Characterization of novel endo-ß-N-acetylglucosaminidases from intestinal Barnesiella intestinihominis that hydrolyze multi-branched complex-type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze N-linked glycans. Many ENGases have been characterized, but few have been identified with hydrolytic activity towards multi-branched complex-type N-glycans. In this study, three candidate ENGases were identified from Barnesiella intestinihominis based on database searches and phylogenetic analysis. A domain search identified the N x E motif in all three candidates, suggesting that they were members of glycosyl hydrolase family 85 (GH85). The three candidate ENGases, named Endo-BIN1, Endo-BIN2, and Endo-BIN3, were expressed in Escherichia coli cells, and their hydrolytic activity towards N-glycans and glycoproteins was measured by high performance liquid chromatography analysis and SDS-PAGE analysis. All ENGases showed hydrolytic activity towards glycoproteins, but only Endo-BIN2 and Endo-BIN3 showed hydrolytic activity towards pyridylaminated N-glycans. The optimum pH of Endo-BIN1, Endo-BIN2, and End-BIN3 was pH 6.5, 4.0, and 7.0, respectively. We measured substrate specificities of Endo-BIN2 and Endo-BIN3 towards pyridylaminated N-glycans, and found that the two Endo-BIN enzymes showed similar substrate specificity, preferring bi-antennary complex-type N-glycans with galactose or α2,6-linked sialic acid residues at the non-reducing ends. Endo-BIN2 and Endo-BIN3 were also able to hydrolyze multi-branched complex-type N-glycans. SDS-PAGE analysis revealed that all Endo-BIN enzymes were capable of releasing complex-type N-glycans from glycoproteins such as rituximab, transferrin, and fetuin. We expect that B. intestinihominis possesses ENGases to facilitate the utilization of complex-type N-glycans from host cells. These findings will have applications in N-glycan remodeling of glycoproteins and the development of pharmaceuticals.

Site-specific, covalent immobilization of PNGase F on magnetic particles mediated by microbial transglutaminase.

In the workflow of global N-glycosylation analysis, endoglycosidase-mediated removal of glycans from glycoproteins is an essential and rate-limiting step. Peptide-N-glycosidase F (PNGase F) is the most appropriate and efficient endoglycosidase for the removal of N-glycans from glycoproteins prior to analysis. Due to the high demand for PNGase F in both basic and industrial research, convenient and efficient methods are urgently needed to generate PNGase F, preferably in the immobilized form to solid phases. However, there is no integrated approach to implement both efficient expression, and site-specific immobilization of PNGase F. Herein, efficient production of PNGase F with a glutamine tag in Escherichia coli and site-specific covalent immobilization of PNGase F with this special tag via microbial transglutaminase (MTG) is described. PNGase F was fused with a glutamine tag to facilitate the co-expression of proteins in the supernatant. The glutamine tag was covalently and site-specifically transformed to primary amine-containing magnetic particles, mediated by MTG, to immobilize PNGase F. Immobilized PNGase F could deglycosylate substrates with identical enzymatic performance to that of the soluble counterpart, and exhibit good reusability and thermal stability. Moreover, the immobilized PNGase F could also be applied to clinical samples, including serum and saliva.

A fluorogenic probe for core-fucosylated glycan-preferred ENGase.

A fluorescence-quenching-based assay system to determine the hydrolytic activity of endo-ß-N-acetylglucosaminidases (ENGases), which act on the innermost N-acetylglucosamine (GlcNAc) residue of the chitobiose segment of core-fucosylated N-glycans, was constructed using a dual-labeled fluorescent probe with a hexasaccharide structure. The fluorogenic probe was evaluated using a variety of ENGases, including Endo-M W251N mutant, Endo-F3, and Endo-S, which recognize core fucosylated N-glycans. The occurrence of a hydrolysis reaction was detected by observing an increased fluorescence intensity, ultimately allowing the ENGase activities to be easily and quantitatively evaluated, with the exception of Endo-S. The obtained results clearly indicated the substrate specificities of the examined ENGases.

Release of bifidogenic N-glycans from native bovine colostrum proteins by an endo-ß-N-acetylglucosaminidase.

Milk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant's gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-ß-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins' biological roles.

