High Stabilization of Enzymes Immobilized on Rigid Hydrophobic Glyoxyl-Supports: Generation of Hydrophilic Environments on Support Surfaces

Alejandro H Orrego,María Romero-Fernández,María Del Carmen Millán-Linares,Javier Rocha-Martin,Justo Pedroche,José M Guisán

doi:10.3390/catal10060676

Abstract

Very rigid supports are useful for enzyme immobilization to design continuous flow reactors and/or to work in non-conventional media. Among them, epoxy-methacrylic supports are easily functionalized with glyoxyl groups, which makes them ideal candidates for enzyme stabilization via multipoint covalent immobilization. However, these supports present highly hydrophobic surfaces, which might promote very undesirable effects on enzyme activity and/or stability. The hydrophilization of the support surface after multipoint enzyme immobilization is proposed here as an alternative to reduce these undesirable effects. The remaining aldehyde groups on the support are modified with aminated hydrophilic small molecules (glycine, lysine or aspartic acid) in the presence of 2-picoline borane. The penicillin G acylase from Escherichia coli (PGA) and alcohol dehydrogenase from Thermus thermophilus HB27 (ADH2) were immobilized on glyoxyl-functionalized agarose, Relizyme and Relisorb. Despite the similar density of aldehyde groups displayed by functionalized supports, their stabilization effects on immobilized enzymes were quite different: up to 300-fold lower by hydrophobic supports than by highly hydrophilic glyoxyl-agarose. A dramatic increase in the protein stabilities was shown when a hydrophilization treatment of the hydrophobic support surface was done. The PGA immobilized on the glyoxyl-Relisorb hydrophilized with aspartic acid becomes 280-fold more stable than without any treatment, and it is even more stable than the PGA immobilized on the glyoxyl agarose.

Highlights

In recent years, the use of very rigid supports for enzyme immobilization has gained interest in the design of heterogeneous biocatalysts for chemical processes [1,2,3,4]
We proposed the modification of the aldehyde groups that have not reacted with the this purpose, we proposed the modification of the aldehyde groups that have not reacted with the enzyme, with small hydrophilic amino ligands as blocking agents enzyme, with small hydrophilic amino ligands as blocking agents to generate microenvironments on the aldehyde-activated to generate microenvironments on the aldehyde-activated support surfaces
The characterization was done by mercury intrusion porosimetry (MIP) and the adsorption isotherms to the agarose 6BCL (AG) [24] and Relizyme

Summary

Introduction

The use of very rigid supports for enzyme immobilization has gained interest in the design of heterogeneous biocatalysts for chemical processes [1,2,3,4]. It allows the design of continuous flow enzymatic reactors with the corresponding intensification of bioprocesses [5,6]. The rigid supports and immobilized enzyme biocatalysts can be completely dried (e.g., with cold acetone) while keeping their porous structure intact [7,8]. Dry immobilized enzyme biocatalysts can be used in solvent-free reaction systems or completely anhydrous reaction media (organic solvents, supercritical fluids, etc.) [7,9,10]. Catalysts 2020, 10, 676 enzyme stabilization, continuous flow reactors and solvent-free reactions is highly desirable for a more 9, x FOR PEER.

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