Abstract
β-d-N-acetyl-hexosaminidase (Hex, EC 3.2.1.52) is an acid hydrolase that catalyzes the cleavage of the β-1,4 bond in N-acetyl-d-galactosamine (Gal-NAc) and N-acetyl-d-glucosamine (Glc-NAc) from the non-reducing end of oligosaccharides and glycoconjugates. It is widely expressed in both the prokaryotic and eukaryotic world, where it performs multiple and important functions. Hex has antifungal activity in plants, is capable of degrading many biological substrates, and can play an important role in the biomedical field for the treatment of Tay-Sachs and Sandhoff diseases. With the aim being able to obtain a device with a stable enzyme, a method of covalent immobilization on polylactic acid (PLA) films was developed for the A isoform of the β-d-N-acetyl-hexosaminidase enzyme (HexA), produced in a recombinant way from Human Embryonic Kidney-293 (HEK-293) cells and suitably purified. An in-depth biochemical characterization of the immobilized enzyme was carried out, evaluating the optimal temperature, thermal stability, pH parameters, and Km value. Moreover, the stability of the enzymatic activity over time was assessed. The results obtained showed an improvement in terms of kinetic parameters and stability to heat for the enzyme following immobilization and the presence of HexA in two distinct immobilized forms, with an unexpected ability for one of them to maintain its functionality for a long period of time (over a year). The stability and functionality of the enzyme in its immobilized form are therefore extremely promising for potential biotechnological and biomedical applications.
Highlights
Introduction βD-N-acetyl-hexosaminidase (Hex, EC 3.2.1.52) is an acid hydrolase whose main function is to catalyze the cleavage of the β-1,4 bond in N-acetyl-D-galactosamine (Gal-NAc) and N-acetyl-D-glucosamine (Glc-NAc) from the non-reducing end of oligosaccharides and glycoconjugates, such as glycoproteins, glycolipids, and glycosaminoglycans (GAGs) [1,2,3]
As for the two peaks eluted with the saline gradient, the first exhibits a higher MUG/MUGS ratio, as expected for hexosaminidase enzyme (HexA), whereas the latter is characterized by a ratio close to 2, in agreement with its HexS assignment [13]
While the HexS peak can only be observed as a shoulder in the CTRL cell profile, it becomes important for HEK-HexA cells, because of the overexpression of the α subunit, which allows the formation of the otherwise rare αα-homodimer
Summary
Introduction βD-N-acetyl-hexosaminidase (Hex, EC 3.2.1.52) is an acid hydrolase whose main function is to catalyze the cleavage of the β-1,4 bond in N-acetyl-D-galactosamine (Gal-NAc) and N-acetyl-D-glucosamine (Glc-NAc) from the non-reducing end of oligosaccharides and glycoconjugates, such as glycoproteins, glycolipids, and glycosaminoglycans (GAGs) [1,2,3]. The animal Hex is certainly the most studied form and its localization is purely lysosomal; while in prokaryotes, some studies have highlighted its presence in the periplasmic membrane and the cytoplasmic granules [3,4,5]. This enzyme is interesting from a biotechnological point of view as it takes part in the chitin degradation cascade, affecting the growth of the fungal and bacterial cell wall, and acts as a powerful defense mechanism in plants in response to fungal infections [3,6,7,8,9]. All the three enzymes are able to break the β-1,4 glycosidic bonds present in substrates such as oligosaccharides or glycoconjugates; only the enzymes containing the α subunit (HexA and HexS), with a positively charged amino acid residue at its binding site, can hydrolyze the β-1,4 bonds of GlcNAc-6-sulfate residues, and only HexA, which is a heterodimer, is able to cleave the β-1,4 Gal-NAc bond of sialic acid present in the GM2 ganglioside [12,19,20]
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