Characterization of novel endo-ß-N-acetylglucosaminidase from Bacteroides nordii that hydrolyzes multi-branched complex type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze the N-linked oligosaccharides. Many ENGases have already been identified and characterized. However, there are still a few enzymes that have hydrolytic activity toward multibranched complex-type N-glycans on glycoproteins. In this study, one novel ENGase from Bacteroides nordii (Endo-BN) species was identified and characterized. The recombinant protein was prepared and expressed in Escherichia coli cells. This Endo-BN exhibited optimum hydrolytic activity at pH 4.0. High performance liquid chromatography (HPLC) analysis showed that Endo-BN preferred core-fucosylated complex-type N-glycans, with galactose or α2,6-linked sialic acid residues at their non-reducing ends. The hydrolytic activities of Endo-BN were also tested on different glycoproteins from high-mannose type to complex-type oligosaccharides. The reaction with human transferrin, fetuin, and α1-acid glycoprotein subsequently showed that Endo-BN is capable of releasing multi-branched complex-type N-glycans from these glycoproteins.

GH18 endo-ß-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans.

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-ß-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.

Evaluation of different PNGase F enzymes in immunoglobulin G and total plasma N-glycans analysis.

Glycoproteins, proteins that are co- and posttranslationally modified by sugars (glycans), have significant roles in pathophysiology of many different diseases. One of the main steps in sample preparation for free N-glycan analysis is deglycosylation or glycan removal. The aim of this study was to compare different peptide N-glycosidase F (PNGase F) enzymes (Rapid PNGase F and two recombinant versions) for deglycosylation of total human plasma glycoproteins and different amounts of human immunoglobulin G (IgG). Deglycosylation with different PNGase F enzymes resulted in different IgG and plasma N-glycosylation hydrophilic interaction liquid chromatography ultra-performance liquid chromatography profiles. Additionally, one recombinant version of PNGase F is more efficient in deglycosylation of complex N-glycans compared with Rapid PNGase F and recombinant version of PNGase F from a different manufacturer. In terms of chromatographic peak intensities and coefficient of variation %Area values, all tested versions of PNGase F enzymes were very reproducible and on the similar level when used in optimal conditions. However, care should be taken in terms of which enzyme is used with which protocol, particularly when scaling up.

Microwave irradiation-assisted high-efficiency N-glycan release using oriented immobilization of PNGase F on magnetic particles.

Peptide-N-glycosidase F (PNGase F) is the most frequently used enzyme to release N-glycan from glycoproteins in glycomics; however, the releasing process using PNGase F is tedious and can range in duration from hours to overnight. Recently, efforts have been made to accelerate this enzymatic reaction, and they include the use of microwave irradiation, ultrahigh pressure, enzyme immobilization, and other techniques. Here, we developed a novel method combining the oriented immobilization of PNGase F on magnetic particles and microwave-assisted enzymatic digestion techniques to achieve highly efficient release of N-glycans. The oriented immobilization of PNGase F on magnetic particles utilizes the affinity of its co-expressed His-tag towards iminodiacetic acid-Nickel modified magnetic particles. Compared with non-oriented immobilization, the oriented immobilization of PNGase F exhibits several advantages including tolerance to high temperature (52 °C) and the ability to retain strong activity after more than five reuses. When used in combination with microwave irradiation, efficient N-glycan removal from ribonuclease B was achieved within 5 min. The proposed strategy was also used to release glycan from fetuin and human serum and has proven to provide a promising deglycosylation method for the characterization of protein glycosylation.

Endoplasmic reticulum stress differentially inhibits endoplasmic reticulum and inner nuclear membrane protein quality control degradation pathways.

Endoplasmic reticulum (ER) stress occurs when the abundance of unfolded proteins in the ER exceeds the capacity of the folding machinery. Despite the expanding cadre of characterized cellular adaptations to ER stress, knowledge of the effects of ER stress on cellular physiology remains incomplete. We investigated the impact of ER stress on ER and inner nuclear membrane protein quality control mechanisms in Saccharomyces cerevisiae. We analyzed the turnover of substrates of four ubiquitin ligases (Doa10, Rkr1/Ltn1, Hrd1, and the Asi complex) and the metalloprotease Ste24 in induced models of ER stress. ER stress did not substantially impact Doa10 or Rkr1 substrates. However, Hrd1-mediated destruction of a protein that aberrantly engages the translocon (Deg1-Sec62) and substrates with luminal degradation signals was markedly impaired by ER stress; by contrast, Hrd1-dependent degradation of proteins with intramembrane degrons was largely unperturbed by ER stress. ER stress impaired the degradation of one of two Asi substrates analyzed and caused a translocon-clogging Ste24 substrate to accumulate in a form consistent with persistent translocon occupation. Degradation of Deg1-Sec62 in the absence of stress and stabilization during ER stress were independent of four ER stress-sensing pathways. Our results indicate ER stress differentially impacts degradation of protein quality control substrates, including those mediated by the same ubiquitin ligase. These observations suggest the existence of additional regulatory mechanisms dictating substrate selection during ER stress.

