THE EXTRACTION AND PURIFICATION OF POLYPHENOL OXIDASE IN WHITE YAM

TABLE OF CONTENT

Contents pages

Title page ..i

Approval page.ii

Dedication.iii

Acknowledgementiv

List of tables and figures.v

Table of contents...vi

Abstractvii

Chapter one

1.1 Introduction. 1

1.2 Historical features.1

1.3 Nutrient composition of yam...1

1.4 Polyphenol oxidase enzymes3

Chapter two

Literature review5

Yam diseases.5

Storage diseases6

Growth and harvest of yam tubers....6

Yam crop productionstages and process of yam production8

Flavonoids10

Functions of flavonoids..10

Tannins11

Biosynthesis and analysis of phenol..12

Biosynthesis of phenol12

Test for phenol..,,13

Uses of phenol.13

Properties of phenol14

Enzymes ......14

Types of enzymes 14

An overview of polyphenol oxidase enzymes.16

CHAPTER THREE

Experimental instrumentation and Materials..18

Flowchart for polyphenol oxidase preparation of... 19 partial purification from white yam...19

Methodology :isolation and purification of enzyme23

Crude enzyme preparation ..23

Acetone precipitation 24

Ammonium sulphate fraction..24

Dialysis ..25

Centrifugation ..26

Sephadex gelfilteration 26

CHAPTER FOUR

Result 127

Definition..27

Result 2..30

PH optimum..34

CHAPTER FIVE

Discussion35

Conclusion ...35

Recommendation.....36

Reference..38

ABSTRACT

Polyphenol oxidase was extracted from peeling of dioscorea rotundata for characterization. The activity of polyphenol oxidase was studied using the partially purified enzyme from 100gof yam peeling of dioscorea rotundata .All operations were carried out from 0 40c cold temperature unless otherwise specified.The preparation of acetone powder was chosen to be first step of purification in other to remove as much endogenous phenolic substrate from the enzymes. The initial purification step outlined by the experimental procedures yielded by final crude extract 6364 fold pure over the initial crude extract of the acetone powder.After obtaining the crude extract,the isolation and purification of the enzyme which undergo some methods and process before the result was gotten shows the extract obtained has higher polyphenol oxidase activity with catechol that with chlorogenic acid variation in substrate concentration catechol were used and their effect on the activity reaction were investigated following the processes of the reaction it was possible to find the Michaels constant km of catechol to be 3.43x103m and the maximum attainable initial velocity vmax to be 0.71 DA 390/min/m/ enzyme while mercaptoxthanol showed a competitive inhibition with ki 3.36x107M the molecular weight of the enzyme was approximately 39000.however,this is to show enzymic browning reaction must be very complex and be the result of the reaction of interaction between not purely understood enzymesand variety of phenolic compound substrate.