Project Materials

Biochemical Molecular Studies on β-Glycosidase from Sorghum (Sorghum Bicolor (L) Moench) and Cassava (Manihot esculenta Crantz)

ABSTRACT

Genes encoding linamarase from cassava root cortex and dhurrinase from sorghum seeds were amplified using the polymerase chain reaction. Crude extracts of linamarase and dhurrinase containing most of the intracellular proteins were used to hydrolyze pnitrophenyl-β-D-glucopyranoside (pNP) and linamarin extracted from cassava cortex. A gene fragment corresponding to linamarase (1,504 bp) was amplified. The native enzyme showed an optimum pH of 6.0 for pNP hydrolysis. The optimum temperature observed from the experimental assay showed that cassava linamarase has maximum activity for the hydrolysis of pNP at 65°C. The Q10, calculated between 40°C and 50°C was 1.17.

Activation energy (Ea) was found to be 17.32 KJ/mol (4.14 kcal mol-1). The KM for linamarase was 5.75 x 10-3 μmol l-1 with a kcat value of 3.41 min-1 and Vmax of 1.96 x 10 -2 μmol l-1 min-1. A 1,695 bp fragment corresponding to dhurrinase gene was also amplified. Dhurrinase enzyme activity was stable at room temperature (25oC) with optimum temperature of 65oC. Temperature coefficient (Q10) calculated between 40oC and 50oC gave a value of 2.53. Activation energy (Ea) of 51.06 KJ/mol was also extrapolated. The pH activity curve put maximal activity for dhurrinase at pH 5.0. The double reciprocal plot of dhurrinase showed a calculated KM of 1.39 x 10-2 μmol l-1, Vmax of 1.01 x 10-1 μmol l-1 min-1 and kcat of 7.27 min-1. Initial velocity data clearly showed that dhurrinase had an index of physiological efficiency (kcat) of 7.27 min-1. Half life of enzyme activity (using linamarin as substrate) extrapolated from steady state data favored dhurrinase (288.79 min), linamarase showing a half life of 117.47 min. Our investigations have shown clearly that dhurrinase holds the capacity to totally detoxify cassava and its products of cyanogenic principles more effectively than linamarase.

