Part:BBa_K590023

Kumamolisin-As_N291D

A mutated Kumamolisin-As enzyme aimed to break down gluten by increased activity with the PQLP peptide, an antigenic epitope in gliadin.

Usage and Biology

This part was constructed by the [http://2011.igem.org/Team:Washington 2011 University of Washington] iGEM team to break down gluten, the primary cause of Celiac's disease. To test BBa _K590021, it was inserted into a protein expression vector, pET29b+. Kumamolisin-As_N291D was then produced and purified as described in the [http://2011.igem.org/Team:Washington/Protocols/50mL_UW 2011 iGEM Team's Small Scale Protein Expression and Purification Protocol]. The purified protein was then tested for activity. For a detailed description of the assay, please see the [http://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay 2011 UW iGEM Purified Enzyme Assay Protocol]. The resulting data is shown below.

The relative activity plot was produced by measuring the quantity of PQLP peptide cleaved per enzyme per hour. Error bars represent a 95% confidence interval from triplicate data.

Mutation(s)	kcat/KM (M-1 s-1)
N291D	(5.21 ± 0.11) x 105
S354N, D358G, D368H	(2.45 ± 0.02) x 105
G319S, D358G, D368H	(2.02 ± 0.02) x 105
G319S, D358G, D368H, N291D (KumaMax)	(3.86 ± 0.04) x 106
wt-Kumamolisin	(3.25 ± 0.05) x 104
SC-PEP	(4.96 ± 0.75) x 103

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1696
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 447
  Illegal NgoMIV site found at 720
  Illegal NgoMIV site found at 1248
  Illegal NgoMIV site found at 1494
  Illegal AgeI site found at 772
  Illegal AgeI site found at 1348
  Illegal AgeI site found at 1588
  
• 1000
  COMPATIBLE WITH RFC[1000]