Review
Oxidation with galactose oxidase: Multifunctional enzymatic catalysis

https://doi.org/10.1016/j.molcatb.2015.06.006Get rights and content

Highlights

  • Galactose oxidase (GAO) produces highly reactive galactoaldehydes.

  • Analytical techniques such as NMR and MS have been developed to reliably analyze products of GAO.

  • Oxidized products have been further derivatized e.g. to uronic acids and deoxy sugars.

  • Oxidized polysaccharides have applied potential from cellulose coatings to aerogels.

  • GAO has been engineered for enhanced expression and altered specificity.

Abstract

Galactose oxidase (EC 1.1.3.9) is a single copper metalloenzyme, having a molecular weight of 65–68 kDa. Galactose oxidase catalyzes the oxidation of primary alcohols to corresponding aldehydes with strict regioselectivity, and the selectivity is high for the galactose C-6 primary hydroxyl group. The oxidation of alcohols to carbonyl compounds is one of the most important reactions in synthetic chemistry, thus enzymatic biocatalysis requiring only molecular oxygen as an oxidant is a valuable alternative to chemical reagents.

We review here the various mono-, oligo- and polysaccharide derivatives that have been synthesized by galactose oxidase-catalyzed reactions, and the several either qualitative or quantitative analytical techniques utilized to follow of the reaction, determine the conversion, and identify the products. The optimal reaction conditions, the formation of side products, and the oxidation of substrates other than carbohydrates are discussed. Finally, we summarize engineering efforts of galactose oxidase that have improved recombinant enzyme expression and yield, or altered substrate specificity.

Introduction

The oxidation of alcohols to carbonyl compounds is one of the most important reactions in synthetic chemistry and many oxidizing reagents exist for this purpose. However, most of these reagents are required in stoichiometric quantities, which usually is expensive and toxic. Thus catalytic oxidation processes are deemed valuable, especially biocatalyses by alcohol oxidases, which require only molecular oxygen as an oxidant and can be performed in aqueous solutions. The selectivity of many biocatalysts also means protecting groups are not needed.

Galactose oxidase (GAO, EC 1.1.3.9) is a single copper metalloenzyme, having a molecular weight of 65–68 kDa. GAO catalyzes the oxidation of primary alcohols to corresponding aldehydes with strict regioselectivity for the galactose C-6 primary hydroxyl group (Fig. 1). Thus the utilization of GAO is feasible in biosensors and other analytical techniques detecting galactose and galactose-containing saccharides, such as lactose. For applications in chemical synthesis, various new mono-, oligo- and polysaccharide derivatives have been prepared by the GAO-catalyzed oxidation. Moreover, the high reactivity of the galactoaldehyde products has enabled further modifications, including cross-link formation required in the preparation of thermally stable hydrogels [1], and novel aerogels [2], from galactomannans and galactoxyloglucan. Notably, certain non-sugar primary alcohols having a structure and configuration similar to the C-4 – C-6 fragment of galactose have also been reported to be substrates of GAO (Fig. 1 [3]).

The mechanism and kinetics of GAO-catalyzed oxidation have been previously extensively studied and reviewed [4], [5]. Accordingly, this review focuses on a new perspective: the utilization of GAO in synthetic applications, such as the modification of galactose-containing mono-, oligo- and polysaccharides. The reaction conditions and the analytical methods used in the characterization and identification of the products are included in the review. Finally, the engineering of galactose oxidase to achieve enhanced expression and altered reactivity are reviewed.

Section snippets

Galactose oxidase

The history of GAO is marked by the re-naming and re-classification of source organisms. GAO was first detected in cultivation media of Polyporus circinatus [6], which was later identified as Dactylium dendroides (NRRL 2903; ATCC 46032), a mycoparasite of P. circinatus [7]. D. dendroides was later reclassified as Fusarium graminearum (Niessen and Vogel 1997). More recently, galactose oxidases (GAOs) have been classified as members of the newly established auxiliary activity (AA) family AA5 in

Formation of reactive aldehydes

It is clear that galactoaldehydes are the main products of GAO-catalyzed oxidation. However, several side products can also form. For example, following 72 h oxidation of methyl α-d-galactopyranoside by GAO, 27% of products could not be identified [25]. In other studies, some of the side products have been identified and characterized, such as an α,β-unsaturated aldehyde [26], [27], [28]. In addition to the formation of GAO-generated side products, the high reactivity of main aldehyde products

Biosensors, diagnostics and environmental analytics

The utilization of GAO in biosensors designed for the detection of lactose in milk and other dairy products has been reviewed recently [81]. In addition to those, various other diagnostic applications of GAO have been developed; some of the most recent applications are reviewed here.

Various immobilization techniques have been used in biosensor studies using GAO. For example, GAO was immobilized within a laponite clay film coated on a Pt electrode surface to construct a multipurpose amperometric

Oxidation of substrates other than carbohydrates

The utilization of GAO variants with modified substrate selectivity is reviewed in Section 6. However, as mentioned in the introduction, the natural form of GAO has also been reported to oxidize compounds other than galactose and its derivatives. Siebum et al. studied 18 potential substrates, of which most were small alcohols (Fig. 3) [3]. Conversions of most of these molecules, determined by 1H NMR, were very low; 1 and 5% for glycerol and 1,2-dihydroxybutane, respectively, and undetectable

Engineering galactose oxidase for enhanced expression and altered specificity

All native forms of GAO that have been characterized to date are encoded by Fusarium species. Early engineering studies of GAO focused on deciphering the reaction mechanism of this enzyme, and thorough reviews on that topic have been published [4], [5]. Accordingly, the current review will focus on engineering efforts to (1) to improve recombinant enzyme expression and yield and (2) to alter the substrate specificity of GAO.

Conclusions

The GAO-catalyzed reaction is shown to be highly useful in various selective oxidations of mono-, oligo- and polysaccharides to aldehyde derivatives. Even though comprehensive analysis of reaction products is challenged by the possible formation of side products, the feasibility of detailed and quantitative characterizations has increased in recent years owing to improved mass spectrometric and NMR techniques and methods. The aldehyde products are of particular interest given they can be

Acknowledgements

We thank Academy of Finland for funding (project numbers 1252183 and 1281628) and Thu Vuong for drawing the 3D structure of galactose oxidase in the graphical abstract.

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