Stabilization of Immobilized Lipases by Intense Intramolecular Cross-Linking of Their Surfaces by Using Aldehyde-Dextran Polymers

Abstract

Immobilized enzymes have a very large region that is not in contact with the support surface and this region could be the target of new stabilization strategies. The chemical amination of these regions plus further cross-linking with aldehyde-dextran polymers is proposed here as a strategy to increase the stability of immobilized enzymes. Aldehyde-dextran is not able to react with single amino groups but it reacts very rapidly with polyaminated surfaces. Three lipases—from Thermomyces lanuginosus (TLL), Rhizomucor miehiei (RML), and Candida antarctica B (CALB)—were immobilized using interfacial adsorption on the hydrophobic octyl-Sepharose support, chemically aminated, and cross-linked. Catalytic activities remained higher than 70% with regard to unmodified conjugates. The increase in the amination degree of the lipases together with the increase in the density of aldehyde groups in the dextran-aldehyde polymer promoted a higher number of cross-links. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of those conjugates demonstrates the major role of the intramolecular cross-linking on the stabilization of the enzymes. The highest stabilization was achieved by the modified RML immobilized on octyl-Sepharose, which was 250-fold more stable than the unmodified conjugate. The TLL and the CALB were 40-fold and 4-fold more stable than the unmodified conjugate.

Immobilization of lipases on hydrophobic supports: immobilization mechanism, advantages, problems, and solutions

“…The first one permitted to hydrolyze an oil in the presence of organic cosolvents avoiding enzyme release (Fernández-Lorente et al, 2011b). Later, using aminated lipases, the modification with aldehyde dextran was used to even increase the enzyme thermostability (Orrego et al, 2018).…”

Stabilization of b-Glucuronidase by Immobilization in Magnetic-Silica Hybrid Supports

“…In this case, the yield was 44 ± 5% and the expressed activity was 100%. Finally, the dextran activated MNPs were mildly oxidized with sodium periodate to create Schiff's bases to which the amines of the enzyme could covalently attach [28]. On further reduction with sodium borohyride the once reversible Schiff's bases are converted into irreversible bonds.…”

Dextran Aldehyde in Biocatalysis: More Than a Mere Immobilization System

“…This one-point modification altered the secondary structure of the enzyme, modifying its functional properties, making the modified enzymes more stable, active, and selective toward different substrates than the unmodified lipase B from Candida antarctica. In another paper [155], lipases from Thermomyces lanuginosus and Rhizomucor miehei and lipase B from Candida antarctica, immobilized via interfacial activation on octyl agarose [156,157], were aminated using the carbodiimide route [158,159]. Then, they were submitted to an intense crosslinking with dexOx, reporting a large increase (ranging from 4 up to 250-fold, depending on the enzyme) on the stability for these immobilized lipases ( Figure 7).…”

“…Then, they were submitted to an intense crosslinking with dexOx, reporting a large increase (ranging from 4 up to 250-fold, depending on the enzyme) on the stability for these immobilized lipases ( Figure 7). This was attributed to the formation of an intense intramolecular covalent crosslinking [155]. Enzymes become inactivated when they interact with hydrophobic interfaces [160], such as gas bubbles [161,162] or organic solvents [163][164][165][166].…”