N-linked Glycan Release Efficiency: A Quantitative Comparison between NaOCl and PNGase F Release Protocols.

There are several methods, both chemical and enzymatic, to release N-linked glycans for structural characterization. One of the most common enzymatic release methods is the use of peptide:N-glycosidase F (PNGase F). A less expensive and quicker alternative has been reported for the release of N-linked glycans chemically using sodium hypochlorite (NaOCl), which hydrolyzes the peptide-glycan bond, yielding the intact glycan with a free reducing terminus. Here, we quantitatively analyzed the efficiency of the NaOCl release protocol compared with the PNGaseF release protocol for small-scale analysis (300 µg) using liquid chromatography-single reaction monitoring-mass spectrometry. We determined that the relative glycan composition of released N-linked glycans from the NaOCl protocol is similar to a typical PNGase F protocol, but the absolute recovery of N-linked glycans is significantly lower with the chemical procedure.

Bacteriophage T4 capsid as a nanocarrier for Peptide-N-Glycosidase F immobilization through self-assembly.

Enzyme immobilization is widely applied in biocatalysis to improve stability and facilitate recovery and reuse of enzymes. However, high cost of supporting materials and laborious immobilization procedures has limited its industrial application and commercialization. In this study, we report a novel self-assembly immobilization system using bacteriophage T4 capsid as a nanocarrier. The system utilizes the binding sites of the small outer capsid protein, Soc, on the T4 capsid. Enzymes as Soc fusions constructed with regular molecular cloning technology expressed at the appropriate time during phage assembly and self-assembled onto the capsids. The proof of principle experiment was carried out by immobilizing ß-galactosidase, and the system was successfully applied to the immobilization of an important glycomics enzyme, Peptide-N-Glycosidase F. Production of Peptide-N-Glycosidase F and simultaneous immobilization was finished within seven hours. Characterizations of the immobilized Peptide-N-Glycosidase F indicated high retention of activity and well reserved deglycosylation capacity. The immobilized Peptide-N-Glycosidase F was easily recycled by centrifugation and exhibited good stability that sustained five repeated uses. This novel system uses the self-amplified T4 capsid as the nanoparticle-type of supporting material, and operates with a self-assembly procedure, making it a simple and low-cost enzyme immobilization technology with promising application potentials.

Characterization of novel endo-ß-N-acetylglucosaminidases from Sphingobacterium species, Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG.

Endo-ß-N-acetylglucosaminidase (ENGase) catalyzes hydrolysis of N-linked oligosaccharides. Although many ENGases have been characterized from various organisms, so far no fucose-containing oligosaccharides-specific ENGase has been identified in any organism. Here, we screened soil samples, using dansyl chloride (Dns)-labeled sialylglycan (Dns-SG) as a substrate, and discovered a strain that exhibits ENGase activity in the culture supernatant; this strain, named here as strain HMA12, was identified as a Sphingobacterium species by 16S ribosomal RNA gene analysis. By draft genome sequencing, five candidate ENGase encoding genes were identified in the genome of this strain. Recombinant proteins, purified from Escherichia coli expressing candidate genes ORF1152, ORF1188, ORF3046 and ORF3750 exhibited fucose-containing oligosaccharides-specific ENGase activity. These ENGases exhibited optimum activities at very acidic pHs (between pH 2.3-2.5). BLAST searches using sequences of these candidate genes identified two fungal homologs of ORF1188, one in Beauveria bassiana and the other in Cordyceps militaris. Recombinant ORF1188, Beauveria and Cordyceps ENGases released the fucose-containing oligosaccharides residues from rituximab (immunoglobulin G) but not the high-mannose-containing oligosaccharides residues from RNase B, a result that not only confirmed the substrate specificity of these novel ENGases but also suggested that natural glycoproteins could be their substrates.

