Extraction, Partial Purification and Characterization of Peroxidase from Malva neglecta

127 Pages·2013·3.36 MB·English

Ministry of Higher Education
and Scientific Research
University of Baghdad
College of Science
Biotechnology Department

Extraction, Partial Purification and Characterization of
Peroxidase from Malva neglecta

A thesis
Submitted to the College of Science-University of Baghdad as a Partial Fulfillment of
the Requirements for the Degree of Master of Science in Biotechnology

By
Rihab Hameed Al-badri
B.Sc. Biotechnology/Al-Nahrain University
College of Science (2009)

Supervised by
Professor
Dr. Ghazi Munim Aziz

October 2012 Zuhadjh 1433
Acknowledgement
Praise to God, mercy and peace are to the prophet Mohammed God
bless him and Grant him, and upon his relatives and faithful
companions.
I would like to express my deep thanks to my supervisor prof. Ghazi
Munim Aziz, for his continuous support and advice during the
practical work and writing of this transcript.
Special thanks for the Dean of college of science/Baghdad University.
My Great thanks and appreciation goes for the head master of
Biotechnology Dept. Dr. Abdul Kareem Al-kazaz and all members
of Biotechnology department for Their Kindness and great help.
I would like to express my sincere thanks and gratefulness for Dr.
Muayad Sabri and Dr. Hassan F. Samir for Their Great help and
support.
Thanks and Gratitude for Miss.Hala Mush'l, Miss. Sahar Rhayem,
and for all the friends that I hope they will forgive me for not
mentioning their names.
Special thanks and Gratitude for my family specially my parents
and appreciate their long patience, and my best friends Ali Jabbar,
Zainab waleed and Sara Qusai and everyone gave a hand to
complete this work.

Rihab
Summary

Summary
Peroxidase has been detected in crude of plant Malva neglecta extract in each
part of plant parts such as roots, leaves, stems as well as whole plant. The
enzymatic activity was found to be higher in the whole plant than other parts.
The guaiacol is used as substrate in the detection of enzymatic activity of
peroxidase. The optimization of extraction process was done by controlling
the type and concentration of buffer, pH of the buffer used, and the ratio of
extraction. The Sodium acetate buffer with 0.2mM and pH 5.0 was found to
be the best buffer for extraction of peroxidase. By using the extraction ratio
for plant tissue of 1:10 (W/V), the specific activity was 10.7 U/mg protein.
peroxidase has been purified from plant Malva neglecta following three
purification steps. These steps include: Ammonium sulfate precipitation with
70% saturation, followed by Ion exchange chromatography using DEAE-
Cellulose and Gel filtration chromatography using Sephadex G-100. These
three purification steps raised the specific activity to 23U/mg protein in the
precipitation step with purification fold 2.1 and enzyme recovery 46.7%, the
specific activity was increased to 40.3U/mg protein in Ion exchange step with
purification fold 3.7 and enzyme recovery 25%, also the specific activity
doubled after gel filtration step to 67.5U/mg protein with purification fold 6.3
and enzyme recovery 18.2%. Characterization results demonstrated that, the
optimal pH for activity and stability was 7 and 7-7.5 respectively, the optimal
temperature for activity and stability was 45 and 30-40˚C respectively. The
activation energy was calculated and it was 14Kcal/mol, The kinetics of
peroxidase such as maximum velocity V and Michalis constant K were
max m
also determined using four different methods including (Linweaver- Burk
plot, Woolf- Augustenson plot, Hanes- Woolf plot and Eadie- Scatchard plot
)using guaiacol and hydrogen peroxide as substrates, the K and V were
m max
I
Summary

-2 -2
measured to be 1.4× 10 mM towards Hydrogen peroxide and 3.2 × 10 mM
-1 -1
towards guaiacol, and 2.5× 10 mM/min and1.6× 10 mM/min respectively.
The effect of some activators such as metallic chlorides (sodium chloride
(NaCl), potassium chloride (KCl), copper chloride (CuCl ), Calcium chloride
2
(CaCl ), and magnesium chloride (MgCl )) on peroxidase activity was
2 2
estimated. Reagents such as EDTA, 2-mercaptoethanol, and Urea are also
used as inhibitors of peroxidase activity. It was observed that the peroxidase
activity increased when incubated with metallic chlorides. The highest
remaining activity was 212% treated with 20mM CaCl , while the specific
2
activity of peroxidase decreased if treated with 20mM of Urea and losses
80% of its activity, and the peroxidase loses its complete activity when it
treated with 0.25M Na So .
2 4
The purified peroxidase was used in preparation of glucose determination kit
and proved its efficiency in achieving of linear relationship between glucose
concentrations; the absorbance was at 460nm when using reaction substances
such as guaiacol and O-dinizidine.
II
List of contents

