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Hydrolysis of Ester using orange peel esterase


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




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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