Specificity of Donor Structures for endo-ß-N-Acetylglucosaminidase-Catalyzed Transglycosylation Reactions.

To demonstrate the structural specificity of the glycosyl donor for the transglycosylation reaction by using endo-ß-N-acetylglucosaminidase from Mucor hiemalis (endo-M), a series of tetrasaccharide oxazoline derivatives was synthesized. These derivatives correspond to the core structure of an asparagine-linked glycoprotein glycan with a ß-mannose unit of a non-natural-type monosaccharide, including ß-glucose, ß-galactose, and ß-talose in place of the ß-mannose moiety. The transglycosylation activity of wildtype (WT) endo-M and two mutants, N175Q and N175A, was examined by using these tetrasaccharide donors with p-nitrophenyl N-acetylglucosaminide (GlcNAc-pNp). The essential configuration of the hydroxy group for the transglycosylation reaction was determined. On the basis of these results, the transglycosylation reaction was investigated by using chemically modified donors, and transglycosylated products were successfully obtained.

MALDI Imaging Mass Spectrometry of N-glycans and Tryptic Peptides from the Same Formalin-Fixed, Paraffin-Embedded Tissue Section.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a unique and well developed tool for probing the protein content of formalin-fixed, paraffin-embedded tissue (FFPE). Integral to this approach is the application of trypsin, and more recently peptide N-glycosidase F, to release tryptic peptides or N-glycans from tissue and report localization of distinct species. This is typically done on serial or adjacent tissue sections, and there is an emerging need to understand the colocalized protein population linked to the exact same regions of N-glycans. Here we describe an approach where N-glycans are first imaged from a tissue section followed by reprocessing of the same tissue section for tryptic peptide MALDI IMS. Strategies for colocalizing peptides to target N-glycans or N-glycan regions are described.

Structural modeling and mutagenesis of endo-ß-N-acetylglucosaminidase from Ogataea minuta identifies the importance of Trp295 for hydrolytic activity.

Endo-ß-N-acetylglucosaminidase from the methylotrophic yeast Ogataea minuta (Endo-Om) is a glycoside hydrolase family 85 enzyme that has dual catalytic activity in the hydrolysis and transglycosylation of complex N-glycans, in common with the enzymes from the eukaryotic species. In this study, we have conducted mutagenesis of Endo-Om at Trp295, to determine the effect on hydrolytic activity. Structural modeling predicted that Trp295 forms an important interaction with the α-1,3-linked mannose residue of the trimannosyl N-glycan core, rather than being directly involved in catalytic activity. Our results showed that an aromatic amino acid is required at position 295 for the hydrolytic activity of this enzyme. Notably, the tryptophan residue is highly conserved in eukaryotic endo-ß-N-acetylglucosaminidases that show activity toward complex oligosaccharides. Accordingly, our results strongly suggested that Trp295 is involved in the recognition of oligosaccharide substrates by Endo-Om.

Chemoenzymatic Glycan Remodeling of Natural and Recombinant Glycoproteins.

N-glycosylation plays important roles in modulating the biological functions of glycoproteins, such as protein folding, stability, and immunogenicity. However, acquiring homogeneous glycoforms of glycoproteins has been a challenging task for functional studies and therapeutic applications. In this chapter, we describe an efficient chemoenzymatic glycan remodeling protocol for making homogeneous glycoproteins that involves enzymatic deglycosylation and subsequent reglycosylation procedures. Two therapeutic glycoproteins, Herceptin (trastuzumab, a therapeutic monoclonal antibody) and erythropoietin (EPO, a glycoprotein hormone), were chosen as the model systems. The detailed protocol includes the deglycosylation of the Herceptin or EPO with a wild-type endo-ß-N-acetylglucosaminidase, to remove the heterogeneous N-glycans, leading to the GlcNAc-protein or Fucα1,6GlcNAc-protein intermediate. Then desired homogeneous N-glycans are attached to the acceptor by using an activated sugar oxazoline as the donor substrate and a specific glycosynthase (mutant of endoglycosidase) as the catalyst to reconstitute a homogeneous glycoform. Using this approach, Herceptin was remodeled to an afucosylated complex glycoform and a Man9GlcNAc2 glycoform, with the former showing significantly enhanced antibody-dependent cellular cytotoxicity. EPO was engineered to carry azide-tagged Man3GlcNAc2 glycans that could be further modified via click chemistry to introduce other functional groups.

