# PNGase F

**PNGase F** (peptide:N-glycosidase F; EC 3.5.1.52), formally peptide-N⁴-(N-acetyl-β-D-glucosaminyl)asparagine amidase, is a bacterial amidase that cleaves N-linked (asparagine-linked) glycans from glycoproteins and glycopeptides, releasing the intact oligosaccharide and converting the modified asparagine residue to aspartic acid.<sup>[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC4367255/)</sup> It is sold by multiple enzyme suppliers as a recombinant reagent for analytical deglycosylation.<sup>[2](https://www.neb.com/en/products/glycobiology/endoglycosidases/endoglycosidases)</sup>

| Key fact | Detail |
|---|---|
| Classification | Peptide:N-glycosidase, EC 3.5.1.52; an amidase, not a true glycosidase<sup>[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC4367255/)</sup><sup> • </sup><sup>[2](https://www.neb.com/en/products/glycobiology/endoglycosidases/endoglycosidases)</sup> |
| Cleavage site | Between the innermost GlcNAc of the chitobiose core and the asparagine residue, liberating the entire sugar chain<sup>[2](https://www.neb.com/en/products/glycobiology/endoglycosidases/endoglycosidases)</sup> |
| Glycan range | Removes almost all N-linked types: high mannose, hybrid, and bi-, tri-, and tetra-antennary complex chains<sup>[3](https://www.neb.com/en-us/faqs/what-is-the-difference-between-pngase-f-and-endo-h)</sup> |
| Key limitation | Does not cleave N-glycans whose core GlcNAc carries an α1-3-fucose, common in plant and insect proteins<sup>[4](https://www.neb.com/en/products/p0708-pngase-f-recombinant)</sup><sup> • </sup><sup>[5](https://www.ncbi.nlm.nih.gov/books/NBK579945/table/CSHLP5087CH50TB1/?report=objectonly)</sup> |
| Chemical result | Asparagine at the site is converted to aspartic acid; the intact glycan and ammonia are released<sup>[6](https://www.thermofisher.com/order/catalog/product/A39245/faqs)</sup><sup> • </sup><sup>[7](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10121237/pdf/jbt-34-1x335dgyl.pdf)</sup> |
| Standard reaction | 37 °C, typically 1 hour under denaturing conditions or 4 to 24 hours on native glycoprotein<sup>[8](https://www.neb.com/en/protocols/pngase-f-protocol)</sup> |
| Source organism | Originally isolated from *Elizabethkingia meningoseptica* (formerly *Flavobacterium meningosepticum*) in 1984; commercial recombinant enzyme is produced in *E. coli*<sup>[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC4367255/)</sup><sup> • </sup><sup>[9](https://staging-250826.genovis.com/wp-content/uploads/instructions-pngase-f-automation.pdf)</sup> |

## Reaction and specificity

PNGase F breaks the bond linking the asparagine residue's amine group to the GlcNAc closest to the protein within the chitobiose core, where that GlcNAc is an N-acetylglucosamine.<sup>[2](https://www.neb.com/en/products/glycobiology/endoglycosidases/endoglycosidases)</sup> Because it severs an amide bond rather than a glycosidic one, it is classified as a glycoamidase: the asparagine is deamidated to aspartic acid, and the released oligosaccharide carries a reducing-end [glucosamine](https://www.edgepedia.org/glucosamine) that hydrolyzes nonenzymatically at physiological pH to give the free glycan and ammonia.<sup>[6](https://www.thermofisher.com/order/catalog/product/A39245/faqs)</sup><sup> • </sup><sup>[10](https://faseb.onlinelibrary.wiley.com/doi/full/10.1096/fj.01-0889rev)</sup>

The enzyme's coverage of N-glycan types is broad but not universal. It removes high-mannose, hybrid, and complex bi-, tri-, and tetra-antennary chains, which makes it the enzyme of choice when the goal is to strip all N-linked carbohydrates without regard to type.<sup>[3](https://www.neb.com/en-us/faqs/what-is-the-difference-between-pngase-f-and-endo-h)</sup> Two structural requirements limit its use. First, the innermost GlcNAc must not be modified with an α1-3-linked fucose, a core modification found in slime molds, plants, insects, and parasites; such glycans are resistant.<sup>[4](https://www.neb.com/en/products/p0708-pngase-f-recombinant)</sup><sup> • </sup><sup>[11](https://www.ncbi.nlm.nih.gov/books/NBK453020/?report=reader)</sup> Second, the enzyme requires at least one amino acid on both the amino-terminal and carboxy-terminal sides of the glycosylated asparagine, so a glycan at the very end of a peptide chain may not be released.<sup>[5](https://www.ncbi.nlm.nih.gov/books/NBK579945/table/CSHLP5087CH50TB1/?report=objectonly)</sup>

