determination of peroxidase from ripe pepper (PDF)

UNIVERSITY OF CAPE COAST
SCHOOL OF BIOLOGICAL SCIENCES
DEPARTMENT OF BIOCHEMISTRY

DETERMINATION AND CHARACTERISATION OF PEROXIDASE IN RIPE
PEPPER
BY
YANKEY JUSTICE KWASI
DECLARE-;
A DISSERTATION SUBMITTED TO THE DEPARTMENT OF
BIOCHEMISTRY OF THE SCHOOL OF BIOLOGICAL SCIENCE, UCC, IN
PARTIAL FULFILMENT OF THE REAQUIREMENTS FOR THE AWARD
OF A BACHELOR OF SCIENCE (HONS) DEGREE IN BIOCHEMISTRY

26/5/2014

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DECLARATION
I YANKEY JUSTICE KWASI HEREBY DECLARES THAT THIS
DISSERTATION IS THE RESULT OF MY OWN WORK AND THAT NO
PART OF IT HAS BEEN PRESENTED FOR ANOTHER DEGREE IN THIS
UNIVERSITY OR ELSEWHERE.
YANKEY JUSTICE KWASI
CANDIDATE SIGNATURE………………………………………….
DATE…………………………………………

SUPERVISOR`S SIGNATURE……………………………………….

DATE……………………………………………

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ABSTRACT
Peroxidase belongs to the class oxidoreductase, that catalysis the redox
reaction between hydrogen peroxide as an electron acceptor by means of
oxygen gas liberation.
In plants, peoxidases play the role of growth regulation and defense
through elimination of reactive oxygen species. It is also observed that the
enzyme also influences the ripening of pepper.
Pepper is an important condiment in foods which is mostly abundant
during rainy season. In order not to go waste it is important to extract the
enzyme and be use for industrial purposes like flavoring, preservation of
foods and designing of glucose determination kits use in laboratory.
O-Dianisidine in the reduced form gets oxides in the presence of the
enzyme and its substrate, hydrogen peroxide.
In the determination of the assay of the enzyme, a phosphate buffer
was prepared which was used during the homogenization of the pepper at
40C, and its centrifugation at 4000g RCF for 40mins. The supernatant was
filtered with a clean-white cloth and used as sample after the confirmatory
test was run on it to confirm peroxdase presence.
The enzyme concentration was estimated to be 17.24mg/ml using the
biuret method. Effect of PH, and temperature was checked and their

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optimum effect was observed at approximately 4.4 and 600C respectively,
for an incubation time, which was found to be approximately 6mins.
Using the effect of substrate on the enzyme activity, the Double
Reciprocal Plot was plotted, which lead to the determination of the Km,
Vmax as well as Kcat as 0.232M, 0.013mMmin-1, and 1.257×10-5s-1
respectively.

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ACKNOWLEGEMENT
Acknowledging a dissertation work represents a silhouette of
magnanimity and help rendered by our family and friends. It gives me
immense pleasure and contentment to acknowledge and thank all those who
in a big and small ways have contributed for this effort.
To begin with, first and foremost I am very grateful to God Almighty
who showered his blessing on me throughout my life.
In the first place with great pleasure, I express my most cordial and
humble thanks to my eminent, respected guide and teaching assistant (T.A.)
Miss Rosemary, Department of Biochemistry, School of Biological Sciences,
for providing her valuable guidance, inspiration, supervision, keen interest,
and moral support from the very early stage till the end of my dissertation
work. Her encouraging words were source of constant motivation to act
with quality and precision and they enriched my growth as a student. I am
indebted to her for all her support. Without her guidance and persistent
help this dissertation would not have been possible.
I am thankful to Mr. Ofori(SRA), Sir Gilbert(SRA), Sir Gilbert(TA), Sir
Anaman(Tithe man), Mad. Priscilla(TA), Sir Kye(SRA) and all the other staff
of the department and other department like the Molecular biology,
Laboratory Technology, Human biology etc. for extending their co-operation
and help.

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My personal and very sincerest acknowledgement to my supportive,
forgiving, generous, hardworking and loving group members, Odei Appiah
Valentine, Offeibea Kwafo , and Addo-Dabankah Benjamin whose efforts I
would forever be grateful.
I sincerely acknowledge the authorities of University of Cape Coast,
especially the Department of Biochemistry for providing the necessary
facilities to carry out my research work.
I would like to thank my family for supporting and encouraging me
to

pursue this research. Without their encouragement, love and concern I

would not have finished this work.
Thank You

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DEDICATION
Dedicated to My family and Axim.

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TABLE OF CONTENTS

CHAPTER
NO.

NAME OF CHAPTER

PAGE NO.

1

INTRODUCTION

11

2

LITERATURE REVIEW

16

3

MOTHEDS AND MATERIALS

25

4

RESULTS

30

5

DISCUSSION

38

6

CONCLUSION

41

7

REFERENCE

42

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LIST OF TABLES
No.
Table

Name of Table

No. of
page

1

Determination of incubation time

30

2

Effect of PH on enzyme activity

31

3

Effect of temperature on enzyme activity

34

4

Effect of substrate on enzyme activity

34

5

Values of Lineweaver-Burk Plot

37

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