Analysis of Golgi-Mediated Protein Traffic in Plant Cells.

In plant secretory pathways, the Golgi apparatus serves as the major sorting hub to receive de novo synthesized protein from the endoplasmic reticulum for further sorting to post-Golgi compartments or for residence in the cisternae of Golgi stacks. Meanwhile, Golgi functions as a pivotal biochemical factory to make modifications of N-glycans and to produce mature glycoproteins. Fluorescent tag-based confocal microscopy in combination with the brefeldin A drug or the genetic tools to disturb Golgi function have been shown as powerful approaches to analyze Golgi-mediated protein traffic in transiently expressed plant protoplasts or in stably expressed transgenic plants. Various endoglycosidases like Endo H and PNGase F have been widely used to monitor Golgi-mediated glycosylation of secretory proteins. Here, using fluorescently tagged Golgi-resident proteins and known glycosylated proteins as examples, we describe detailed protocols to analyze Golgi-mediated protein traffic and glycosylation in transiently expressed protoplasts derived from Arabidopsis suspension culture cells and in stably expressed transgenic plants.

Deglycosylating enzymes acting on N-glycans in fungi: Insights from a genome survey.

BACKGROUND: N-Glycosylation, one of the most prominent post-translational modifications of proteins, is found in all domains of life, i.e. eukaryotes, bacteria and archaea, and has been shown to play a crucial role in modulating the physicochemical/physiological properties of carrier proteins. Deglycosylating enzymes that act on N-glycans are widely used in analyzing the structures/functions of N-glycans. Fungi are known to produce various deglycosylating enzymes, some of which are fungi-specific. While such enzymes likely are biologically relevant in fungal biology, their properties as well as their functions have not been explored in detail. SCOPE OF REVIEW: In this review, we summarize the current knowledge of fungal deglycosylating enzymes and discuss their biological significance. MAJOR CONCLUSIONS: As of this writing, five types of deglycosylating enzymes that act on N-glycans are known to occur in fungi; (1) the cytosolic peptide: N-glycanase (PNGase), (2) the acidic PNGase, (3) the glycoside hydrolase family (GH) 85 endo-ß-N-acetylglucosaminidase (ENGase), (4) the GH18 cytosolic ENGase, and (5) the GH18 secreted ENGase. Interestingly, genome surveys indicate that the loss of cytosolic PNGase activity in certain fungi coincide with the occurrence of GH18 cytosolic ENGase, implying that the GH18 ENGase serves to replace the deglycosylation function of the cytosolic PNGase in those filamentous ascomycete fungi. GENERAL SIGNIFICANCE: Our review concludes that fungi promise to be valuable organisms for developing an understanding of the biological functions of PNGases/ENGases.

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-ß-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus.

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-ß-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes.

The ENGases: versatile biocatalysts for the production of homogeneous N-linked glycopeptides and glycoproteins.

The endo-ß-N-acetylglucosaminidases (ENGases) are an enzyme class (EC 3.2.1.96) produced by a range of organisms, ranging from bacteria, through fungi, to higher order species, including humans, comprising two-sub families of glycosidases which all cleave the chitobiose core of N-linked glycans. Synthetic applications of these enzymes, i.e. to catalyse the reverse of their natural hydrolytic mode of action, allow the attachment of N-glycans to a wide variety of substrates which contain an N-acetylglucosamine (GlcNAc) residue to act as an 'acceptor' handle. The use of N-glycan oxazolines, high energy intermediates on the hydrolytic pathway, as activated donors allows their high yielding attachment to almost any amino acid, peptide or protein that contains a GlcNAc residue as an acceptor. The synthetic effectiveness of these biocatalysts has been significantly increased by the production of mutant glycosynthases; enzymes which can still catalyse synthetic processes using oxazolines as donors, but which do not hydrolyse the reaction products. ENGase biocatalysts are now finding burgeoning application for the production of biologically active glycopeptides and glycoproteins, including therapeutic monoclonal antibodies (mAbs) for which the oligosaccharides have been remodelled to optimise effector functions.