## Comparison with other deglycosylating enzymes

**Endo H** is the usual point of comparison. Endo H cleaves between the two GlcNAc residues of the chitobiose core, leaving one GlcNAc still attached to the protein, and acts only on high-mannose and some hybrid glycans, not complex ones.<sup>[3](https://www.neb.com/en-us/faqs/what-is-the-difference-between-pngase-f-and-endo-h)</sup><sup> • </sup><sup>[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC3869378/)</sup> This narrower specificity is analytically useful: sensitivity to Endo H indicates an immature, ER-stage glycan, whereas resistance after Endo H but sensitivity to PNGase F indicates processing through the medial Golgi.<sup>[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC3869378/)</sup> PNGase F is described as the only enzyme among the common set that cleaves tetraantennary chains.<sup>[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC3869378/)</sup>

For α1-3-fucosylated glycans, alternatives exist. PNGase A, first identified in almond emulsion in 1977, will cleave glycans with core α1-3-fucose, though its efficacy on mammalian glycoproteins is poorly defined.<sup>[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC4367255/)</sup><sup> • </sup><sup>[5](https://www.ncbi.nlm.nih.gov/books/NBK579945/table/CSHLP5087CH50TB1/?report=objectonly)</sup> More recently characterized bacterial enzymes include PNGase F-II from *E. meningoseptica*, which accepts either α1,3- or α1,6-fucose on the core GlcNAc but strongly prefers denatured protein, and PNGaseRc from *Rudaea cellulosilytica*, which accommodates both fucose linkages on native protein at neutral pH.<sup>[13](https://portlandpress.com/essaysbiochem/article-pdf/doi/10.1042/EBC20220256/945295/ebc-2022-0256c.pdf)</sup> PNGase F remained the sole prokaryotic PNGase known from its 1984 discovery until candidate PNGase genes were identified in genome projects, including ones from *Deinococcus radiodurans* and *Bacteroides fragilis*.<sup>[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC4367255/)</sup>

## Practical use

**Denaturing conditions** give the fastest, most complete release. A typical protocol combines 1 to 20 µg of glycoprotein with denaturing buffer, heats at 100 °C for 10 minutes, then adds buffer, 10% NP-40, and PNGase F and incubates at 37 °C for 1 hour.<sup>[8](https://www.neb.com/en/protocols/pngase-f-protocol)</sup> Denaturation matters because protein secondary and tertiary structure can prevent the enzyme from reaching its substrate.<sup>[14](https://www.neb.com/faqs/i-tried-the-pngase-f-on-my-glycoprotein-and-didn-t-see-removal-of-the-carbohydrate-what-could-be)</sup> PNGase F is inhibited by SDS, so the non-ionic detergent NP-40 must be included in denaturing reactions; NEB notes that the reason this detergent counteracts the inhibition is not known.<sup>[8](https://www.neb.com/en/protocols/pngase-f-protocol)</sup><sup> • </sup><sup>[4](https://www.neb.com/en/products/p0708-pngase-f-recombinant)</sup>

**Native (non-denaturing) conditions** preserve the folded protein but are slower: 2 to 5 µl of enzyme with 1 to 20 µg of glycoprotein incubated at 37 °C for 4 to 24 hours, with longer incubation and more enzyme often required.<sup>[8](https://www.neb.com/en/protocols/pngase-f-protocol)</sup> A denatured parallel digest serves as the positive control for full deglycosylation.<sup>[8](https://www.neb.com/en/protocols/pngase-f-protocol)</sup>

Completion is most simply assessed by mobility shifts on SDS-PAGE; the deglycosylated protein's mass drops by roughly 2 kDa per glycan chain cleaved.<sup>[8](https://www.neb.com/en/protocols/pngase-f-protocol)</sup><sup> • </sup><sup>[15](https://trungtamthuoc.com/usp-en/oligosaccharide-analysis)</sup> Unit definitions differ by supplier: NEB defines one unit as the amount removing more than 95% of the carbohydrate from 10 µg of denatured RNase B in 1 hour at 37 °C in a 10 µl volume,<sup>[4](https://www.neb.com/en/products/p0708-pngase-f-recombinant)</sup> while the USP defines one unit as release of N-linked oligosaccharides from 1.0 nmol of denatured ribonuclease B per minute at pH 7.5 at 37 °, equal to 1 IUB milliunit.<sup>[15](https://trungtamthuoc.com/usp-en/oligosaccharide-analysis)</sup>