TABLE OF CONTENTS

Cover page……………………………………………………………………………i
Title page………………………………………………………………………………ii
Declaration……………………………………………………………………..…….iii
Certification……………………………………………………………………………. iv
Dedication……………………………………………………………………………. v
Acknowledgements……………………………………………………………………..vi
Abstract………………………………………………………………………………. viii
Table of Content………………………………………………………………………… ix
List of Tables………………………………………………………………………….. xii
List of Figures…………………………………………………………………………. .xiii
List of Plates……………………………………………………………………………xiv
CHAPTER ONE
1.0 Introduction…………………………………………………………………………1
1.1 Hypothesis…………………………………………………………………………..3
1.2 Justification………………………………………………………………………….4
1.3 Research Questions………………………………………………………………….4
1.4 Aim…………………………………………………………………………….…….5
1.5 Objectives……………………………………………………………………….……5
1.6 Significance of the Study……………………………………………………………5
CHAPTER TWO
2.0 Literature review………..………………………………………………………… …6
2.1 Sorghum…………………………………. …………………………………….……6
2.1.1 Origin of Sorghum………………………………………………………….………6
2.1.2 Biology of Sorghum……………………………………………………….……….6
2.1.3 Ecology of Sorghum………………………………………………………………10
2.2 Sorghum Products…………………………………………………………………..11
2.3 Sorghum Cultivation in Nigeria……………………………………………………..11
2.4 Cassava…………………………………………………………………..………….13
2.4.1 Origin of Cassava……………………………………………………….…………13
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2.4.2 Ecology of Cassava….…………………………………………………………15
2.4.3 Biology of Cassava..……………………………………………………………15
2.5 Cassava Products…………………………………………………………………20
2.6 Cassava in Africa…………………………………………………………………21
2.7 Plant Toxins and Cyanogens……………………………………………………..22
2.7.1 Cyanogenic Crops………………………………………………………………23
2.7.2 Biology of Cyanogens and Enzymes in Plant Tissue…………………………..24
2.7.3 Biosynthesis (Cyanogenesis) and Structure……………………………………25
2.7.4 Chemistry and Breakdown……………………………………………………..27
2.7.5 Cyanogens in Food and Health……………………….………………………..28
2.8 β-glycosidases…………………………………………………………………….31
2.9 Theory of Methods……………………………………………………………….32
2.9.1 Plant Genomic DNA Extraction………………………………………………..32
2.9.2 Polymerase Chain Reaction…………………………………………………….33
2.9.3 Gel Electrophoresis and Visualization………………………………………….35
CHAPTER THREE
3.0 Materials and Methods………………………………………………………..…..36
3.1 Materials……………………………………………………………………..…….36
3.1.1Research Location…………………………………………..……………….……36
3.1.2 Reagents…………………….……………………………………………………36
3.1.3 Plant Materials…………………………………………………………………….36
3.1.3.1 Sorghum…………………………………………………………………………36
3.1.3.2 Cassava……………………………………………………………………….…36
3.2 Methods……………………………………………………………………………..36
3.2.1 Genomic DNA Extraction………………………………………………………..36
3.2.2 DNA Quality Confirmation………………………………………………………37
3.2.3 Polymerase Chain Reaction………………………………………………………38
3.2.4 Preparation of Crude Enzyme…………………………………………………….39
3.2.5 Assay of Enzyme Activity………………………………………………………..39
3.2.6 Effect of pH on β-glycosidase activity……………………………………………40
3.2.7 Effect of Temperature on β-glycosidase activity…………………………….……40
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CHAPTER FOUR
4.0 Results…………………………………………………………………………..….41
4.1 Molecular Characterization of Genes Coding Linamarase and Dhurrinase….……..41
4.1.1 Cassava genomic DNA Extraction………………………………………….……41
4.1.2 Sorghum Genomic DNA Extraction…………………………………………..…43
4.2 Gene Amplification…………………………………………………………………43
4.3 Linamarase and Dhurrinase Assay…..……………………………………………..45
4.3.1 Temperature Studies on Linamarase……………………………………………..45
4.3.2 Temperature Studies on Dhurrinase………………………………………………48
4.4 pH Optimum for Enzymatic Hydrolysis………………………………………..….50
4.4.1 pH Studies on Linamarase………………………………………………………..50
4.4.2 pH Studies on Dhurrinase…………………………………………………….…50
4.5 Initial Velocity Studies……………………………………………………………53
4.5.1 Determination of KM for Linamarase with p-nitrophenyl-β-D-glucopyranoside as
substrate………………………………………………………………………..53
4.5.2 Determination of KM for Dhurrinase with p-nitrophenyl-β-D-glucopyranoside as
substrate………………………………………………………………………..55
4.6 Determination of Half Life of Enzyme Activity…………………………………..55
4.6.1Steady State………………………………………………………………………55
4.6.2 Determination of Linamarase Half Life………………………………………….55
4.6.3 Determination of Dhurrinase Half Life using Linamarin as substrate……………55
CHAPTER FIVE
5.0 Discussion …………………………………………………………………………..60
CHAPTER SIX
6.0 Summary, Conclusion and Recommendation…………………………….………….67
6.1 Conclusion……………………………………………………………………….…67
6.2 Recommendations…….………………………………………………….. ………..68
References…………………………………………………………………………..69
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CHAPTER ONE

1.0 INTRODUCTION

Cyanogenic compounds capable of releasing cyanide on chemical breakdown are widespread in the plant kingdom (Bak et. al., 2006). More than 2,650 plant species are known to be capable of producing sufficient quantities of cyanogenic compounds that may be important in animal and human health (Bak et. al., 2006). These compounds are present in important food and feed crops, including cassava, lima beans, sorghum, almonds, plums, apricots and peaches, to mention but a few. The hydrolysis of these compounds, either by enzyme action or by strong acid is known to release cyanide, which can be very dangerous, if this release occurs in an animal or human body. In order to render them safe and edible, the toxicity associated with these important food crops is reduced by various processing methods, notable among them being various forms of fermentations.

