Difference between revisions of "Part:BBa K2317007"

Latest revision as of 02:42, 2 November 2017

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 wider substrate range and higher catalysis activity.
Sequence and Features

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 wide 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