Part:BBa_K2317007

TfdB-JLU

TfdB-JLU is a novel 2,4-dichlorophenol hydroxylase whose amino acid sequence exhibits less than 48% homology with other known TfdBs. Compared to wild type TfdB, TfdB-JLU has a wilder substrate range and higher catalysis activity.
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 262
  Illegal NgoMIV site found at 925
  Illegal NgoMIV site found at 951
  Illegal NgoMIV site found at 1036
  Illegal NgoMIV site found at 1576
  Illegal NgoMIV site found at 1767
  
• 1000
  COMPATIBLE WITH RFC[1000]

Experiment

Because substract and production of tfdB-JLU share close maximum absorbance, along with tfdB-JLU is a NADPH dependent enzyme, we determined enzyme activity by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH.

1. Overexpression and purification of TfdB-JLU

1.1 Digestion assay

  To certify succeed in constructing pET28a-tfdB-JLU, digestion assay using BamHI and XbaI was performed. The product digested by BamHI and Xbal was around 1800bp as predicted 1776bp of tfdB-JLU.

Figure 1. Digestion assay of pET28a-tfdB-JLU

1.2 Overexpression and purification of TfdB-JLU
  We used optimized protocol to overexpress and purify enzyme, the purified enzyme migrated as a single band with an Mw of 63 kDa on SDS-PAGE, close to the predicted 66.9 kDa Mw of TfdB-JLU plus the 6xHis-tag.

Purified TfdB-JLU was eluted in 250mM imidazole elution buffer (lane 7, lane 8)
Lane 1: crude extract
Lane 2: flow through
Lane 3: 20 mM imidazole washing buffer
Lane 4: 50 mM imidazole washing buffer
Lane 5, 6: 100 mM imidazole washing buffer
Lane 7, 8: 250 mM imidazole elution buffer
Lane 9: 500 mM imidazole buffer

Figure 2. SDS–PAGE analysis of the expressed TfdB-JLU enzyme

1.3 substrate activity and preference of TfdB-JLU   TfdB-JLU has a wild substract preference among phenolic components. The activities of chlorophenol hydroxylases were determined by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH (Ledger et al. 2006). Unless otherwise indicated, standard enzyme activity assays were performed by incubating the purified enzyme with 0.1 mM 2,4-DCP and 0.2 mM NADPH in 50 mM sodium phosphate buffer (pH 7.5) at 25℃ in 1ml.

For determining substrate specificity, the enzyme was incubated in 50 mM sodium phosphate buffer, pH 7.5, with 0.1 mM substrate under standard conditions. Relative activity is expressed as a percentage of the maximum enzyme activity towards 2,4-DCP with FAD.

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Reference

[1] Ledger T, Pieper DH, Gonzalez B, Chlorophenol hydroxylases encoded by plasmid pJP4 differentially contribute to chlorophenoxyacetic acid degradation. Appl Environ Microbiol 2006 72:2783–2792