Origin

Escherichia coliJM109 carrying the plasmid encoding acylglucosamine 2-epimerase (AGE) gene from hog kidney.※1

Reaction

N-acetyl-D-glucosamine N-acetyl-D-mannosamine
AGE can tightly bind to renin and inhibit the renin activity.※1-※2

Appearance

Lyophilized powder.

Activity

More than 30 units/mg protein.

Unit definition

One unit of enzyme activity is the quantity that produced 1 μmol of N-acetyl-D-glucosamine per minute at 25 ºC at pH8.9 using N-acetyl-D-mannosamine as a substrate.

Storage

Stable for one year when stored below 5 ºC.
For prolonged storage, keep at -20 ºC.

Properties

Molecular weight	Approx. 93,000 Da (sedimentation equilibrium)
Subunit	46,600 (SDS-PAGE)
Optimum pH	6.8
Optimum temperature	47℃
pH stability	6.0～8.0
Thermal stability	below 37 ºC
Activator	ATP (or dATP) and MgCl2
Substrate specificity	N-acetyl-D-glucosamine and N-acetyl-D-mannosamine
Michaelis constant	7.4x10-6M (N-acetyl-D-glucosamine) 6.3x10-6M (N-acetyl-D-mannosamine) 1.8x10-7M (ATP)

Assay method

Assay system is composed of 10 µl of 1 M Tris-HCl (pH7.4), 40 µl of 0.1 M N-acetyl-D-mannosamine, 4 µl of 0.1 M ATP, 10 µl of 0.1　M MgCl2, and 36 µl of enzyme solution. Reaction is carried out at 37 ºC for 30 minutes, then stop by boiling for 3 minutes. Determination of the reaction product (N-acetyl-D-glucosamine) can be carried out by N-acetyl-D-glucosamine-PMP(1-phenyl-3-metyl-5-pyrazorone) HPLC method.※1-※3
[Note] Dilute the enzyme with 20 mM potassium phosphate buffer(pH7.2) -1 mM EDTA-0.02% 2-mercaptoethanol-0.1%(w/v)bovine serum albumine.

References

1. ※1. I. Maru, Y. Ohta, K. Murata and Y. Tsukada, J. Biol. Chem., 271, 16294 (1996)
2. ※2. S. Takahashi, T. Ohsawa, R. Miura and Y. Miyake, J. Biochem., 93, 265 (1983)
3. ※3. S. Honda, E. Akao, S. Suzuki, M. Okuda, K. Kakehi and J. Nakamura, Anal. Chem., 180, 351 (1989)

Package

1 unit, 5 units