat surfaces and particle size and shape) as well as the surface properties (e.g., porosity, roughness, specific surface area per pore size, surface chemistry, surface charge, etc.)…”

Enzymatic Catalysis at Nanoscale: Enzyme-Coated Nanoparticles as Colloidal Biocatalysts for Polymerization Reactions

“… 15 , 16 , 34 , 35 Depending on the immobilization technique, the immobilization can enhance the properties of enzymes in terms of thermal stability, tolerance to extreme pH, and organic solvents. 36 Therefore, immobilized enzymes can appear to have higher activities than native enzymes under drastic conditions due to the enhanced stability. 34 , 36 − 42 On the other hand, the apparent activity of conventional immobilized enzymes can also be lower than that of their native counterparts, mainly because of the hindered substrate accessing or unfavorable conformational transition of the enzyme on the support.…”

“… 36 Therefore, immobilized enzymes can appear to have higher activities than native enzymes under drastic conditions due to the enhanced stability. 34 , 36 − 42 On the other hand, the apparent activity of conventional immobilized enzymes can also be lower than that of their native counterparts, mainly because of the hindered substrate accessing or unfavorable conformational transition of the enzyme on the support. 13 , 43 − 46 Particularly in the case of colloidal supports, the catalytic performance of the immobilized enzymes also strongly depends on the colloidal stability of the colloidal support.…”

“…In general, proteins and enzymes can be immobilized onto solid supports using various techniques, ,, such as (a) “multipoint” covalent bonding via click chemistry − or coupling reactions, yielding amide − or imine bonds, − (b) cross-linking, (c) affinity immobilization, , (d) entrapment, , and (e) adsorption. ,,, Depending on the immobilization technique, the immobilization can enhance the properties of enzymes in terms of thermal stability, tolerance to extreme pH, and organic solvents . Therefore, immobilized enzymes can appear to have higher activities than native enzymes under drastic conditions due to the enhanced stability. ,− On the other hand, the apparent activity of conventional immobilized enzymes can also be lower than that of their native counterparts, mainly because of the hindered substrate accessing or unfavorable conformational transition of the enzyme on the support. ,− Particularly in the case of colloidal supports, the catalytic performance of the immobilized enzymes also strongly depends on the colloidal stability of the colloidal support. , The catalytic performance of the enzymes immobilized onto NPs can decrease drastically, due to hindered accessibility of the substrates to their active sites, when the NPs are not stable and aggregate or sediment in the reaction media. Hence, to create a functional enzymatically active colloidal system, a robust enzyme immobilization on the NPs as well as a colloidally stable system is required.…”

General Preparation of Heme Protein Functional Fe3O4@Au‐Nps Magnetic Nanocomposite for Sensitive Detection of Hydrogen Peroxide

“…Unfortunately, DET between Hb and electrode surface is generally difficult because the redox‐active center is embedded in its protein shell deeply and enzyme usually loses its bioactivity on the bare electrode . To facilitate DET and retain the bioactivity of immobilized enzyme, biocompatible materials have been used to immobilize Hb, such as surfactants , biopolymers , hydrogels , polyelectrolytes and nano‐materials .…”