In the present work, polyphenol oxidase (PPO) enzyme was purified from potato peel using three-phase partitioning (TPP). In this method, ammonium sulfate and t-butanol were added to precipitate the protein/enzyme from the crude aqueous extract. The PPO enzyme precipitated as an interfacial layer between the upper organic solvent phase and lower aqueous phase. Different purification parameters such as crude extract to t-butanol ratio, ammonium sulfate concentration, temperature, and pH were optimized for TPP. About 69% PPO enzyme activity was recovered in a single step of TPP with 9.2-fold purification. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of partially purified PPO enzyme showed molecular weight in the range of about 30-40 kDa. The PPO enzyme was then investigated for the fabrication of a portable, cost-effective, and disposable colorimetric paper biosensor or colorimetric "test strips" for detection of phenolic contaminants. PPO and a chromophore reagent (3-methyl-2-benzothiazolinone hydrazine) generated a range of color in the presence of phenolic compounds (catechol, phenol, p-cresol, 4-methyl catechol) within 15 min, and limit of detection was found to be 0.5 μM. The biosensor worked in a broad range of pH from 3 to 11 and showed good storage stability at 25 °C and 4 °C for 30 days with no significant loss of activity. The biosensor was also applied on environmental water and urine sample to show reliability of biosensor.
Keywords: Biosensor; Catechol; Phenols; Pollutants; Polyphenol oxidase; Three-phase partitioning.