Coding

Part:BBa_K3895005

Designed by: Rui Zhu   Group: iGEM21_SZ_SHD   (2021-10-01)


Keratinase kerBlMKU3

kerBlMKU3 is a serine keratinase originally secreted by Bacillus licheniformis MKU3. It can work at 37°C for 48 h in nutrient broth or on feather medium [1].

T--SZ SHD--KUO.png

6x His-tags were added to both sides of the sequence for purification, and constructed into PET-28a(+) (BBa_K3895009). The length of kerBlMKU3 is 1202 bp.

Figure 1. Plasmid construction of kerBlMKU3.
Figure 2. PCR and gel electrophoresis of kerBlMKU3.
Name of keratinase IPTG concentration/mM Temperature/°C Time/h
KerBIMKU3 0.4 37 4

Modeling

The biological activity of a protein is not only determined by the primary structure of the protein molecule but also closely related to its specific spatial structure. Elucidating the process of protein folding in functional and structural details will be of great significance to evaluate their keratinotic functions. According to the amino acid sequences in the enzyme active sites and associated catalytic mechanisms, proteases can be classified into seven broad groups: serine, cysteine, threonine, aspartic and glutamic proteases, metalloproteases and asparagine peptide lyases. According to the nature of their active site, keratinases belong to serine- and metalloproteases or serine metalloproteases. Moreover, the enzymatic degradation of keratin is a multistage process that requires the following steps: adsorption of the keratinases to the surface of macromolecule by electrostatic and hydrophobic interactions, followed by catalytic action [2]. Hence we used Swiss Model to predict their hydrophobic region and serine groups.


Figure 1. Swiss Model showing serine residues of kerBlMKU3.
Figure 2. Swiss Model showing hydrophobisity of kerBlMKU3.
Figure 3. Ramachandran Plot of kerBlMKU3.KU3 Ramachandran Favoured is 91.25%


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 733
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 583
  • 1000
    COMPATIBLE WITH RFC[1000]



References

Radha, S., & Gunasekaran, P. (2007). Cloning and expression of keratinase gene in Bacillus megateriumand optimization of fermentation conditions for the production of keratinase by recombinant strain. Journal of Applied Microbiology, 103(4), 1301–1310. https://doi.org/10.1111/j.1365-2672.2007.03372.x

Vidmar, Beti, and Maša Vodovnik. “Microbial Keratinases: Enzymes with Promising Biotechnological Applications.” Food technology and biotechnology vol. 56,3 (2018): 312-328. doi:10.17113/ftb.56.03.18.5658

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