HYDROLYSIS OF CASSAVA STARCH BY AMYLASES FROM MAIZE

By

Martins

Presented To

Department of Biochemistry

ABSTRACT
Crude maize amylase was extracted from twenty (20) cultivars of maize grains obtained from International Institute of Tropical Agriculture (IITA), Ibadan. Cassava starch was extracted from eight (8) varieties of hydrid cassava tubers from IITA, Ibadan. Maize amylase activity was determined by the Dinitrosalicyclic acid (DNS) method using 1% pure soluble corn starch as substrate. The effects of pH, Ca2+ ion on enzyme activity with highest activity was used to hydrolyze the cassava starch from the eight varieties of cassava. Amylase activity was found in
ungerminated and germinating maize. The highest activity was detected on the third day of germination in all maize varieties. The pH profile produced three peaks at pH 5.5, 7.5 and 9.5 suggesting three isoenzyme forms suspected to be -amylase, --amylase and limit dextrinase, Ca2+ (0.05M) activated amylase activity two fold, while Ca2+ (0.1M) inhibited it two-fold. The highest amylase activity was determined with maize variety coded Y-Syn-VAR-IF2. All eight varieties of cassava starch were hydrolyzed by maize amylase. The highest amount of reducing sugar (glucose) was obtained from the cassava variety coded TME 419. The rate of hydrolysis of cassava starch was as pure soluble corn starch.

TABLE OF CONTENTS
PAGE
Title Page i
Certification ii
Dedication iii
Acknowledgements iv
Abstract v
Table of Contents vi
List of Figures ix
CHAPTER ONE: INTRODUCTION
11 Introduction … … … … … … … … … 1
12 Biology of Cassava and Maize … … … … … … 2
121 Cassava … … … … … … … … … 2
122 Maize … … … … … … … … … 6
13 Starch … … … … … … … … … 8
131 Starch properties … … … … … … … … 8
132 Test for starch … … … … … … … … 10
133 Cassava starch … … … … … … … … 11
134 Starch synthesis … … … … … … … … 12
135 General biosynthesis of polysaccharides … … … … … 12
136 Biosynthesis of a glycosidic bond … … … … … 14
137 Structural alterations to sugar nucleotide before polysaccharide assembly … 16
138 Starch hydrolysis (degradation) … … … … … … 17
139 Acid hydrolysis … … … … … … … … 17
1310 Enzyme hydrolysis of starch … … … … … … … 19
14 Amylases … … … … … … … … … 19
141 General properties of amylases … … … … … … 19
142 Sources of amylases … … … … … … … … 20
143 Plant amylases… … … … … … … … … 20
144 Amylases production in germinating cereals … … … … 21
145 Maize amylases … … … … … … … … 22
15 Aims and objectives of research … … … … … … 23
CHAPTER TWO: MATERIALS AND METHODS
21 Materials … … … … … … … … … 24
211 Sample collection and location(s) … … … … … … 24
2111 Cassava varieties … … … … … … … … 24
2112 Maize varieties … … … … … … … … 24
212 Chemicals/Reagents/Samples … … … … … … 25
213 Apparatus … … … … … … … … … 26
22 Methods … … … … … … … … … 26
221 Preparation of buffers … … … … … … … 26
222 Extraction of cassava starch … … … … … … 27
223 Glucose calibration curve … … … … … … … 27
224 Enzyme extraction … … … … … … … 27
225 Method of enzyme assay … … … … … … … 27
226 Effect of germination on maize amylase activity … … … 27
227 Effect of pH on maize amylase activity … … … … … 28
228 Germination of maize … … … … … … … 28
229 Determination of activity in 20 varieties of hydrid maize … … … 28
2210 Assay of amylase activity in maize … … … … … … 28
2211 Effect of Ca
2+
ions on amylase activity … … … … … 29
2212 Determination of protein concentration in all enzyme extracts … … 29
2213 Protein determination … … … … … … … 29
2214 Maize amylase specific activity … … … … … … 30
2215 Activity of maize amylase on cassava starch … … … … 30
2216 Effect of pH on amylase activity using cassava starch as substrate … … 30
2217 Effect of substrate concentration on enzyme activity … … … 30
23 Statistical Analysis … … … … … … … 31
CHAPTER THREE: RESULTS
31 Effect of germination on amylase activity … … … … … 32
32 Effect of pH on amylase activity … … … … … … 32
33 Amylase activity before imbibition at pH 55, 75 and 95 … … … 33
34 Amylase activity on day 3 after imbibition at pH 55, 75 and 95 … … 34
35 Amylase activity on day 5 after imbibition at pH 55, 75 and 95 … … 35
36 Protein determination … … … … … … … 39
37 Maize amylase specific activity … … … … … … 41
38 Determination of maize variety with the highest amylase activity … … 45
39 Hydrolysis of cassava starch by enzyme extract from VARIF2 … … 45
310 Effect of Ca
2+
on enzyme activity … … … … … 45
311 Effect of substrate (starch) concentration on maize enzyme activity … 45
312 Effect of pH on amylase activity using 1% cassava starch … … … 46
CHAPTER FOUR: DISCUSSION AND CONCLUSION
41 DISCUSSION … … … … … … … … 50
42 CONCLUSION … … … … … … … … 55
REFERENCES … … … … … … … … … 57
APPENDICES … … … … … … … … … 62

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