Recent advances in immobilization strategies for glycosidases

Abstract

Glycans play important biological roles in cell-to-cell interactions, protection against pathogens, as well as in proper protein folding and stability, and are thus interesting targets for scientists. Although their mechanisms of action have been widely investigated and hypothesized, their biological functions are not well understood due to the lack of deglycosylation methods for large-scale isolation of these compounds. Isolation of glycans in their native state is crucial for the investigation of their biological functions. However, current enzymatic and chemical deglycosylation techniques require harsh pretreatment and reaction conditions (high temperature and use of detergents) that hinder the isolation of native glycan structures. Indeed, the recent isolation of new endoglycosidases that are able to cleave a wider variety of linkages and efficiently hydrolyze native proteins has opened up the opportunity to elucidate the biological roles of a higher variety of glycans in their native state. As an example, our research group recently isolated a novel Endo-β-N-acetylglucosaminidase from Bifidobacterium longum subsp. infantis ATCC 15697 (EndoBI-1) that cleaves N-N′-diacetyl chitobiose moieties found in the N-linked glycan (N-glycan) core of high mannose, hybrid, and complex N-glycans. This enzyme is also active on native proteins, which enables native glycan isolation, a key advantage when evaluating their biological activities. Efficient, stable, and economically viable enzymatic release of N-glycans requires the selection of appropriate immobilization strategies. In this review, we discuss the state-of-the-art of various immobilization techniques (physical adsorption, covalent binding, aggregation, and entrapment) for glycosidases, as well as their potential substrates and matrices.

Mass spectrometry for protein sialoglycosylation

“…For large‐scale glycan analysis, the release procedure requires long times and high enzyme expense, thus an improved method is needed to reduce process costs and complete the release of glycans from a variety of glycoproteins . Several approaches, such as microwave irradiation, pressure‐cycling technology, and enzyme immobilization, have been developed to rapidly cleave oligosaccharides from glycoproteins .…”

Improvement of covalent immobilization procedure of β‐galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study

“…According to Karav et al, covalent binding immobilization is successful when the support is activated with reactants that modify it. Despite decreasing enzyme loss by leaching, enzymatic activity diminishes due to steric hindrance which could be avoided with the use of a spacer arm, such as GA, that allows the enzyme to move.…”

Immobilization of Levansucrase: Strategies and Biotechnological Applications

“…Other method is the covalent binding immobilization, which involves the binding of enzymes to water insoluble supports [30]. Covalent binding of enzymes to supports occurs in their side chain amino acids.…”