Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent

Alejandro H Orrego,Javier Rocha-Martin,María Millán-Linares,José Guisán,María Yust,Maria Romero-Fernández

doi:10.3390/catal8080333

Abstract

Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 °C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts.

Highlights

The use of enzymes in industrial processes has been gaining interest in recent decades [1,2,3].These biocatalysts allow chemical reactions to be performed under milder conditions and without side reactions [4]
We propose the use of 2-picoline borane (2-PB) as an alternative reducing agent for protein immobilization into aldehyde-activated supports, using GA as a model support. 2-PB is a selective and non-toxic reducing agent that is increasingly used in green chemistry for reductive amination processes [33,34,35]
The step in the immobilization procedure was the reduction of the reversible Schiff base that was formed between the amino groups of the lysine residues on the enzyme surface, and the aldehyde groups of the support (Figure 1)

Summary

Introduction

The use of enzymes in industrial processes has been gaining interest in recent decades [1,2,3]. These biocatalysts allow chemical reactions to be performed under milder conditions and without side reactions [4]. Scientists have made enormous efforts to develop methods to increase the stability of proteins. These approaches range from molecular biology [5] to physico-chemical [6,7] strategies. Protein immobilization is a physicochemical strategy that has proven to be a simple and cost-efficient methodology to generate improved biocatalysts [7,8].

Methods

Results

Conclusion