Cassava (Manihot esculenta Crantz) is a perennial starchy root crop that is widely grown in the tropical regions of the world. Cassava gives a carbohydrate production per hectare which is about 40% higher than rice and 25% more than maize, thus cassava is the cheapest source of calories for both human nutrition and animal feeding (Tonukari, 2004). Global production of cassava is around 152 million tons per year. Half of the 16 million hectares of cultivated cassava worldwide is devoted to small-scale cassava cultivation in Africa, 30 percent is grown in Asia and 20 percent in Latin America (CIAT, 2001). Small scale farming is characterized by cultivation using traditional methods with little or no inputs and frequent intercropping practices. Cassava’s role as a – 16 – traditional human food is changing to an efficient industrial crop in some parts of Africa, for instance in Nigeria (Nweke, 2004) and many parts in Asia and Latin America.

Cassava produces potentially toxic levels of cyanogenic glycosides (Linamarin [95%] and lotaustralin [5%]) which are synthesized in the leaves (Koch et. al., 1992) and translocated to all other parts of the plant including the edible tuberous roots (McMahon et. al., 1995). The breakdown of cyanogenic glycosides by the β–glycosidase linamarase results in hydrogen cyanide (HCN) production when cassava tissues are mechanically damaged. HCN in cassava tissues has been medically proven to be a potential health hazard for consumers if the plant is inadequately processed (Tylleskär et. al., 1992; McMahon et. al., 1995).

Sorghum is a genus of numerous species of grasses, one of which, Sorghum bicolor (L) Moench (commonly called sorghum), is raised for grain and many of which are used as fodder plants either cultivated or as part of pasture. Sorghum is an important food crop in Africa, Central America, and South Asia and it is the “fifth most important cereal crop grown in the world”. (National Academy of Sciences, 1996) Guinea corn (sorghum) is the most widely grown of all the cereal crops produced by farmers in the Savanna zones of Nigeria. Annual production in this part of the country is estimated at over four million tonnes of grains. This crop is used almost entirely for human consumption and provides the staple food for most of the population, particularly in the ecological zones most suited for its cultivation.

The cyanogenic glycoside dhurrin constitutes 30% of the dry weight of sorghum shoot tips but is absent from the seed and roots. The co-distribution of dhurrin and its synthesizing system in the upper portion of the shoot shows that production and storage sites are located within the same cells (Adewusi, 1990). Sorghum seedlings contain a specific β-glycosidase capable of converting dhurrin into p-hydroxybenzaldehyde, cyanide, and glucose. Upon damage to sorghum tissues, the enzyme and its substrate, which are compartmentalized in intact tissues, come into contact and release a toxic aglycone or a derivative (HCN) (Siritunga and Sayre, 2003). Under these conditions, dhurrin is hydrolyzed by an endogenous β-glycosidase (dhurrinase) to produce phydroxymandelonitrile, which subsequently disassociates to free HCN and phydroxybenzaldehyde.

Cassava (Manihot esculenta Crantz) is the most important source of dietary carbohydrates for 750 million people around the world, with its starchy root being the main harvested organ (National Academy of Sciences, 1996). Sorghum (Sorghum bicolor (L) Moench), king of millets is one of the staple food crops of the world, especially the drier parts of tropical Africa and Asia. It is a principal source of energy, protein, vitamins and minerals for millions of the poorest people in the regions where it is cultivated (National Academy of Sciences, 1996). Both crops contain potentially toxic compounds called cyanogenic glycosides, linamarin and dhurrin respectively, which are broken down by their specific enzymes linamarase and dhurrinase upon disruption of the plant cells to form hydrogen cyanide. In humans, the symptoms of acute cyanide intoxication from inadequately prepared cassava or sorghum can include: rapid respiration, drop in blood pressure, rapid pulse, dizziness, headache, stomach pains, vomiting, diarrhoea, mental
confusion, twitching and convulsions (Bokanga et. al., 1994). Therefore, proper processing methods which ensure total liberation of cyanogens must be developed.

