Part:BBa_K1216003

beta-N-Acetylglucosaminidase (nagZ) from Escherichia Coli

nagZ encodes β-N-Acetylglucosaminidase, a cytoplasmatic hydrolase which is involved in the murein tripeptide recycling pathway of E.coli^[1].

3D representation of the β-N-Acetylglucosaminidase from [http://www.rcsb.org/pdb/explore/explore.do?structureId=4GVG RCSB]

Usage and Biology

β-N-Acetylglucosaminidase can be used as a reporter enzyme in a histochemical reaction with substrates like 5-Bromo-4-Chloro-3-Indolyl-N-Acetyl-β-D-Glucosaminide, which will after hydrolysis release a blue chromophore^[2].

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 25
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 76

Characterization

The final construct was sequenced.

Figure 1. Liquid culture from E.Coli overexpressing NagZ after reacting with pNP-GlcNac.

[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used NagZ in their project as reporter enzyme. To test the functionality of the enzyme, liquid culture of E.Coli overexpressing NagZ was incubated with pNP-GlcNac.

Figure 2. Enzymatic reaction of NagZ with pNP-GlcNac.

Cell lysate would have been tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-N-acetyl-β-D-glucosaminide. Unforunately this did not work until the wiki freeze.

Figure 4. Enzymatic reaction of NagZ with 4-MU-N-acetyl-β-D-glucosaminide.

References

Cheng et al. (2000). Molecular Characterization of the b-N-Acetylglucosaminidase of Escherichia coli and Its Role in Cell Wall Recycling. Journal of Bacteriology. 182: 4836-4840.
[http://www.inalcopharm.com/cart.php?m=product_detail&p=6 Inalcopharm datasheet]