Glycosidases are ubiquitous intracellular and extracellular enzymes responsible for the hydrolysis of glycosidic linkages. Glycosidases are divided into two types: exoglycosidases and endoglycosidase. These enzymes are specific for the anomeric configuration and hydrolyze glycosidic bonds with either retention or inversion of the anomeric configuration. Glycosidases are available from natural sources, such as microbial or fungal cultures, as well as higher organisms (plant seeds, mollusks, etc.).

Glycosidases have different functions, most of which are required as degradative enzymes for the digestion of carbohydrates to monosaccharides. Some glycosidases are required for the catabolism of polysaccharides, in response to physiological requirements and the turnover of complex carbohydrates. In contrast to these degradative enzymes, some intracellular glycosidases participate in a biosynthetic pathway, such as α-glucosidases, α-mannosidases, and N-acetylglucosaminidases.

Many glycosidases can utilize specially engineered substrates both as glycosyl acceptors and donors. By using these substrates carrying various functional groups in the molecule, structurally modified carbohydrate products can be prepared and the regioselectivity or the yield of the enzymatic reaction are influenced. Amerigo Scientific offers a range of glycosidases and kits for release of N- and O-linked glycans from glycoprotein therapeutics, cell lines, biological fluids, or any glycoprotein moieties of you interest.

Endoglycosidases

Endoglycosidases are enzymes that cleave internal linkages in a glycosidic chain, releasing an oligosaccharidic residue, for instance, removing the entire intact oligosaccharide portion from a glycoprotein. Endoglycosidases that work on N-linked glycans and O-linked glycans are effectively used for the structural studies of complex carbohydrates. Endo-β-N-acetylglucosaminidases from Flavobacterium meningosepticum cleave both high-mannose and complex glycoproteins, including Endo F1, Endo F2, and Endo F3. All three enzymes hydrolyze glycosidic bond of the β1,4 di-N-acetylchitobiose core structure adjacent to asparagine. O-Glycosidase is a endoglycosidase that removes the Core 1 O-Linked glycan (Gal-β(1-3)GalNAc-α) from serine or threonine amino acids.

These endoglycosidases are highly stable in clean preparations with no glycerol, NaCl, or other additives such as EDTA. All enzymes are tested for the absence of proteolytic or unexpected glycosidic activity.

Product Name Application Source
Endoglycosidase F1 Endo F1 cleaves high mannose and some hybrid type N-glycans from peptides and proteins. Recombinant gene from Elizabethkingia miricola in E. Coli
Endoglycosidase F2 For selective release of biantennary and high mannose (at a 40X reduced rate) glycans from peptides and proteins.
Endoglycosidase F3 For selective release of triantennary and α(1-6) fucosylated biantennary N-glycans from peptides and proteins.
Endoglycosidase H Endo H cleaves asparagine-linked hybrid or high mannose oligosaccharides but not complex oligosaccharides. Recombinant gene from Streptomyces plicatus in E. Coli
O-glycosidase For cleaving only unsubstituted Gal-β(1-3)GalNAc-α disaccharides attached to the serine or threonine residues of glycoproteins or glycopeptides. Recombinant from Streptococcus pneumoniae in E. Coli
Enzymatic CarboRelease Kit This kit includes the enzymes and buffers required to deglycosylate glycoproteins, removing all N-linked oligosaccharides and many O-linked sugars.  
Enzymatic DeGlycoMx Kit This kit is developed for protein deglycosylation and includes our DeGlycoMx, a premixed cocktail of the enzymes required to remove all N-linked oligosaccharides and most O-linked sugars from glycoprotein.  
Endo-Beta-Galactosidase Endo-β-galactosidase cleaves internal β(1-4) galactose linkages in unbranched, repeating poly-N-acetyllactosamine structures. Recombinant gene from Bacteroides fragilis in E. Coli
Ceramide glycanase kit For deglycosylating a variety of glycosphingolipids by cleaving the β-glycosyl linkage. Hirudo medicinalis

PNGase F

Although peptide N glycosidase F (PNGase F) is an amidase, it is generally classified as an endonucleosidase due to its similar activity of cleaving intact N-linked glycans. PNGase F is suitable for release of all types (high-mannose, hybrid, and complex) N-linked glycans from glycoproteins and glycopeptides, but does not remove oligosaccharides containing α(1-3) linked core fucose commonly found on plant glycoproteins.

