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STUDIES ON THE INHIBITORY EFFECT ON TYROSINE ON POLYPHENOL OXIDASE IN WATER YAM

SCIENCE LABORATORY TECHNOLOGY RESEARCH PROJECT TOPICS AND MATERIALS

Abstract

Yam (Dioscorea SPP) is an important food in West Africa. Nigeria alone produces 21,814 millions tons of yams per year, making it the worlds  largest yam producer. It is estimated that after six months in storage, up to 56% of the crop is lost due to rot. Polyphenol oxidase, a copper-containing metalloprotein, catalyzes the oxidation of phenolic compounds to quinines, which pr5oduce brown pigments in wounded tissues. This enzymes mechanism causes post harvest losses and mainly affects tropical roots and tubers. Characterization of the polyphenol oxidase could help to develop or to choose more effective methods for controlling browning of roots and tubers. Polyphenol oxidase (PPO) was isolated from water yam (Dioscorea alata) and purified by acetone precipitation and dialysis. The optimum pH for the acetone purified PPO catalyzed oxidation of catechol was found to be pH 6.8.

Catechol is used to provide quinine compounds that are used to produce melanins. Tyrosine is used to synthesize melanins in humans. The application of lineweaver-burke analysis of enzymatic activity; water yam polyphenol oxidase (PPO) Vmax is 0.5 absorbance units per minute while km is 0.25m. Vmax is not measured in “absorbance units per minute”. Those units were chosen to simplify the experiment.

 

Table of contents

Title page                                                       i

Certification                                                    ii

Dedication                                                      iii

Acknowledgement                                           iv

Abstract                                                         v

Table of content                                              vi

Chapter one

1.0 Introduction                                              1-4

1.1 Aims and Objectives                                   5-6

Chapter two

2.0 Literature review                                        6

2.1 Origin of yam                                            6

2.2 General morphology of yam tuber                7

2.3 Prospects and constraints of production and

Marketing                                                  8

2.4 Utilization                                                  10

2.4.1 Prospect and constraints of the utilization of

Yam                                                        11-12

2.5 Distribution                                               13

2.6 Polyphenol oxidase                                     13

2.7 Enzymatic browning                                   14

2.7.1 Browning control                                     15-16

2.7.2 Chemical antibrowning effectors                17-18

2.8 Enzyme inhibition                                       19

2.8.1 Competitive inhibition                              19-21

2.8.2 Noncompetitive inhibition                         22

2.8.3 Uncompetitive inhibition                                  23

2.8.4 Substrate and product inhibition                24-25

2.8.5 Allosteric enzymes                                   26

2.8.6 Cooperativity (Homoallostery)                   26

Chapter three

3.0 Materials and methods                                27

3.1 Source of materials                                    27

3.2 Methods                                                    28

3.2.1 Extraction and purification of PPO              28

3.2.2 PPO assay                                               29

3.2.3 Browning and POD inhibitor                      30

3.3 To determine the nature of inhibition of

tyrosine on polyphenol oxidase in water yam  30-32

Chapter four

4.0 Results and discussion                                33

4.1 Discussion                                                 33-34

4.2 Results of determinations in

Spectrophotometer                                    35

4.3 Results of inverse of determinations

of spectrophotometer                                        36

Chapter five

5.0 Conclusion and recommendation                  37

5.1 Conclusion                                                37

5.2 Recommendation                                       37-38

References                                                      39-45

CHAPTER ONE

1.0 INTODUCTION:

Yam (Dioscorea Spp),which is an annual or perennial climbing plant with edible underground tuber, is native to warmer regions of both southern and northern hemispheres (IITA,2004).Yam is an important food crop for many people in the yam zone of west Africa and a very important source of income in rural and marginal areas (Phaction,2002, Vernier, 1998).it has long served as the principal source of food and nutrition for many of the worlds poorest and undernourished house holds, and generally valued for their stable yields under conditions in which other crops may fail (Alexadratos,1995, Scott et al. 2000).

