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Purification and Characterization of Novel Nucleases from a Thermophilic Fungus Kyle S University of Massachusetts Amherst ScholarWorks@UMass Amherst Masters Theses 1911 - February 2014 2012 Purification and Characterization of Novel Nucleases from a Thermophilic Fungus Kyle S. Landry University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/theses Part of the Agriculture Commons, Biochemistry Commons, Biotechnology Commons, Environmental Microbiology and Microbial Ecology Commons, Food Biotechnology Commons, Food Microbiology Commons, and the Other Plant Sciences Commons Landry, Kyle S., "Purification and Characterization of Novel Nucleases from a Thermophilic Fungus" (2012). Masters Theses 1911 - February 2014. 804. Retrieved from https://scholarworks.umass.edu/theses/804 This thesis is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Masters Theses 1911 - February 2014 by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. PURIFICATION AND CHARACTERIZATION OF NOVEL NUCLEASES FROM A THERMOPHILIC FUNGUS A Thesis Presented By KYLE S. LANDRY Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE May 2012 Food Science © Copyright by Kyle S. Landry 2012 All Rights Reserved PURIFICATION AND CHARACTERIZATION OF NOVEL NUCLEASES FROM A THERMOPHILIC FUNGUS A Thesis Presented By KYLE S. LANDRY Approved as to style and content by: _________________________________________________ Robert E Levin, Chair _________________________________________________ Ronald G Labbe, Member _________________________________________________ Robert Wick, Member _____________________________________________ Eric Decker, Department Head Department of Food Science ACKNOWLEDGMENTS I wish to express my appreciation to my advisor Dr. Robert E. Levin for his guidance and encouragement throughout the course of my studies. I would also like to thank Dr. Ronald G. Labbe of the Department of Food Science and Dr. Robert Wick of the Department of Plant, Soil and Insect Science for serving as members of my thesis committee and also for all of the support they have provided. I would also like to express great appreciation towards Ruth Witkowsky and the countless time and support she has provided during my time here. Her untiring effort to maintain a safe and productive environment should not go unnoticed. Finally, I would like to thank my family for their support and continual belief that I can do anything I set my mind to. iv ABSTRACT PURIFICATION AND CHARACTERIZATION OF NOVEL NUCLEASES FROM A THERMOPHILIC FUNGUS MAY, 2012 KYLE S. LANDRY, B.S., FRAMINGHAM STATE COLLEGE M.S., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Robert E. Levin A thermophilic fungus was isolated from composted horse manure. The organism was as a Chaetomium sp. by sequencing the highly conserved ITS region of the fungus and comparing to known regions in a genomic database and was referred to as TM-417. TM-417 was found to have an optimal growth temperature of 45 oC and an optimal pH of 7.0. An extracellular DNase and RNase was found to be produced by the isolate and were purified 145.58-fold and 127.6-fold respectively using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined electrophoretically using 4-15% gradient polyacrylamide gels. Both DNase and RNase were dependent on metal co-factors for activity. The metal ion Mg2+ was the preferred ion for the DNase, whereas for the RNase, Zn2+ and Mn2+ yielded an increase in enzyme activity over that with Mg2+. The purified DNase demonstrated maximum activity at pH 6.0 with no activity at pH 2.0 or 10.0. The RNase exhibited two peaks of maximum activity, on at pH 3.0 and the other at pH 7.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the purified DNase was 65oC. The optimal temperature for the RNase was 70oC. The molecular of the DNase and RNase were determined to be 56 kDa and 69kDa respectively v using a Sephadex G-75 column. A standard curve was generated using several standard proteins of known molecular weight. vi Table of Contents Page LIST OF TABLES ............................................................................................................................... xi LIST OF FIGURES ............................................................................................................................ xii LIST OF DIAGRAMS ........................................................................................................................ xv ABBREVIATIONS ........................................................................................................................... xvi INTRODUCTION ............................................................................................................................... 1 CHAPTER 1. LITERATURE REVIEW ................................................................................................................... 3 Thermophilic Fungi ...................................................................................................................... 3 History ...................................................................................................................................... 3 Ecology ..................................................................................................................................... 4 Nutritional Requirements ........................................................................................................ 6 Isolation ................................................................................................................................... 7 Taxonomic Identification of Thermophilic Fungi ............................................................................ 8 Thermophilic Fungi in Biotechnology ...................................................................................... 9 General Characteristics of Nucleases ......................................................................................... 11 Mechanism ............................................................................................................................ 11 Site Specificity ........................................................................................................................ 13 Sugar Specificity ..................................................................................................................... 13 Strand Specificity ................................................................................................................... 14 Metal Ion Dependent and Independent Nucleases ............................................................... 14 Mode of Attack .......................................................................................................................... 15 Endonucleases ....................................................................................................................... 15 Exonucleases .......................................................................................................................... 17 Fungal Nucleases ........................................................................................................................ 18 Classification of Ribonucleases .................................................................................................. 19 Bovine Pancreatic RNase ....................................................................................................... 19 Classification of Deoxyribonucleases ......................................................................................... 20 Deoxyribonuclease I ............................................................................................................... 21 Deoxyribonuclease II .............................................................................................................. 22 vii Catalytic Properties .................................................................................................................... 23 Divalent Metal Ions ................................................................................................................ 23 Inhibition of DNase ................................................................................................................ 25 Assay and Purification Techniques ............................................................................................ 26 Challenge of DNase Studies ................................................................................................... 26 Various Assay Techniques ...................................................................................................... 27 Purification Techniques ......................................................................................................... 29 2. MATERIALS AND METHODS ...................................................................................................... 31 Fungal Methods ......................................................................................................................... 31 Culture Isolation..................................................................................................................... 31 Initial DNase Screening .........................................................................................................
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