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Information to Users INFORMATION TO USERS This manuscript has been reproduced hx>m the microfilm master. UMI films the text directly fi’om the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter 6ce, while others may be fi-om any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing fi’om left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 CHARACTERIZATION, PURIFICATION, AND MOLECULAR CLONING OF HYDROXYNITRILE LYASE FROM CASSAVA (MANIHOT ESCULENTA CRANTZ ) DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Wanda Lynn Broyles White, B.S. ***** The Ohio State University 1996 Dissertation Committee: Approved by; Dr. Richard T. Sayre, Adviser Dr. Michael Evans Dr. Terrence Graham Adviser Plant Biology Dept. UMI Number: 9710682 UMI Microform 9710682 Copyright 1997, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT Cassava {Manihot esculenta Crantz) is a staple food crop in Africa. The cyanogenic glycoside, linamarin, is stored in cassava cells and probably functions as an herbivore deterrent. When cassava roots are prepared for human consumption, linamarin and its degradation products, acetone cyanohydrin eind hydrogen cyanide, can remain in the food products. Exposure to these cyanogens has been linked to epidemics of neurological disorders in Africa. Acetone cyanohydrin is the main contributor to cyanide exposure from cassava consumption. We purified hydroxynitrile lyase, which converts acetone cyanohydrin to acetone and hydrogen cyanide, from cassava leaf apoplast extracts and whole leaves. The enzyme was a homodimer of 29 kD subunits. The pH and temperature optima were 5.0 and 30°C, respectively. The enzyme displayed typical Michaelis-Menten kinetics. Activity was saturated by 20 mM acetone cyanohydrin and the Km was 0.925 mM. Hydroxynitrile lyase maintained over 50 percent of its optimal activity at pH 4.0-5.5 and temperatures 4-45°C. Also, the enzyme was not permanently inactivated by 24 hours of exposure to low pHs (4.0-6.0). ii Anti-hydroxynitrile lyase antibodies were raised and used to localize the enzyme. Using FITC-labeled secondary antibodies ^ hydroxynitrile lyase was found in the cell walls of leaf epidermal, parenchyma, and mesophyll cells. Western blots demonstrated the presence of hydroxynitrile lyase in leaves and not stems or roots. The gene encoding hydroxynitrile lyase was isolated from a cassava leaf cDNA library. The 1.1 kb cDNA clone encoded a 258 amino acid protein. The derived amino acid sequence had 78 percent identity to rubber tree (Hevea brasiliensis) hydroxynitrile lyase and 30 percent identity and 56 percent similarity to two pathogen induced response proteins from rice {Oryza sativa). Southern blot analysis supported the presence of one copy of the hydroxynitrile lyase gene in the cassava genome. Little expression of the hydroxynitrile lyase gene (1.3 kb mRNA) was found in cassava stems and roots. Ill ACKNOWLEDGMENTS I would like to thank the members of my committee, Mike Evans, Terrence Graham, and Luther Waters, and my adviser, Richard Sayre for their efforts in the successful completion of my project. Ron Hutchison, Diana Arias, Stuart Ruffle, and meiny other members of the lab helped me along the way with experiments. I hope I have given as much help back to the lab as I have received. Dr. Graham and his group, Ming-Ching Hsieh, especially, helped me use the HPLC and taught me plant pathology techniques. I would like to thank Drs. Sack, Coplin, Davis, McDonald, and Yang for the use of equipment. Thanks also to their students who helped me use it; Yakang Lin, Greg Bell, Shalu Mittal, and Matt Geisler. Finally I would also like to thank those who prepared me for my work here at OSU. I thank the faculty of Rhodes College, especially members of the Biology Department, for teaching me invaluable lessons about science and, more importantly, personal growth. I thank my family for their support throughout the years and their sacrifices for my education. I thank my husband. Trey, for his constant compeuiionship and encouragement. iv VITA May 11, 1969.................. Bom-Johns on City, Tennessee 