Fig 3: SDS-PAGE analysis by Coomassie Brilliant Blue staining
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The dark protein bands are observed slightly above the 33 kDa ladder which is equal to the expected size of the enzyme catechol-2,3-dioxygenase of 35 kDa. {ref.https://www.ncbi.nlm.nih.gov/pubmed/8713131}
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<strong>2-HMS Assay:</strong>
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In this assay, 2-Hydroxymuconate Semialdehyde (2-HMS) is detected, which is the degradation product of catechol, catalysed by enzyme catechol-2,3-dioxygenase (xylE). This product has a characteristic λmax at 380 nm. Here in this assay, we used 415 nm and a range of catechol concentration (0.1 - 0.4 mM) for 2-HMS detection at intervals of 5 mins where total volume of test was 100 uL with the prepared culture suspension (18-24 hrs old) of 0.5 OD.
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pBAD_xylE with Arabinose as substrate (pBAD is a arabinose induced promoter)
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Absorbance readings at 415 nm at different time intervals for range of catechol from 0.1 mM to 0.4 mM
From the above 3D graphs of Absorbance v/s Time of the reaction system, with respect to increasing concentration of catechol in the range of 0.1 mM to 0.4 mM, it can be interpreted that-
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<li>With advancement in time, the absorbance of the system increases.</li>
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<li>With increasing catechol concentration, the absorbance value increases till 0.35 mM catechol concentration.</li>
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<li>After the concentration of 0.35 mM of catechol, the absorbance readings are found to decrease, which can be suggested from the fact that catechol at high concentrations is toxic for the cell growth.</li>
The part codes for the enzyme catechol-2,3-dioxygenase which cleaves the oxidative ring of catechol to form 2-hydroxymuconate semialdehyde. It also contains a 6X His-tag which is a useful marker for purification and identification of the protein of interest.
Structure:
Fig1: 3D-Structure of enzyme catechol-2,3-dioxygenase
