Immobilization of Lipase B from Candida antarctica in Octyl-Vinyl Sulfone Agarose: Effect of the Enzyme-Support Interactions on Enzyme Activity, Specificity, Structure and Inactivation Pathway

Abstract

Lipase B from Candida antarctica was immobilized on heterofunctional support octyl agarose activated with vinyl sulfone to prevent enzyme release under drastic conditions. Covalent attachment was established, but the blocking step using hexylamine, ethylenediamine or the amino acids glycine (Gly) and aspartic acid (Asp) altered the results. The activities were lower than those observed using the octyl biocatalyst, except when using ethylenediamine as blocking reagent and p-nitrophenol butyrate (pNPB) as substrate. The enzyme stability increased using these new biocatalysts at pH 7 and 9 using all blocking agents (much more significantly at pH 9), while it decreased at pH 5 except when using Gly as blocking agent. The stress inactivation of the biocatalysts decreased the enzyme activity versus three different substrates (pNPB, S-methyl mandelate and triacetin) in a relatively similar fashion. The tryptophane (Trp) fluorescence spectra were different for the biocatalysts, suggesting different enzyme conformations. However, the fluorescence spectra changes during the inactivation were not too different except for the biocatalyst blocked with Asp, suggesting that, except for this biocatalyst, the inactivation pathways may not be so different.

Upstream and Downstream Bioprocessing in Enzyme Technology

“…Physical and covalent bonds are among the support binding methods [ 23 , 24 ]. The physical unions are carried out on hydrophobic materials, weak Van der Wals unions, or by encapsulation using polymers, biopolymers, or a combination of both [ 25 ].…”

“…Another strategy to stabilize and reuse the enzyme is immobilization, which consists of fixing the enzyme on a material (binding to support) or forming cross-linked enzyme aggregates (CLEAs) between the same molecules [ 21 , 22 ]. Physical and covalent bonds are among the support binding methods [ 23 , 24 ]. The physical unions are carried out on hydrophobic materials, weak Van der Wals unions, or by encapsulation using polymers, biopolymers, or a combination of both [ 25 ].…”

Propelling of Enzyme Activity by Using Different Triggering Strategies: Applications and Perspectives

“…Conceptually, an immobilized enzyme can be defined as an enzyme that is physically confined or localized to a particular solid support, whose catalytic activities are maintained, and can be used repeatedly and continuously. , In scientific research, many methods of enzyme immobilization have been reported, including adsorption, covalent bonding, cross-linking, and entrapment; each has advantages and disadvantages. ,, Owing to the improved stability, reusability, and high enzyme retention of the proposed supports, more and more types of materials have been used as supports to immobilize various enzymes . To date, many carriers have been used for immobilization including natural − or synthetic polymers, copolymers, hydrogels, beads, − blends, nanofibers, − nanoparticles, − composites, − metal–organic frameworks, , etc. We refer readers to several recent review articles that comprehensively review these areas (method and support). ,− Although the scientific literature is full of reports on new carrier supports and new approaches to immobilize enzymes, most of them are not even suitable for enzyme immobilization as the immobilization method and carrier used are insufficient to improve the enzyme properties of the matrix.…”

Metal–organic framework-supported ionic liquids for lipase immobilization: design, characterization, and investigation of catalytic performance

“…, zeolites, mesoporous silica, polymers) have been used as host materials for enzyme immobilization. 17–20 Their large specific surface area is beneficial for enzyme capacity, and their porosity may improve the diffusion of the substrate.…”

“…15,16 Additionally, because of their large specific surface area and porosity, many porous materials (e.g., zeolites, mesoporous silica, polymers) have been used as host materials for enzyme immobilization. [17][18][19][20] Their large specific surface area is beneficial for enzyme capacity, and their porosity may improve the diffusion of the substrate.…”