Team:TU Darmstadt/Protocols/pNP Assay

pNP-Assay

pNP-assays are a common way to quantify hydrolytic activity. We use para-Nitrophenylbutyrate (pNPB) as a substrate. As the catalysts we use the enzymes FsC, pNB-Est13 or our transformed and induced bacteria.

Conditions: T = 34°C pH = 7.4

Method: Measurement of absorption at 405nm every minute over 30 minutes, after addition of enzyme. The released pNP-group has a typical absorbtion at 405nm wavelength. The absorbtion is meassured by an ELISA reader capable of 96 well plates.

The mechanism of pNPB degradation. The release of pNP is meassured by the characteristic absorption at 405 nm

Enzyme

Reagents

• A 1xPBS buffer (1M) pH 7.4 at 34°C (8g of NaCl, 0.2g of KCl, 1.44g of Na₂HPO₄ and 0.24g of KH₂PO₄ in 1L dest. H₂O
  
• B One of the primarily named substrates in an organic solvent
  
• 4-Nitrophenyl butyrate: 8,8µL in 1mL acetonitril and additional dilutions between 5mM and 50µM
  
• Bis(4-nitrophenyl) succinate: 36mg in 1mL DMSO diluted to a concentration of 5mM
  
• 4-Nitrophenyl (2E)-3-phenylacrylate: 27mg in methanol diluted to a concentration of 1mM
  
• C Enzyme stock solution
  

Procedure

1. 1mL of reagent A was added to 10µL of B and mixed by inversion.

   If Bis(4-nitrophenyl) succinate would be added, add 900µL of reagent A to 100µL of reagent B.
   

2. Now 100µL of the new solution were pipetted into several wells of a grainer 96 well plate.
   
3. To half the amount of wells xµL of enzyme solution were added to get the desired concentration of the respective enzyme.

   The volume of each enzyme-solution added was 6.6µL of Est13 or 6 µL of FsC respectively. So Est13 had a concentration of 50nM while FsC had a concentration 5nM.

4. The amount of absorption was measured 30 times, each minute one measuring on every filled well was run and recorded.

Bacteria

Reagents

• A 1xPBS buffer (1M) pH 7.4 at 34°C (8g of NaCl, 0.2g of KCl, 1.44g of Na₂HPO₄ and 0.24g of KH₂PO₄ in 1L dest. H₂O
  
• B 4-Nitrophenyl butyrate: 8.8 µL in 1mL acetonitril diluted to a concentration of 10mM
  
• C Bacteria with different concentrations of arabinose for induction (0.5%, 0.2%, 0.02% and 0%)

Procedure

1. A bacteria suspension with a OD of 0.1 is prepared from each bacterial culture induced by different arabinose concentrations (reagent C) in reagent A
   
2. 100µL of these four suspensions are added to several wells of grainer 96 well plate
   
3. To these 10µL of diluted reagent B are pipetted
   
4. From this moment on each minute to a total count of 30 minutes the respective suspensions' absorptions is recorded