Part:BBa_K620001

WT-F87A (p450)

A wild-type p450 with a mutation at the 87th amino acid. It is confirmed to degrade bisphenol A (BPA), but as it is a promiscuous protein, it can likely bind and degrade other organic molecules.

Usage and Biology

Plasmid map of K620001 in pSB1C3 (submission plasmid).

GCMS-electrospray of BPA showing characteristic peak at 227g/mole

This image shows the GCMS of BPA before degradation, in which the mass of BPA (227g/mole) is clearly visible.

GCMS of BPA after reaction with the WT-F87A P450 that shows a degraded peak at 205 g/mole

This image shows BPA after a reaction was conducted with this p450. The p450 was expressed in cells and extracted. Then a reaction was prepared with 5 microliters of 20mM BPA in DMS0, 12 microliters of a 5 micromolar p450 solution, 20 microliters glucose, and 2 microliters GDH, in 161 microliters 1M KPI buffer. This reaction was performed for 4 hours and analyzed using GCMS. Here, there is an apparent large 205g/mole peak, and the 227 g/mole peak is greatly reduced. This indicates that BPA is being reduced to several structures weighing 205 g/mole.

HPLC of BPA, without enzyme added

HPLC of BPA degraded, with p450 Wt-F87A

As shown in the HPLC results, the plain BPA HPLC has only one peak, while the HPLC analysis of BPA degraded with the WT-F87A cytochrome p450 has two. 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 AgeI site found at 75
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1219
  Illegal SapI.rc site found at 2242
  Illegal SapI.rc site found at 2842