Part:BBa_K3748002

Alpha amylase from Bacillus subtilis

Alpha amylase is a secreted protein that acts extracellularly. This enzyme do the catalysis of the endohydrolysis of (1->4)-alpha-D-glucosidic linkages in polysaccharides containing three or more alpha-(1->4)-linked D-glucose units. The product of the reaction are shorter chain saccharides that are then accesible to enter the cell where they can be used as a source of energy. Its native sequence is compose of its signal peptide and the sequence for the active enzyme. This enzyme is naturally secreted in B.subtilis.

Alpha amylase has been successfully secreted in S.cerevisiae, using pRNR1. Alpha amylase activity has been tested in a qualitative assay, the halo test, as well as in a quantitative one, in a colorimetric assay. If you want to know more go to https://2021.igem.org/Team:Groningen/Experiments.

Figure 1. Triplicates of halo test in sample SP027, alpha amylase from B.subtilis. Different products of the cells were used to compare the activity, a) Yeast cells, b) cells’ supernatant from liquid cultures and c) lysate from cells by bead beating them during 10s.

Figure 2. Increase in absorbance over time as a result of p-nitrophenol generation due to the cleavage of ethylidene-pNP-G7 (polysaccharide) by alpha amylase in the supernatant of S.cerevisiae overnight culture.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 730
  Illegal SpeI site found at 22
  Illegal PstI site found at 86
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 730
  Illegal SpeI site found at 22
  Illegal PstI site found at 86
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 730
  Illegal BglII site found at 1202
  Illegal BglII site found at 1372
  Illegal BamHI site found at 1222
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 730
  Illegal SpeI site found at 22
  Illegal PstI site found at 86
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 730
  Illegal SpeI site found at 22
  Illegal PstI site found at 86
  
• 1000
  COMPATIBLE WITH RFC[1000]