Since several decades yam has been embedded in the population habit and has a socio-cultural significance (IITA, 2006). There are over 600 yam species grown throughout the world , but in west Africa the most economically important species are the white yam (Dioscorea  rotundata),yellow yam (Dioscorea cayenensis) and water yam (Dioscorea data ) (vernier, 1998).

Dioscorea alata is wide spread in distribution being grown is wide spread in distribution being grown in tropics and subtropics of Africa, America, Asia and Caribbean. The tubers are nearly always single and very large weighing upto 60kg and measuring upto 2 meter in length. The dry matter of the yam tuber varies between 20-40% of which inturn consist of 60-80% of starch depending on the variety (Lebot et al., 2005).

Despite its importance, yam including D. alata belongs to the neglected crops and many constraints limit its production. Due to the perishability of the crop, the tubers cannot be kept for more than a few weeks after harvesting, 50% of the crops may be lost within 6 months due to rot or germination if no stabilization processes are used, and this explains the volatility in fresh yam prices over the years (Vernier, 1998). Also, the high water content and fragility of fresh tubers affects transport and marketing costs.

L-tyrosine is a conditionally essential aminor acid because under normal conditions the body synthesis sufficient quantities from phenylalanine. For those with phenylketonuria, however, a severe deficiency in the evizyme phenylalanine hydroxylase prevents conversion of phenylalanine to tyrosine, making tyrosine an essential amino acid for this population (Glaeser et al., 1979).

Tyrosine is incorporated into proteins of all life forms and is a precursor for synthesis of thyroxin, melanin, and the neurotransmitters dopamine and norepinephrine. Food sources of tyrosine include fish, soy products, poultry, eggs, dairy products, lima beans, almonds, peanuts, sesame seeds, pumpkin seeds, wheat germ, oats, avocados, and bananas. Clinical conditions for which tyrosine supplementation may be of therapeutic benefit include depression, hypertension, stress, cognitive function and memory, parkinson’s disease, phenylketonuria, and narcolepsy (Melamed and Glaeser, 1980).

Polyphenol Oxidase (PPOs) are a group of copper-proteins, widely distributed phylogenetically from bacteria to mammals, that catalyze the oxidation of phenolics to quinines which produce brown pigments in wounded tissues. PPO has been implicated in the formation of pigments, oxygen scavenging and defense mechanism against plant pathogens, and herbivory insects. Phenolic compounds serve as precursors in the formation of physical polyphenolic barriers, limiting pathogen translocation. The quinines formed by PPOs can bind plant proteins, reducing protein digestibility and their nutritive value to herbivores (Mayer, A.M. 1987). On the other hand, the oxidation of phenolic substrates by PPO is thought to be the major cause of the brown coloration of many fruits and vegetables during ripening, handling, storage and processing. This problem is of considerable importance to the food industry as it effects the nutritional quality and appearance, reduces the consumer’s acceptability and, therefore, causes significant economic impact, both to food producers and to food processing industry. It is estimated that over 50% of losses in fruits occur as a result of enzymatic browning and tropical and subtropical fruits and vegetables are the most susceptible to these reactions (Marshall et al., 2000).

PPO is regarded to be a critical enzyme in food technology, and it has been intensively studied in several plants. It is known that plant PPOs are synthesized as preproteins and contain putative plastid transit peptides at the N-terminal region, which target the enzyme into chloroplast and thylcaloid lumen. PPO from some plants has been described as a multiple gene family (Mayer, 2006).

Plant PPOs have broad substrates specificities and are able to oxidize a variety of mono, di or polyphenols. Phenolic compounds are natural substances that contribute to the sensorial properties (color, taste, aroma and texture) associated with fruit quality (Yoruk and Marshall, 2003).

1.1 Aims and objectives

  • To extract the enzyme polyhenol oxidase from yam.
  • To dialyze the enzyme that was extracted
  • To study the inhibitory effect of tyrosine on polyhenol oxidase in water yam.

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