1. Phenol Management
By: Van Huynh, Summervir Cheema
California State University, East Bay (Department of Chemistry and Biochemistry)
ACS SEED 2013

2. Bisphenol A (BPA)
• Common in everyday
materials (plastic & cans)
causing continuous
exposure
• Similar to hormones in the
human body (e.g. estrogen)
• Disruption of the endocrine
system results in serious
health issues.

3. Strategy for Phenol Removal in Water Samples
Chemisorption
Chitosan Collected into a more contained solid waste
Oxidation Polymerization
Polyphenol Oxidase (PPO) converts phenols into quinones Quinones Melanins (Slow process)
Phenol Contamination in the Environment
Phenol (BPA) In waste water or leakage from landfills
Literature Examples: Yoshida, Mitsuru, et al. "Oxygenation of bisphenol A to quinones by polyphenol oxidase in
vegetables." Journal of agricultural and food chemistry 50.15 (2002): 4377-4381.
Sun, Wei Qiang, et al. "Tyrosinase reaction/chitosan adsorption for removing phenols from wastewater." Biotechnology
progress 8.3 (1992): 179-186.

4. Polyphenol Oxidase (PPO)
• PPO are enzymes located at the
plastids and the mitochondria
• Catalyze the reaction between
phenols and oxygen to create
colored compounds called
quinones which gradually turn to
melanins
• Responsible for the browning
reaction of cut vegetables and
fruits
Phenols
Example:
Catechol
(Substrate)
PPO
(enzyme)
ortho-
benzoquinone

5. Chitosan
• Produced from crustacean’s shells
and seashells
•Cheap to obtain and vastly available
•Insoluble in basic or neutral
solutions, only soluble in acidic
solutions
•Amino-group (-NH2) can react with
and capture quinones

6. Preparation of Crude Plant Extracts
Trader Joe’s Gold Chopped potato (99 g) was placed in a
blender with 200mL extraction buffer
Extraction buffer: 0.1 M sodium phosphate pH 6.8 with
1 % v/v Triton X100 and 1 % w/v polyvinylpyrrolidone
The material was filtered through a cheese cloth and
centrifuged. All solutions were kept at 4C.

7. Method Development with Catechol
Catechol
Catechol,
Potato
Catechol,
Potato,
Chitosan
Catechol,
Chitosan
Technical Details: 50 mM catechol in 0.1 M sodium phosphate buffer pH 6.8 with or without 0.01 mL potato extract
(corresponding to 5 mg raw potato), and 0.025 g chitosan flakes. Incubation time one hour. Ten-fold more potato
extract corresponds to 50 mg raw potato.

8. Method Development with Catechol
Catechol
Catechol,
Potato
Catechol,
Potato,
Chitosan
Catechol,
Chitosan Catechol
Catechol,
10X Potato
Catechol,
10X Potato,
Chitosan Water
Technical Details: 50 mM catechol in 0.1 M sodium phosphate buffer pH 6.8 with or without 0.01 mL potato extract
(corresponding to 5 mg raw potato), and 0.025 g chitosan flakes. Incubation time one hour. Ten-fold more potato
extract corresponds to 50 mg raw potato.

9. BPA Removal
Technical Details:1 mM bisphenol A in 0.1 M sodium phosphate buffer pH 6.8 with or without 0.01 mL potato extract
(corresponding to 5 mg raw potato), and 0.025 g chitosan flakes. Ten-fold more potato extract corresponds to 50 mg
raw potato.
BPA
Potato,
BPA
Potato, BPA,
Chitosan
BPA,
Chitosan BPA
10 fold Potato,
BPA
10 fold Potato,
BPA, Chitosan
10 fold
Potato

10. BPA oxidation can be monitored with UV/VIS
spectroscopy
-0.1
0.4
0.9
1.4
1.9
2.4
2.9
250 300 350 400 450 500
Absorbance
Wavelength (nm)
Buffer: 0.4 M sodium phosphate pH 7.2
1 mM BPA in buffer
0.6 mL Potato extract and 2.4 mL buffer
0.6 mL Potato extract and 1 mM BPA in
buffer after 30 min. incubation
380nm

