Endo-M is one of the enzymes known as endo-β-N-acetylglucosaminidases (endo-β-GlcNAc-ases). This enzyme was found by Yamamoto et al.¹⁾, in the culture fluid of Mucor hiemalis isolated from soil. Endo-M hydrolyzes the N,N'-diacetylchitobiose moiety in oligosaccharides bound to the asparaginyl residue of various glycoproteins through the N-glycosidic linkage. The efficacy of this enzyme comes from the fact that one N-acetylglucosamine residue remains bound to the protein while cleaving the N,N'-diacetylchitobiose moiety. The enzyme is thus able to transfer the intact oligosaccharide to suitable acceptors. Unlike the conventional endo-β-GlcNAc-ase, it has been found that Endo-M is an enzyme with a broad substrate specificity, cleaving not only the high-mannose type and hybrid type of asparagine-linked oligosaccharides but also the complex type oligosaccharides in glycoproteins. Therefore, Endo-M is expected to be applied to various fields.

Yamamoto et al.²⁾ incubated an asialotransferrin glycopeptide with Endo-M in the presence of GlcNAc, followed by pyridylaminating (PA) oligosaccharides in the supernatant. In this experiment, they observed by HPLC that two separate PA-oligosaccharides had formed. One was the oligosaccharide released by hydrolysis, and the other was the released oligosaccharide that was transfered to GlcNAc. As acceptors, diacetylchitobiose and dansyl-asparaginyl N-acetylglucosamine [DNS-Asn(GlcNAc)] were also found to be effective. The enzyme was also capable of transferring high-mannose oligosaccharide to the acceptor diacetylchitobiose.
Haneda et al.³⁾ have transferred oligosaccharides with 9-fluorenylmethoxycarbonyl-asparaginyl-N-acetyl-glucosaminide [Fmoc-Asn(GlcNAc)] by incubating sialotransferrin glycopeptide, asialotransferrin glycopeptide and Man₆GlcNAc₂-Asn-peptide with Endo-M. Furthermore, synthetic hCG (β12-16)-GlcNAc-peptide has been subjected to transglycosylate with a sialo complex type oligosaccharide. An alternative synthetic method of peptide containing GlcNAc has been developed by Inazu et al.⁴⁾ This method uses Fmoc-Asn(GlcNAc), which was synthesized from aspartic acid containing an N-terminal group protected by an Fmoc group, and azide of GlcNAc instead of Fmoc-Asn-OH, and it applies a mixed acid anhydride method using dimethylthiophosphic acid (Mpt-MA) which generally shows poor responses toward the hydroxyl group. By combining this method with Endo-M, many glycopeptides can be designed and easily prepared. Yamamoto⁵⁾ has compiled the outline of this methodology as the Chemo-Enzymatic Synthesis in his review. Endo-M can also be used to create new functions, by introducing glyco-chains, to the substances that originally do not have the specific functions.⁶⁾
As a specific example, it is also possible to synthesize functional undecasaccharide by transferring a biotin and azidoethyl group to an acceptor oligosaccharide as shown below.

*1 unit will catalyze the release of 1 μmol of Fmoc-Asn(GlcNAc) from Fmoc-SGN per min. at pH6.0 at 37°C

Related products
A1614 Nomega-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)-Nalpha-(tert-butoxycarbonyl)-L-asparagine
A2172 2-Azidoethyl 2-Acetamido-2-deoxy-beta-D-glucopyranoside
G0297 N-GlcNAc-Biotin
S0523 Sialylglycopeptide

Yamamoto et al. have recently purified and isolated endo-α-N-acetylgalactosaminidase (Endo-α) found in the culture fluid of Bifidobacterium longum.⁷⁾ Endo-α can recognize the structure of Galβ1-3GalNAc disaccharide α-linked with a hydroxyl group. It releases Galβ1-3GalNAc by hydrolysis. When a compound possessing an hydroxyl group coexists as an acceptor, the released Galβ1-3GalNAc is transferred to the acceptor.⁸⁾ Discovered by Yamamoto et al., Endo-α can transfer Galβ1-3GalNAc to various compounds such as monosaccharides, peptides, and proteins, using core 1 contained in mucin-type oligosaccharide chains as a donor.

Ashida et al. have reported the oligosaccharide transfer reaction using Endo-α.^8a) According to the report, Galβ1-3GalNAcα-pNP was treated with Endo-α to produce Galβ1-3GalNAc and it transferred to monosaccharides (glucose, galactose, and mannose), disaccharides (maltose and sucrose), and sugar alcohols (mannitol and sorbitol).

Thus, by using Endo-M and Endo-α properly, it is possible to transfer both N-linked and O-linked oligosaccharides. As a tool for the enzymatic synthesis of glycoconjugates, it is expected that many applications will be realized in the various fields.

*1 unit will hydrolyze 1 μmol of Galβ1-3GalNAcα-pNP to Galβ1-3GalNAc and pNP per min. at pH5.0 at 37°C

These products were merchandised as the fruition of NEDO project.
Endo-M was merchandised under licensed from patent-holding companies of Takara Bio Inc. and Kirin Brewery Co., LTD.