Coding

Part:BBa_K1175007

Designed by: Nicholas K. Goldner   Group: iGEM13_WLC-Milwaukee   (2013-09-17)

beta-galacturonidase (YesZ) from Bacillus Subtilis 168

yesZ The enzyme acts on the terminal end of side chains of Rhamnogalacturonan I Pectin, releasing the free galactose. This gene has been isolated from the bacterium Bacillus subtilis subtilis 168. The enzyme yesZ has beta-galactosidase (beta-galacturonidase) activity, cleaving (1→4)-β-D-galactans to produce single galactose molecules from RG I Pectin.

Usage and Biology Pectin is a component of the primary cell wall of plants. As galactose can be metabolized by the human body and by animals, it is an alternative to the usual target of nutrition/energy boosts in biotechnology, glucose.

(1) http://www.ncbi.nlm.nih.gov/pubmed/17449691 (2) http://subtiwiki.uni-goettingen.de/wiki/index.php/YesZ (3) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1694227/ (4) http://aem.asm.org/content/73/12/3803 (5) http://www.tandfonline.com/doi/full/10.1080/15583724.2011.615962#tabModule

yesZ

The Beta-Galactosidase yesZ is globular and has three aspects that are important to its function. It contains a zinc ion binding site at the C residues 153, 155, and 158 in yellow. The putative nucleophile and acid base cleavage sites are at the E residues 155 and 296 in red. The active site where substrate binding occurs is through the amino acid residue sequence ETSPSYAASL from residues 296- 305.

yesZ Enzyme Activity

 The kinetic parameters fo the hydrolysis of pNPGal by wild type YesZ Kcat = 81.4 ± 4 s-1, and Km= 3.0 ± 0.2 mM, and Kcat/Km= 27 ± 2 mM -1 s-1. Figure A describes the time dependent inactivation of the YesZ enzyme using DNP2FGal. The time dependent inactivation observed as a single exponential decay to a non-zero value when DNP2FGal was incubated with the enzyme. Studies of inactivation kinetics were performed by pre-incubating 100 microliters of the enzyme at 37 oC with DNP2FGal at a range of concentrations in a total volume of 140 microliters.">

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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 260
    Illegal NgoMIV site found at 395
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 559


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