## Applications in glycomics and proteomics

Released N-glycans are the starting material for glycan profiling: glycomics workflows release N-glycans with PNGase F (or PNGase A, or hydrazinolysis), derivatize them, and analyze them by MALDI or ESI mass spectrometry.<sup>[16](https://pmc.ncbi.nlm.nih.gov/articles/PMC5498624/)</sup> In glycoproteomics, digestion with PNGase F converts each occupied N-glycosylation site's asparagine to aspartic acid, adding a mass shift that marks the site; in heavy-oxygen water (H₂¹⁸O) the shift incorporates ¹⁸O from the surrounding water, enabling stable-isotope mapping of N-linked sites, provided chemical deamidation is minimized.<sup>[16](https://pmc.ncbi.nlm.nih.gov/articles/PMC5498624/)</sup> Analysis of N-glycan composition of recombinant therapeutic proteins by PNGase F digestion has become part of product characterization and release specifications in pharmacopeial methods.<sup>[15](https://trungtamthuoc.com/usp-en/oligosaccharide-analysis)</sup>

**Formulation matters for mass spectrometry.** Standard PNGase F is stored in 50% glycerol, which is not tolerated by HPLC and mass spectrometry instruments, so glycerol-free formulations are used for those workflows.<sup>[6](https://www.thermofisher.com/order/catalog/product/A39245/faqs)</sup> A variant with a chitin-binding domain tag (Remove-iT PNGase F) allows the enzyme to be removed after the reaction, leaving the protein and released glycans ready for analysis.<sup>[17](https://www.neb.cn/en-gb/-/media/nebuk/files/brochures/glycoproteomics_tech_guide.pdf?hash=D41BB22DC8D639CCA3DD471D5A80B99D&rev=032daa66b66640a9a6637fc4b2629269)</sup>

**Speed has improved.** Conventional N-glycan release requires several hours to overnight digestion at relatively high enzyme-to-substrate ratios (about 1:250 to 1:500).<sup>[18](https://pmc.ncbi.nlm.nih.gov/articles/PMC7168572/)</sup> Rapid PNGase F formulations with detergent-based buffers achieve complete release in about 15 minutes, with glycan profiles comparable to overnight protocols, and short incubations of about 5 minutes have been shown sufficient to deglycosylate every N-glycan site of a monoclonal antibody without bias.<sup>[19](https://www.sigmaaldrich.com/GD/en/technical-documents/technical-article/protein-biology/protein-mass-spectrometry/pngase-fast-asms)</sup><sup> • </sup><sup>[20](https://www.neb.cn/en/-/media/nebus/files/brochures/rapid_pngase_f_trifold.pdf?hash=1147ED8777BB5567025EB133761ABB48&rev=5c7ec10fa8cf4bed90dd4bfff82472b1)</sup>

## Production and origin

The first PNGase (PNGase A) was identified in almond emulsion in 1977, and the first prokaryotic one, PNGase F, was isolated from *Elizabethkingia meningoseptica* in 1984 and named after the organism.<sup>[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC4367255/)</sup> Commercial PNGase F is produced recombinantly: Promega describes its enzyme as cloned from *Elizabethkingia miricola*,<sup>[21](https://worldwide.promega.com/resources/protocols/product-information-sheets/n/pngase-f-protocol/)</sup> and Genovis describes PNGase F expressed in *E. coli* from a recombinant gene.<sup>[9](https://staging-250826.genovis.com/wp-content/uploads/instructions-pngase-f-automation.pdf)</sup> Bacterial PNGase F can be produced cost-effectively at large scale in prokaryotic hosts in an insoluble form, yielding after refolding an unglycosylated active enzyme of high purity suitable for proteomics.<sup>[22](https://pmc.ncbi.nlm.nih.gov/articles/PMC4231336/)</sup> The enzyme's natural biological role in the bacterium has remained elusive; because protein N-glycosylation is rare in bacteria, PNGase activity has been speculated to provide a niche in host-bacterium interactions.<sup>[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC4367255/)</sup>

