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Hydrolysis of Ester using orange peel esterase
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Materials Required

 

  • 0.1M phosphate buffer

 

  • Screw cap tube

 

  • Blender

 

  • 0.2M p- nitrophenyl acetate

 

  • Potassim permanganate solution in reagent spray bottle

 

  • Acetone

 

  • TLC plate

 

  • TLC chamber

 

  • Ferric chloride – potassium ferricyanide solution in reagent bottle

 

  • Vaccum filter

 

  • TLC solvent (70:30 petroleum ether and ethyl acetate)

 

  • Orange peel

 

  • Shaker

 

Procedure

 

1. The orange peel is collected from orange with a citrus zester

 

2. 40ml of 0.1M phosphate buffer is poured into it and the resulting mixture is homogenized via blender

 

3. The mixture is vacuum filtered over a thin layer of celite 545.

 

4. The blender was rinsed with 5- 10 ml of buffer and filtrate the material

 

5. The clear orange filtrate is collected in a beaker. The extract is stable for at least 72 hour at 2-8°C.

 

6. Two 5ml of screw topped vial is taken and the following two reactions are set up:

 

a) 6 drops pNPA solution + phosphate buffer, pH 7.0 for VT = 3ml

 

b) 6 drops pNPA solution + Orange peel filtrate for VT = 3ml

 

7. The two reaction mixtures are capped and allowed to stir

 

8. The reactions are monitored by TLC every 5 minutes

 

 

 

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