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

Ricardo Rodrigues de Melo,Hélia Harumi Sato,Cesar Mateo,Roberto Ruller,Nadia Krieger,Robson Carlos Alnoch,Emanuel Maltempi de Souza,Adriana Ferreira Lopes Vilela

doi:10.3390/molecules22071088

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.

Highlights

A simple, cheap and efficient immobilization strategy represents one of the main bottlenecks in the implementation of many industrial-scale enzymatic processes
Thisthe work proposed the of Agarose-based beads were chosen as a baseTherefore, matrix for construction of use different glutaraldehyde for the activation and formation of covalent linkages as a viable alternative to the alkaline heterofunctional supports
By obtaining a basic structure containing epoxy/diol groups, the new heterofunctional supports were prepared using the following strategy: (i) the epoxy groups formed were functionalized with different bifunctional reagents; and (ii) the diol groups were firstly oxidized with sodium periodate (NaIO4 ), and activated with amino groups using ethylenediamine (EDA)

Summary

Introduction

A simple, cheap and efficient immobilization strategy represents one of the main bottlenecks in the implementation of many industrial-scale enzymatic processes. Covalent immobilization on solid supports has been shown to be able to stabilize a variety of enzymes. Supports activated with different functional groups (epoxy, aldehyde, glutaraldehyde, and others) have been developed and applied. The use of these supports only permit the random immobilization or the immobilization through only one region (e.g., richest in lysine region for aldehyde supports). This makes it so that only one kind of catalyst can be obtained using a functional covalent group; the product of the rigidification through one region or the average of the properties of the different enzyme molecules by random immobilization [7,8,9,10]

Methods

Results

Conclusion