Ludger Acetyl Esterase enzyme structure

    LudgerZyme Acetyl Esterase (sialate-O-acetylesterase) Kit


    LZ-ACASE-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.


      Product guide

      Certificate of stability

    • Part Number
    • LZ-ACASE-KIT
    • Amount of Enzyme
    • 50 µL









    Kit includes enzyme plus reaction buffer.
    Sufficient for up to 50 samples.





Example of exoglycosidase sequencing using LZ-ACASE-KIT followed by HILIC HPLC profiling


Ludger Exoglycosidase Acetyl Esterase Data

Procainamide labelled N-glycans released from erythropoietin were treated with O-acetyl esterase to expose and quantify glycans decorated
with O-acetylation on sialic acid.
 1





LudgerZyme Acetyl Esterase Kit


Acetyl esterase (sialate-O-acetylesterase) is a recombinant protein from Tannerella forsythia, ATCC 43037 strain, expressed in Escherichia coli. The enzyme removes acetyl groups attached via an O- group, mainly 9-, 8- and 7-. It can be used for monitoring of diacetylation of sialic acids on products such as erythropoietin (EPO)

Application: Acetyl esterase (sialate-O-acetylesterase) can be used to remove 9-, 8- and 7-O-acetyl groups from released sialic acids, released glycans or glycoproteins.

Source: Recombinant from Tannerella forsythia in E. Coli.

EC: 3.1.1.53

Contents:
Ludger sialate-o-acetylesterase - Kit Contents
LudgerZyme Acetyl Esterase (Supplied in PBS pH7.5 buffer containing 10 mM Tris-HCl)
LudgerZyme Acetyl Esterase RXN 10x buffer (500 mM sodium acetate pH5.5)

Number of Samples: Sufficient for up to 50 samples.

Amount of Sample: Up to 10 µg glycoprotein, up to 2.5 µg released glycans and up to 1 µg free sialic acid per digestion.

Suitable Samples: Acetyl esterase (sialate-O-acetylesterase) can act upon complex glycoprotein samples, such as erythropoietin (EPO), bovine submaxillary mucin and oral epithelial cell-bound glycans, and on N- and O-glycans released from a glycoprotein. Either fluorescently labelled or unlabelled glycans are suitable. It can also be used on released sialic acids.

Unit Definition: One unit (U) of acetyl esterase is defined as the amount of enzyme required to produce 300 µmole of 4-nitrophenol and acetate in 1 minute at 30°C in a buffer containing 50 mM Tris-HCl, 140 mM NaCl, pH 8.5, from 4-nitrophenyl acetate, a chromogenic esterase substrate.

Molecular Weight: 76.3 kDa.

Suggested usage:
1. To digest <10 µg of glycoprotein, make up a total reaction volume of 10 µL by dissolving the glycoprotein in 1 µL of LudgerZyme Acetyl Esterase RXN 10x buffer, 1 µL of LudgerZyme Acetyl Esterase and water to a final volume of 10 µL.
Use the same method for digestion of <2.5 µg released glycans or <1 µg free sialic acid.
The reaction may be scaled-up linearly to accommodate larger amounts of glycoprotein, released glycans or free sialic acids.

2. Incubate your samples in a water bath, oven or any other constant heating source at 37°C for 7 to 16 hours.
Note: Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate.
The samples are now ready for clean-up and subsequent analysis by HPLC.
Note: Optionally, the samples can be stored frozen at this point for analysis at a later date.

Storage: Store at 4°C. Protect from sources of heat and light. When stored correctly, the enzyme should be stable for 24 months from date of purchase. Exposure to ambient temperatures (20 - 26°C) over 3 days does not result in a reduction of enzymatic activity.





Companion Products:

Process controls - View our N-glycan Nomenclature Table

  • CM-SRP-01
  • Sialic acid reference panel, contains Neu5Gc, Neu5Ac and its O-acetylated derivatives

Glycan clean-up - removal of enzymes from glycan mixtures following exoglycosidase treatment

  • LC-EXO-A6
  • LC-EXO-96
  • LudgerClean Post-Exoglycosidase Clean-up Spin Columns (6 samples)
  • LudgerClean Post-Exoglycosidase Clean-up Plate (96 samples)

Labelling and derivatisation of released glycans - View our Glycan Labelling Summary Table

  • LT-KAB-A2
  • LT-KAB-VP24
  • LT-KAB-VP96
  • LT-KPROC-24
  • LT-KPROC-96
  • LT-KPROC-VP24
  • LT-KAA-A2
  • LT-KAA-VP24
  • LT-PERMET-96
  • LT-PERMET-VP96
  • LudgerTag 2-AB Glycan Labelling Kit, sodium cyanoborohydride (24 samples)
  • LudgerTag 2-AB Glycan Labelling Kit, picoline borane (24 samples)
  • LudgerTag 2-AB Glycan Labelling Kit, picoline borane (96 samples)
  • LudgerTag Procainamide Glycan Labelling Kit, sodium cyanoborohydride (24 samples)
  • LudgerTag Procainamide Glycan Labelling Kit, sodium cyanoborohydride (96 samples)
  • LudgerTag Procainamide Glycan Labelling Kit, picoline borane (24 samples)
  • LudgerTag 2-AA Glycan Labelling Kit, sodium cyanoborohydride (24 samples)
  • LudgerTag 2-AA Glycan Labelling Kit, picoline borane (24 samples)
  • LudgerTag Permethylation Kit (96 samples)
  • LudgerTag Permethylation Kit, without methyl iodide (96 samples)

Alternative enzymes

  • E-S001
  • E-S005
  • E-S007
  • Sialidase Au α(2-3,6,8,9)
  • Sialidase Cp α(2-3,6)
  • Sialidase Sp α(2-3)






References:

1. Phansopa C, Kozak RP, Liew LP, Frey AM, Farmilo T, Parker JL, Kelly DJ, Emery RJ, Thomson RI, Royle L, Gardner RA, Spencer DI, Stafford GP. Characterization of a sialate-O-acetylesterase (NanS) from the oral pathogen Tannerella forsythia that enhances sialic acid release by NanH, its cognate sialidase. Biochem J. 2015 Dec 1;472(2):157-67. doi: 10.1042/BJ20150388. Epub 2015 Sep 16.

2. Thomson RI, Gardner RA, Strohfeldt K, Fernandes DL, Stafford GP, Spencer DIR, Osborn HMI. Analysis of Three Epoetin Alpha Products by LC and LC-MS Indicates Differences in Glycosylation Critical Quality Attributes, Including Sialic Acid Content. Anal Chem. 2017 Jun 20;89(12):6455-6462. doi: 10.1021/acs.analchem.7b00353. Epub 2017 Jun 9.