11. BPA oxidation can be monitored with UV/VIS
spectroscopy
-0.1
0.4
0.9
1.4
1.9
2.4
2.9
250 300 350 400 450 500
Absorbance
Wavelength (nm)
Buffer: 0.4 M sodium phosphate pH 7.2
1 mM BPA in buffer
0.6 mL Potato extract and 2.4 mL buffer
0.6 mL Potato extract and 1 mM BPA in
buffer after 30 min. incubation
Our spectra are similar to:
Yoshida, Mitsuru, et al.
"Oxygenation of bisphenol A to
quinones by polyphenol
oxidase in vegetables." Journal
of agricultural and food
chemistry 50.15 (2002): 4377-
4381.
380nm

12. Potato extract control:
0.6 mL potato extract in
3 mL 0.1 M sodium
phosphate buffer pH 6.8
(corresponding to 0.3 g
potato)
Potato extract and BPA:
BPA control:
0.8 mM BPA in 0.1 M
sodium phosphate
buffer pH 6.8
Slope
ΔAbs/min =
0.0057/min;
13 nanomol BPA are
oxidized in one minute
by 1 mL potato extracttime
Absorbanceat380nm

13. Chitosan Flakes
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
250 300 350 400 450 500 550
absorbance
wavelength (nm)
BPA+ potato extract+chitosan
flakes (1 day)
BPA + potato extract

14. Optimization
How much potato
extract ?
How much Chitosan?
Reaction conditions
(pH, T)

15. Optimization
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
30 35 40 45 50
Abs(380nm)/min(initialrate)
µL potato extract
Potato Extract
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
5.9 6.1 6.3 6.5 6.7 6.9 7.1 7.3Abs/min
pH
pH Variation
BPA and potato
extract

16. Preparation of Magnetic Chitosan Beads
0.5 g Chitosan flakes were dissolved in
50 mL 1.5% v/v acetic acid.
1 g magnetite (iron(II/III)oxide) was
added.
Black, viscous solution was dripped
into a 1M KOH, 25% ethanol solution.
Singh, Abhay Narayan, et al. "Glutaraldehyde-activated chitosan matrix for immobilization of a novel
cysteine protease, Procerain B." Journal of agricultural and food chemistry 59.11 (2011): 6256-6262.
Šafaříková, Mirka, and Ivo Šafařík. "One-step partial purification of Solanum tuberosum tuber lectin using
magnetic chitosan particles." Biotechnology letters 22.11 (2000): 941-945.

17. Chitosan beads
UV/VIS spectra Magnetic Chitosan Beads
0
0.5
1
1.5
2
2.5
3
250 300 350 400 450 500
absorbance
wavelength (nm)
0.6 mL potato extract in 1 mM
BPA and 60 mM sodium
phosphate buffer pH 7.2
addition of magnetic chitosan
beads (10 minutes)
addition of magnetic chitosan
beads (20 minutes)

18. Extra Slides

19. Acknowledgements
•Thank You for
Everything

20. Preparation of Crude Plant Extracts
•99g chopped potato was placed in a blender with 200mL extraction buffer (0.1 M sodium
phosphate pH 6.8 with 1 % v/v Triton X100 and 1 % w/v polyvinylpyrrolidone).
•The material was filtered through a cheese cloth and centrifuged. All solutions were kept at 4C.
Plant matter /
extraction
buffer
Total protein
content in
extract
Total
protein
content per
g potato
PPO activity
in extract
PPO activity
per g potato
0.5 g/mL
6.24 ± 0.136
mg/mL
12.61 ± 0.27
mg/g
14.75 ± 1.44
IU/mL
29.8 ± 2.9
IU/g
Bradford Assay Activity Assay
•One IU (International Unit) corresponds to the formation of one micromole quinone per one
minute.

21. Purification of PPO from Potato
Purification Stage Separation Principle Specific Activity (IU/mg total
protein)
Crude Extract None 2.7
Ammonium sulfate precipitation
and dialysis
Solubility 3.4
Ion exchange chromatography
using SE-Sephadex
Charge 8.6
Activity was determined with the substrate catechol. One IU (International Unit) corresponds to the
formation of one micromole quinone per one minute. Protein content was determined with the
Bradford assay.