1.1 Null Hypothesis

On account of the unique expression of linamarase and dhurrinase in their respective crops and the properties of the enzymes in catalysis of their substrates, a novel approach can be developed for the total hydrolysis of cyanogenic glycosides from cassava in the fermentation process leading to the production of cyanide free products. I therefore assume that the biochemical and molecular parameters measured for linamarase and dhurrinase are dissimilar.

1.2 Justification of Study

Cassava produces potentially toxic levels of cyanogenic glycosides (Linamarin (95%) and lotaustralin (5%) which are synthesized in the leaves and translocated to all other parts of the plant including the edible tuberous roots (McMahon et. al., 1995). The breakdown of cyanogenic glycosides by β–glycosidase enzymes results in hydrogen cyanide (HCN) production when cassava tissues are mechanically damaged. Hydrogen Cyanide in cassava tissues has been medically proven to be a potential health hazard forconsumers if the plant is inadequately processed (McMahon et. al., 1995). Sorghum also expresses cyanogenic glycosides but mainly at the seedling stage and only at the shoot
tips. Cassava, with the exception of the seeds, expresses in every tissue and at all times.

There exist similarities between the actions of β–glycosidase in these two crops on their respective glycosides resulting in the release of hydrogen cyanide. It will be significant to identify any homologous sequences in genes encoding these glycosidases in local varieties of these crops. Considering that intoxication could lead to rapid respiration, drop in blood pressure, rapid pulse, dizziness, headache, stomach pains, vomiting, diarrhoea, mental confusion and convulsions; even death may occur in some cases, the purpose therefore, is to determine the comparative nature of these toxic principles in their respective crops; the ultimate goal being to be able to produce cyanide free cassava products. The molecular analyses will go a long way to increase the understanding of β-glycosidase gene structure in different plant species.

1.3 Research Questions

In an effort to establish the possible relationship between Cassava linamarase and Sorghum dhurrinase, the following research questions have been considered.

i. What is the size of the gene in both plants and does this correspond to information in the gene bank?

ii. Do the sequences correspond to the gene bank and what similarities exist between the two sequences?

iii. What are the kinetic properties of the native enzyme extracts and how do these properties relate to each other?

1.4 Aim of the Study

Compare the biochemical and molecular parameters for linamarase (α-Hydroxyisobutyronitrile β-D-glucopyranoside) and dhurrinase (4- Hydroxymandelonitrile- β-D-glucoside)

1.5 Objectives of the Study

i. To isolate and quantify total DNA from Cassava (Manihot esculenta Crantz) and Sorghum (Sorghum bicolor (L) Moench)

ii. To amplify the gene encoding linamarase (α-Hydroxyisobutyronitrile β-Dglucopyranoside) and dhurrinase (4-Hydroxymandelonitrile- β-D-glucoside) from the isolated total DNAs by PCR.

iii. To determine kinetic properties of the native enzymes isolated from Sorghum (Sorghum bicolor (L) Moench) and Cassava (Manihot esculenta Crantz)

1.6 Significance of Study

The results of a study such as this are important in contributing further to the body of knowledge on the gene structure and origin of β-glycosidases in plants. More significantly, it will increase the availability of basic enzymological data obtained by molecular characterization. Also, the use and trial of modified versions of certain assays are expected to contribute to the process of defining more robust, yet simple and easily applicable assays for isolation and amplification of genes. There is also the possibility of contributing to knowledge which could aid in gene expression studies, leading to the selection of local crop varieties for cyanogens.