New England Biolabs, P0773S, Endoglycoceramidase I (EGCase I)

Related Categories Endoglycosidases Specification Unit Definition One unit of R. triatomea EGCase I was defined as the amount of enzyme required to hydrolyze 1 μmol of ganglioside GM1a per minute at 37°C. Reaction Conditions 1X EGCase I Reaction Buffer Incubate at 37°C 1X EGCase I Reaction Buffer 50 mM sodium acetate 0.1% Triton® X-100 (pH 5.2 @ 25°C) Storage Buffer 50 mM NaCl 20 mM Tris-HCl 1 mM EDTA pH 7.5 @ 25°C Heat Inactivation 65°C for 20 minutes Molecular Weight Apparent: 50 kDa Unit Assay Conditions Two fold dilutions of EGCase I are incubated with 10 nmoles of GM1a substrate in 1X EGCase I Reaction Buffer in a 10 μl reaction. The reaction mix is incubated for 5 minutes at 37°C. Released glycans are labeled with 2-AB, and reaction products are analyzed by ultra-performance hydrophilic interaction liquid chromatography with fluorescence detection. FAQ Q: Can the enzyme be stored at other temperatures? A: EGCase I enzyme may be stored at 4°C for up to 1 month; -20°C for longer periods. Q: How do I optimize the reaction? A: Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Typical reaction conditions are as follows: incubate 2 nmol of glycosphingolipid with 6 mU (1 μL) of EGCase I for 16 hours at 37°C in 50 mM sodium acetate buffer, pH 5.2 containing 0.1% Triton X-100. Total reaction volume is 20 μL. More complex substrates may require additional EGCase I for optimal results. Q: What additional substrates are hydrolyzed by EGCase I? A: See substrates listed in Table 1 below, which was adapted from: Albrecht, S. et al. (2016) Analytical Chemistry 88, 4795-4802. Table 1. Substrate specificity of recombinant Endoglycoceramidase I The table below lists the quantities of glycans released from glycolipid standards (2 nmol) using 2 mU R. triatomea rEGCase I following incubation at 37°C for 16 hours. Substrate Glycolipid Structure Hydrolysis % Gangliosides GM3 Neu5Acα2-3Galβ1-4Glcβ1-1'Cer 100 GM1a Galβ1-3GalNAcβ1-4(Neu5Acα2-3) Galβ1-4Glcβ1-1'Cer 100 FGM1 Fucα1-2Galβ1-3GalNAcβ1-4 (Neu5Acα2-3)Galβ1-4Glcβ1-1'Cer 100 GD3 Neu5Acα2-8Neu5Acα2-3Galβ1-4 Glcβ1-1'Cer 100 GD1a Neu5Acα2-3Galβ1-3GalNAcβ1-4 (Neu5Acα2-3)Galβ1-4Glcβ1-1'Cer 100 GD1b Galβ1-3GalNAcβ1-4(Neu5Acα2-8 Neu5Acα2-3)Galβ1-4Glcβ1-1'Cer 100 GT1b Neu5Acα2-3Galβ1-3GalNAcβ1-4 (Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1'Cer 100 Globosides Gb4 GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1'Cer 100 Cerebrosides GlcCer Glcβ1-1'Cer 100 GalCer Galβ1-1'Cer 5-10 Sulfatide HSO3-3Galβ1-1'Cer 0 Psychosine Galβ1-1'sphingosine 0 LacCer Galβ1-4Glcβ1-1'Cer 100 Q: Can I purchase large amounts of an existing Enzyme for Innovation? A: Yes, if you have use for large amounts of an Enzyme for Innovation, please contact NEB at EnzymesForInnovation@neb.com. Please note that there may be limited availability and thus extended lead times required for large orders. Additional QC testing may also be necessary to meet your specific application and needs. Q: What are Enzymes for Innovation? A: Enzymes for Innovation (EFI) is a project initiated by NEB to provide unique enzymes to the scientific community in the hopes of enabling the discovery of new and innovative applications. These enzymes have interesting properties and unique specificities that are not commercially available elsewhere at the quality that you would expect from NEB. Enzymes for Innovation graduates are enzymes that have transitioned from having unknown or exploratory applications to becoming the cornerstone of a defined application. If you have an idea for an “Enzyme for Innovation” with a suggested application that may be useful, please email us at EnzymesForInnovation@neb.com. For more information, please view video.