1991.......................... B.S. Biology, Rhodes College 1991-present.................. Graduate Teaching Associate, The Ohio State University PUBLICATIONS White, W.L.B., and R.T. Sayre (1993) Peurtial purification and characterization of hydroxynitrile lyase from cassava. In: Roca, W. and Thro, A., eds. Proceedings of the first international scientific meeting of the Cassava Biotechnology Network, Cartegena, Colombia, 25-28 August 1992. Cali, Colombia: Centro International de Agriculture Tropical, 379-383. White, W.L.B., McMahon, J.M., and R.T. Sayre (1994) Regulation of cyanogènesis in cassava. Acta Hortlculturae 375: 69-78. McMcihon, J.M., White, W.L.B., and R.T. Sayre (1995) Cyanogenesis in cassava. Journal of Experimental Botany 288: 731-741. White, W.L.B., and R.T. Sayre (1995) The characterization of hydroxynitrile lyase for the production of safe food products from cassava (Manihot esculenta Crantz). In: D.L. Gustine and H.E. Flores, eds. Phytochemicals and Health. Proceedings of the Tenth Annual Penn State Symposium in Plant Physiology, May 18-20, 1995. (American Society of Plant Physiologists, Rockville, Maryland) pp. 303-4. FIELDS OF STUDY Major Field: Plant Biology TABLE OF CONTENTS Page Abstract.............................................. 11 Acknowledgments....................................... Iv Vita.................................................. V List of Tables........................................ Ix List of Figures....................................... x List of Abbreviations................................. xll Chapters : 1. Introduction..................................... 1 1.1 General Information about Cassava.......... 1 1.2 Cyanogenesis In Cassava.................... 5 1.3 The Role of Cyanogenesis................... 12 1.4 Cyanogenesis and Human Consumption......... 15 1.5 The Goals of This Research Project......... 21 2. Purification and Characterization of Hydroxy- nltrlle Lyase from Cassava....................... 23 2.1 Introduction................................ 23 2.2 Methods..................................... 25 2.2.1 Assay for Hydroxynitrile Lyase Activity.......................... 25 2.2.2 Purification of Hydroxynitrile Lyase from Apoplast Extracts...... 27 2.2.3 Purification of Hydroxynitrile Lyase from Whole Leaves........... 29 2.2.4 Determination of Native Molecular Weight............................ 30 2.3 Results...................................... 30 2.3.1 Spontaneous Degradation of Acetone Cyanohydrin Versus Enzymatic Breakdown......................... 30 2.3.2 Purification of Hydroxynitrile Lyase from Leaf Apoplast Extracts.. 31 VI 2.3.3 Characterization of Hydroxynitrile Lyase: Stability and Optimization of Activity....................... 36 2.3.4 Purification of Hydroxynitrile Lyase from Whole Leaf Extracts 40 2.3.5 Determination of Native Molecular Weight............................ 40 2.3.6 Determination of Kinetic Properties 45 2.4 Discussion................................... 47 Immunolocalization of Hydroxynitrile Lyase....... 51 3.1 Introduction................................ 51 3.2 Methods..................................... 53 3.2.1 Western Blot....................... 53 3.2.2 Fluorescent Immunolocalization of Hydroxynitrile Lyase in Leaves 54 3.3 ^^esults...................................... 55 3.3.1 Localization and Quantitation of Hydroxynitrile Lyase in Cassava Organs............................ 55 3.3.2 Cellular Localization of Hydroxy­ nitrile Lyase in Cassava Leaves.... 55 3.4 Discussion................................... 57 Cloning of the Hydroxynitrile Lyase cDNA......... 60 4.1 Introduction................................ 60 4.2 Methods..................................... 62 4.2.1 Screening the cDNA Library......... 62 4.2.2 DNA Sequencing and Sequence Analysis.......................... 67 4.2.3 DNA Isolation and Southern Blot.... 68 4.3 Results...................................... 68 4.3.1 Hydroxynitrile Lyase cDNA and Derived Amino Acid Sequence....... 69 4.3.2 Southern Blot...................... 71 4.4 Discussion.................................. 73 Expression
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