Items Titles Page
No.
Summary I
List of contents III
List of figures X
List of tables XII
Abbreviations XIII
Introduction 1
Chapter one: Literature Review
1.1 Medicinal plants 3
1.2 Malva 4
1.3 Morphology of malva neglecta 4
1.4 Classification of Malva 6
1.5 History 6
1.6 Cultivation and uses 7
1.7 Species list of Malva plant 8
1.8 Enzymes 9
III
1.9 Peroxidase 10
1.10 Classes of peroxidase 12
1.11 Isoenzymes of peroxidase 14
1.12 Assay of peroxidase 15
1.13 Structure of peroxidase 16
1.14 Reaction mechanism 19
1.15 Extraction of peroxidase 22
1.16 Purification of peroxidase 24
1.17 Characterization of peroxidase 26
1.17.1 Molecular weight 26
1.17.2 Effect of pH on peroxidase activity and stability 27
1.17.3 Optimum temperature of peroxidase activity and stability 28
1.17.4 Isoelectric point 29
1.17.5 Peroxidase Inhibitors 30
1.18 Peroxidase applications 30
Chapter two: Materials and methods
2.1 Materials and methods 34
IV
2.2 Sample collection 35
2.3 Peroxidase extraction 36
2.3.1 Buffers and solutions 36
2.3.1.1 Potassium Phosphate buffer (0.1M, pH 7) 36
2.3.1.2 Potassium Phosphate buffer (0.2M, pH 7) 36
2.3.1.3 Sodium acetate buffer (0.1M, pH 5 and 6) 37
2.3.1.4 Sodium acetate buffer (0.2M, pH 5 and 6) 37
2.3.1.5 Tris- HCl buffer (0.1M, pH 8 and 9) 37
2.3.1.6 Tris- HCl buffer (0.2M, pH 8 and 9) 37
2.3.2 Method 37
2.3.3 Comparison between dry and fresh plant as a source of peroxidase 37
2.3.4 Detection of peroxidase in different plant parts 38
2.4 Determination of optimum conditions of peroxidase extraction 38
2.4.1 Determination the optimum buffer of extraction 38
2.4.2 Determination the optimum ratio of extraction 38
2.5 Determination of peroxidase activity 39
2.5.1 Materials and buffers 39
V
2.5.1.1 Guaiacol solution (0.05M stock solution) 39
2.5.1.2 Hydrogen peroxide solution (0.02M) 39
2.5.1.3 Sodium acetate buffer (0.1M, pH 5.5) 39
2.5.2 Method 39
2.6 Determination of protein concentration 41
2.6.1 Materials and buffers 41
2.6.1.1 Bovine serum albumin (mg/ml) (stock solution) 41
2.6.1.2 Coomassie brilliant blue G-250 (protein dye reagent) 41
2.6.1.3 Tris- HCl buffer (0.015M, pH 7) 41
2.6.2 Method 41
2.6.2.1 Preparation the standard curve of BSA 41
2.6.2.2 Determination of protein concentration in enzymatic solutions 43
2.7 Purification of enzyme 44
2.7.1 Ammonium sulfate precipitation 44
2.7.2 Ion exchange chromatography 44
2.7.2.1 Materials and buffers 44
2.7.2.1.a Potassium phosphate buffer (5mM, pH 7) 44
VI
2.7.2.1.b HCl solution (0.25M) 46
2.7.2.1.c NaOH solution (0.25M)- NaCl buffer (0.25M) 46
2.7.2.1.d Potassium phosphate buffer (5mM, pH 7) and salt gradient (0.1- 46
1M)
2.7.2.2 Preparation of ion exchange column (DEAE-Cellulose) 46
2.7.2.3 Separation of enzyme through ion exchange resin (DEAE- 47
Cellulose)
2.7.3 Gel filtration chromatography 47
2.7.3.1 Materials and buffers 47
2.7.3.1.a Potassium phosphate buffer (0.2M, pH 7) 47
2.7.3.1.b Preparation of sephadex G-100 gel 48
2.7.3.2 Enzyme separation through sephadex G-100 column 48
2.8 Characterizations of peroxidase 49
2.8.1 Optimum pH of enzyme activity 49
2.8.2 Optimum pH of enzyme stability 49
2.8.3 Optimum temperature of enzyme activity 50
2.8.4 Optimum temperature of enzyme stability 50
2.8.5 Determination of activation energy 50
2.8.6 Determination of K and V values 51
m max
VII
2.8.7 Effect of inhibitors and activators on peroxidase 52
2.9 Preparation of glucose determination kit 53
2.9.1 Buffers used 53
2.9.2 Method 53
Chapter three: Results and Discussion
3.1 Determination of peroxidase activity in fresh and dry plant 54
3.2 Determination of peroxidase activity in different plant parts 55
3.3 The extraction buffer 56
3.4 The optimum ratio for extraction 58
3.5 Peroxidase purification 60
3.5.1 Precipitation with ammonium sulfate 60
3.5.2 Ion exchange chromatography 60
3.5.3 Gel filtration chromatography 64
3.6 Characterization of peroxidase 66
3.6.1 The optimum pH of enzyme activity 66
3.6.2 The optimum pH of enzyme stability 68
3.6.3 Optimum temperature of enzyme activity 69
VIII