New Heterofunctional Supports Based on Glutaraldehyde-Activation: A Tool for Enzyme Immobilization at Neutral pH

Abstract

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candida rugosa lipase, metagenomic lipase, β-galactosidase and β-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.

New Strategy for the Immobilization of Lipases on Glyoxyl–Agarose Supports: Production of Robust Biocatalysts for Natural Oil Transformation

“…Recently, a strategy was proposed to obtain covalent enzyme derivatives using heterofunctional glutaraldehyde supports at neutral pH without the need for enzyme amination [ 60 ]. However, even when that point may be improved by optimization of the strategy presented in this paper, as it is, it allows the production of Gx derivatives with stabilization factors at 70 °C up to 15-fold higher and, yet, without the need for additional experimental steps to introduce new groups on the support.…”

Biocatalysis for Rare Ginsenoside Rh2 Production in High Level with Co-Immobilized UDP-Glycosyltransferase Bs-YjiC Mutant and Sucrose Synthase AtSuSy

“…The M315F and AtSuSy onto glyoxyl-Ni 2+ -chelate supports by two-step of co-immobilization retained high activities (nearly 99%) after seven times of desorption (Figure 1B,C), nearly no enzyme activity was leached. Most importantly, the co-immobilization of M315F and AtSuSy with the two-step ways significantly improved the binding stability of the co-immobilized bi-enzyme, though histagged enzymes specifical binding with Ni 2+ -chelate groups and covalently cross-linking between the glyoxyl groups and the adsorbed proteins [29].…”

“…Macroporous resin LX1000HG (contained hydroxyl group) modification was according to ref. [29,32], and the procedure was also shown in Figure S7. It was firstly modified with epichlorohydrin to obtain epoxy-activated resin LX1000HG.…”

Performance Comparison of Commercial and Home-Made Lipases for Synthesis of Poly(δ-valerolactone) Homopolymers

“…By the immobilization of lipase onto a hydrophobic support material, it is possible to obtain fully exposed active sites [8]. This may result in increased specificity, stability, and activity [8,10].…”