Isolation, Partial Purification and Characterization of A Protease from Ginger Zingiberaceae Officinale Roscoe

Abstract

Protease from Zingiberaceae officinale Roscoe (cv. Haliya indang) was isolated, purified to homogeneity in a three step procedure involving ammonium sulphate fractionation, gel filtration, and ion-exchange chromatography.
The partially purified enzyme was characterized. The enzyme was found to be made up of two sub-units designated ‘ginger protease I and II (GPI & GPII); the subunits had specific activity of 173.88±5.6 and 131.54±3.7mg gelatin/min/mg  protein respectively, with  purification folds  of and 2.57 over crude extract.
The molecular weights of 23.97 and 25.05 kDa were obtained by SDS-PAGE. The enzyme subunits had similar optimum temperature of 40^oC, while retaining high activity at 46^oC for GPI and 58^oC  for GPII suggesting GPI to be more heat labile.
The subunits incubated at different temperatures showed highest stability at 40^oC, with an optimum pH of 6.0 and 5.5, retaining significant activity within a pH range of 5.5-7.5 for GPI, and 5.0-7.5 for GPII. Mn²⁺, Zn²⁺ inhibited the subunits mildly while Hg²⁺ had a severe inhibition on the subunits. The subunits were also severely inhibited by Iodoacetate (IA), a cysteine protease inhibitor.

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Table Of Contents

Cover page……………………….i
Fly leaf…………………………….ii
Title page……………………… ii
Declaration…………………..iii
Certification…………………… iv
Acknowledgment……………… v
Abstract………………………….. vii
List of Tables/Figures……….xii
CHAPTER ONE

1. 0 INTRODUCTION………………….. 1
2. 1 Statement of research problem.2
3. 2 Aim…………………………………….. 3
4. 3 Justification………………………..3
5. 4 Specific objectives……………….4

CHAPTER TWO

• LITERATURE REVIEW…………………….. 5
• Protease enzyme………………………………. 5
• Physiological functions of proteases……6
• Classes of proteases………………………….. 7
• Applications of proteases………………….. 8
• Cysteine proteases…………………………….9
• Ginger in Nigeria………………………………12
• Chemical constituents of ginger………….13
• Zingibain…………………………………………..14

CHAPTER THREE

3. 0 MATERIALS AND METHODS……………16
4. 1 Chemicals and Reagents……………………..16
5. 2 Equipment and analytical instruments…. 16
6. 3 Collection of samples………………………….. 17
7. 4 Preparation of ginger powder……………….. 17
8. 5 Preparation of buffers………………………17
9. 6 Extraction and purification of protease enzyme…..18
10. 7 Determination of protein concentration……………… 19
11. 8 Enzyme activity assays…………………………………….. 20
12. 9 Sodium Deodocyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)20
13. 10.. Molecular weight determination………………… 21
14. 11.. Determination of temperature stability………..21
15. 12.. Determination of optimum temperature……..22
16. 13.. Determination of optimum pH……………………22
17. 14 Determination of storage Stability of Protease….23
18. 15.. Inhibition studies………………………………………..23
19. 16.. Effect of metal ions…………………………………….. 23
20. 17.. Effect of different compounds…………………….. 24
21. 18.. Substrate specificity test……………………………..24

CHAPTER FOUR
4.0… RESULTS……….25
CHAPTER FIVE

5. 0 DISCUSSION…..49

CHAPTER SIX
SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS……. 53
APPENDIX……. 59

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Introduction

Background Of Study
Enzymes, especially proteases have become an important and indispensable part of the processes used in many applications by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption.
Enzyme technology has evolved to become an integral part of the food industry (Demir et al, 2007). Proteases have found applications in various industrial and medical uses.
Several enzymes have been discovered, isolated and developed from various sources; plants, animals, bacteria and fungi.
Presently, approximately 100 different biocatalytic processes are implemented in pharmaceutical, chemical, agricultural, and food industries and its applications continue to grow very rapidly (Bente, 2002).
This technology has helped in processing food products. In spite of these successes, however, the vast potential of biocatalysis is yet to be fully realized.
Bioactive agents from plants are one of the most intensive areas of natural product research today and this vast area is far from being exhausted.
Biologically active plants with a broad range of therapeutic properties and rarely associated with adverse side effects, are exploited for economic benefits.

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References

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