Product Name Source
Recombinant PNGase F enzyme Recombinant from Elizabethkingia miricola in E. coli
PNGase F (Peptide N Glycosidase F) From Elizabethkingia meningosepticum
Recombinant PNGase F Recombinant from Elizabethkingia miricola

Sequence Examples of Glycans Cleaved by Endoglycosidases 1
Sequence Examples of Glycans Cleaved by Endoglycosidases 2Sequence Examples of Glycans Cleaved by Endoglycosidases

Exoglycosidases

Exoglycosidases are enzymes that cleave a single glycosidic residue at the nonreducing end of an oligosaccharide chain. Exoglycosidases usually show strong substrate specificity for the monosaccharide portion and its anomeric structure. They are named after the monosaccharide and the anomeric structure that they hydrolyze, such as α-focusidase or β-galactosidase. Since exoglycosidases release monosaccharides only from the non-reducing termini of a glycan chain, the monosaccharide sequence of the chain can be easily determined by sequential exoglycosidase digestion.

All exoglycosidases are tested for the absence of proteolytic or unexpected glycosidic activity and can be used for:

  • cleaving specific terminal-monosacharides from glycans
  • detailed structural characterization of glycans (monosaccharide type, linkage, and sequence)
Product Name Application Source
Sialidase Au Alpha-(2-3,6,8,9) α(2-3,6,8,9) Sialidase cleaves all non-reducing terminal sialic acid residues from complex carbohydrates and glycoproteins. In addition, the enzyme will cleave branched sialic acids (linked to an internal residue). Recombinant from Arthrobacter ureafaciens in E. coli
Sialidase Cp Alpha-(2-3,6) Sialidase Cp cleaves all non-reducing terminal non-branched α(2-3) and α(2-6) sialic acid residues from complex carbohydrates and glycoproteins. Recombinant from Clostridium perfringens in E. coli
Sialidase Sp Alpha-(2-3) Sialidase Sp cleaves the non-reducing terminal α(2-3) unbranched sialic acid residues from complex carbohydrates and glycoproteins. Recombinant from Streptococcus pneumoniae in E. Coli
Sialate O-acetylesterase kit To remove 9-, 8- and 7-O-acetyl groups from released sialic acids, released glycans or glycoproteins. Used for characterisation of highly sialylated biotherapeutics such as EPO, FSH and blood clotting factors. Recombinant from Tannerella forsythia in E. Coli
Beta-(1 -4)-galactosidase Non-reducing terminal β(1-4)-Galactose. Number of antennae does not affect cleavage rate. Fucose linked to the penultimate N-acetylglucosamine will block cleavage of the galactose. Recombinant from Streptococcus pneumoniae in E. coli
Beta-(1 -3,4,6)-galactosidase Cleaves all β1-3 and β1-4 linked non-reducing, terminal galactose. β1-6 linked galactose is released at a slower rate.  
Alpha-(1 -3,6)-galactosidase Alpha Galactosidase from E. coli cleaves α(1-3) and α(1-6) linked, non-reducing terminal galactose from complex carbohydrates and glycoproteins. Recombinant from E. coli
Beta-M-acetylglucosaminidase N-acetylglucosaminidase cleaves all non-reducing terminal β-linked N-acetylglucosamine residues from complex carbohydrates and glycoproteins. Recombinant from Streptococcus pneumoniae in E. coli
Alpha-( 1 -2,3,6)-mannosidase Cleaves all α(1-2,3,6) linked mannose From Jack Bean
Alpha-(1,6) core mannosidase α(1-6) core mannosidase cleaves unbranched non-reducing terminal mannose, α(1-6) linked to the β-linked core mannose of the conserved mannosylchitobiose core of N-linked oligosaccharides. Recombinant from Xanthamonas manihotis in E. Coli
Alpha-( 1 -3,4)-fucosidase α(1-3,4) Fucosidase cleaves branched non-reducing terminal fucose, linked α(1-3) or α(1-4) to the N-acetylglucosamine of terminal Gal-GlcNAc disaccharide structures. Xanthomonas manihotis

Sequence Examples of Glycans Cleaved by ExoglycosidasesSequence Examples of Glycans Cleaved by Exoglycosidases

Glycan Release kits

Amerigo Scientific offers the Liberate™ glycan release kits for chemical release of glycans.

Product Name Source
Liberate Hydrazinolysis N- and O-glycan release kit For release of N- and O-linked glycans from glycoprotein therapeutics.
Liberate Orela O-glycan release kit For release of O-linked glycans from glycoprotein therapeutics.
Liberate MR-glycan release kit For immobilizing, clean up and PNGase F mediated release of glycans from glycoproteins.

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