## References

1. [Identification and Characterization of a Novel Prokaryotic Peptide:N-Glycosidase from Elizabethkingia meningoseptica, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC4367255/)
2. [Endoglycosidases, New England Biolabs](https://www.neb.com/en/products/glycobiology/endoglycosidases/endoglycosidases)
3. [FAQ: What is the difference between PNGase F and Endo H?, NEB](https://www.neb.com/en-us/faqs/what-is-the-difference-between-pngase-f-and-endo-h)
4. [PNGase F, Recombinant (P0708), NEB](https://www.neb.com/en/products/p0708-pngase-f-recombinant)
5. [Table of enzymes for glycan analysis, Essentials of Glycobiology, NCBI Bookshelf](https://www.ncbi.nlm.nih.gov/books/NBK579945/table/CSHLP5087CH50TB1/?report=objectonly)
6. [PNGase F Glycan Cleavage Kit FAQs, Thermo Fisher](https://www.thermofisher.com/order/catalog/product/A39245/faqs)
7. [Do I Need to Trypsin Digest Before Releasing IgG Glycans With PNGase-F?, PMC](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10121237/pdf/jbt-34-1x335dgyl.pdf)
8. [PNGase F Protocol, NEB](https://www.neb.com/en/protocols/pngase-f-protocol)
9. [SmartEnzymes PNGase F automation instructions, Genovis](https://staging-250826.genovis.com/wp-content/uploads/instructions-pngase-f-automation.pdf)
10. [Suzuki et al. (2002). Cytoplasmic peptide:N-glycanase (PNGase) in eukaryotic cells, FASEB Journal](https://faseb.onlinelibrary.wiley.com/doi/full/10.1096/fj.01-0889rev)
11. [N-Glycans, Essentials of Glycobiology, NCBI Bookshelf](https://www.ncbi.nlm.nih.gov/books/NBK453020/?report=reader)
12. [Freeze & Kranz. Endoglycosidase and Glycoamidase Release of N-Linked Glycans, Current Protocols, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC3869378/)
13. [Characterised PNGase enzymes, Essays in Biochemistry (Portland Press)](https://portlandpress.com/essaysbiochem/article-pdf/doi/10.1042/EBC20220256/945295/ebc-2022-0256c.pdf)
14. [FAQ: PNGase F did not remove the carbohydrate, NEB](https://www.neb.com/faqs/i-tried-the-pngase-f-on-my-glycoprotein-and-didn-t-see-removal-of-the-carbohydrate-what-could-be)
15. [Oligosaccharide Analysis, USP 2025 General Chapters](https://trungtamthuoc.com/usp-en/oligosaccharide-analysis)
16. [Glycomic and glycoproteomic analysis of glycoproteins—a tutorial, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC5498624/)
17. [Glycoproteomics Technical Guide, NEB](https://www.neb.cn/en-gb/-/media/nebuk/files/brochures/glycoproteomics_tech_guide.pdf?hash=D41BB22DC8D639CCA3DD471D5A80B99D&rev=032daa66b66640a9a6637fc4b2629269)
18. [Analysis of carbohydrates and glycoconjugates by MALDI mass spectrometry: 2009–2010 update, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC7168572/)
19. [Rapid Glycan Prep & Analysis by LC-MS Workflow, Sigma-Aldrich](https://www.sigmaaldrich.com/GD/en/technical-documents/technical-article/protein-biology/protein-mass-spectrometry/pngase-fast-asms)
20. [Rapid PNGase F Trifold, NEB](https://www.neb.cn/en/-/media/nebus/files/brochures/rapid_pngase_f_trifold.pdf?hash=1147ED8777BB5567025EB133761ABB48&rev=5c7ec10fa8cf4bed90dd4bfff82472b1)
21. [PNGase F Protocol, Promega](https://worldwide.promega.com/resources/protocols/product-information-sheets/n/pngase-f-protocol/)
22. [Discovery and characterization of a novel extremely acidic bacterial N-glycanase, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC4231336/)

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*Topic: Encyclopedia › Life and health › Biological foundations › Biochemistry and metabolism › Enzyme classes and activities › Proteolytic and peptidase enzymes › Applied and research proteases › Proteases as laboratory reagents*

*Initially written Sep 17, 2026 · Reviewed: Sep 30, 2026; Oct 21, 2026 · Edited: Oct 15, 2026; Oct 21, 2026 · Last review: Oct 21, 2026*

*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*

License: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license