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A COMPARATIVE STUDY TO DETERMINE IF TURNIPS CAN BE USED AS AN ALTERNATIVE SOURCE OF PEROXIDASES TO HORSERADISH $9.53   Add to cart

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A COMPARATIVE STUDY TO DETERMINE IF TURNIPS CAN BE USED AS AN ALTERNATIVE SOURCE OF PEROXIDASES TO HORSERADISH

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A COMPARATIVE STUDY TO DETERMINE IF TURNIPS CAN BE USED AS AN ALTERNATIVE SOURCE OF PEROXIDASES TO HORSERADISH

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  • August 12, 2021
  • 36
  • 2021/2022
  • Thesis
  • Katrine wallis
  • Unknown
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Henna Yahya 4837224




A COMPARATIVE STUDY
TO DETERMINE IF
TURNIPS CAN BE USED AS
AN ALTERNATIVE SOURCE
OF PEROXIDASES TO
HORSERADISH




BY HENNA YAHYA
Coventry University
Priory St,
Coventry
CV1 5FB


1

,Henna Yahya 4837224


Contents
1. Introduction .......................................................................................................................................... 4
2. Methodology......................................................................................................................................... 8
2.1 Materials ............................................................................................................................... 8
2.2 Preparation of sodium phosphate buffer at pH 7 .................................................................. 8
2.3 Bradford Method .................................................................................................................. 8
2.3.1 Protein estimation using Bradford Method ....................................................................... 8
2.3.2 Determination of protein concentration of horseradish and turnip extracts ........ 8
2.4 Enzyme assay using ABTS as substrate ............................................................................... 9
2.5 Determination of horseradish peroxidase activity ................................................................ 9
2.6 Peroxidase extraction from vegetables ............................................................................... 10
2.7 Ammonium Sulphate Precipitation .................................................................................... 10
2.7.1 Preparation of ammonium sulphate pilot study ................................................. 10
2.7.2 Ammonium sulphate pilot study ....................................................................... 10
2.7.3 Ammonium sulphate precipitation of vegetable extract .................................... 11
2.8 Gel Filtration of turnip and horseradish ............................................................................. 12
2.9 Calculations for purification table ...................................................................................... 12
2.10 SDS-PAGE ....................................................................................................................... 13
2.11 Effect of temperature on peroxidases ............................................................................... 13
3. Results ................................................................................................................................................ 14
3.1 Determination of protein concentration .............................................................................. 14
3.2 Commercial horseradish peroxidase activity ...................................................................... 14
3.3 Turnip and horseradish peroxidase activity in crude extract .............................................. 15
3.4 Ammonium sulphate precipitation ..................................................................................... 16
3.4.1 Ammonium sulphate pilot study ....................................................................... 16
3.4.2 Ammonium sulphate precipitation of horseradish and turnip ........................... 19
3.5 Gel Filtration of turnip and horseradish ............................................................................. 19
3.6 Purification table ................................................................................................................ 20
3.7 SDS-PAGE ......................................................................................................................... 22
3.8 Effect of temperature on activity of peroxidase ................................................................. 24
4. Discussion .......................................................................................................................................... 26
4.1 Bradford Method ................................................................................................................ 26
4.2 Enzyme assay using ABTS as substrate ............................................................................. 26
4.3 Extraction of peroxidases from vegetable sources ............................................................. 27
4.4 Ammonium sulphate pilot study ........................................................................................ 27
4.5 Ammonium sulphate precipitation of vegetable extracts ................................................... 28
4.6 Gel filtration of vegetable extracts ..................................................................................... 28
4.7 Purification table ................................................................................................................ 29
4.8 SDS-PAGE ......................................................................................................................... 30
4.9 Effect of temperature on peroxidase activity ...................................................................... 31
4.10 Limitations and Improvements ......................................................................................... 31
4.11 Conclusion........................................................................................................................ 32
References .............................................................................................................................................. 34
FINAL WORD COUNT: 5434




2

,Henna Yahya 4837224



ACKNOWLEDGEMENTS

In the name of Allah, the most gracious, the most merciful



I would like to thank my project supervisor Dr Katrine Wallis for being a supportive

supervisor and always promptly responding to my numerous emails. I would also like

to thank the lab technicians Angela and Patrick for telling me where all the equipment

was in the lab



I especially want to thank Hafsah, Zoe and Ash for their continuous support and for

putting up with my constant moaning about horseradish and turnips. Without these

three I wouldn't have got through the data collection period.




3

, Henna Yahya 4837224



ABSTRACT

Horseradish is a recognised source of peroxidase and has many commercial uses and

research is being done on other sources of peroxidases. Peroxidase was extracted and

assessed from turnip and horseradish. Both were purified using ammonium sulphate

precipitation and gel filtration. Enzyme activity was measured using an artificial

substrate known as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS).

Yield of horseradish peroxidase came out to be six times higher than turnip

peroxidase. Horseradish peroxidase was purified by 18.66 folds whereas turnip

peroxidase was purified by 5.40 folds. SDS-PAGE revealed the molecular mass of

turnip peroxidase to be 37 kDa. Thermal stability was carried out on turnip and

horseradish peroxidase in order to assess the activity of these peroxidases at different

temperatures.



1. INTRODUCTION


Peroxidases are heme-containing enzymes which are found in various plants and

microorganisms. Peroxidases catalyse many reactions in the presence of peroxides

such as hydrogen peroxide:




Some of these reactions include: oxidation of phenols and amines, detoxification of

soil and neutralisation of contaminated water (Hamid and Rehman 2009). Peroxidases

can be used in diagnostic kits for determining the quantity of uric acid, glucose or

cholesterol and can be simply used for detection of toxins, pathogens and potential

cancer causing materials in the bladder and prostate (Hamid and Rehman 2009). Some



4

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