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Directed Evolution of Cyanide Degrading Enzymes

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Directed Evolution of Cyanide Degrading Enzymes DIRECTED EVOLUTION OF CYANIDE DEGRADING ENZYMES A Dissertation by MARY ABOU NADER Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Michael Benedik Committee Members, Carlos Gonzalez Matthew Sachs Wayne Versaw Head of Department, U. J. McMahan December 2012 Major Subject: Microbiology Copyright 2012 Mary Abou Nader ABSTRACT Cyanide is acutely toxic to the environment. However, this simple nitrile is used in several industrial applications especially the mining industry. Due to its high affinity to metals, cyanide has been used for years to extract gold and other precious metals from the ore. Cyanide nitrilases are considered for the detoxification of the industrial wastewaters contaminated with cyanide. Their application in cyanide remediation promises cheaper and safer processes compared to chemical detoxification. However, application of these enzymes in industry requires improving their characteristics. The goal of this dissertation is to better understand cyanide nitrilases, in particular the cyanide dihydratase from of Bacillus pumilus and Pseudomonas stutzeri and to improve their activity and stability. The lack of any high resolution structure of these enzymes calls for isolating or screening for mutants showing enhancement in enzyme properties. Described first is a simple and efficient method utilizing in vivo recombination to create recombinant libraries incorporating the products of PCR amplification. This method is useful for generating large pools of randomly mutagenized clones after error- prone PCR mutagenesis. Several parameters were investigated to optimize this technique; length of homology region, vector treatment, induction time and ratio of fragment to vector. Using error-prone PCR for random mutagenesis, several CynDpum mutants were isolated for higher catalysis at pH 7.7. Three point mutations, K93R, D172N and E327K ii increased the enzyme’s thermostability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 suggesting an effect on the active site. However, the A202T mutation located in the dimerization or the A surface rendered the enzyme inactive by destabilizing it. No significant effect on activity at alkaline pH was observed for any of the purified mutants. Lastly, an important region for CynDstut activity was identified in the C-terminus. This same region increased the stability of CynDpum compared to the wild-type enzyme. Also, CynDpum-stut hybrid was found to be highly more stable than CynDpum. This same hybrid exhibited 100% activity at pH9, a pH where the parent enzyme is inactive, and retained 40% of its activity at pH 9.5 making it a true pH tolerant mutant. iii DEDICATION To my parents, my sisters, my husband and my precious Blueberry for their endless love, support and encouragement. “Scientific research constitutes for you, as it does for many, the way for the personal encounter with truth, and perhaps the privileged place for the encounter itself with God, the Creator of heaven and earth." Pope John Paul II iv ACKNOWLEDGEMENTS I would like to express my gratitude for my graduate advisor, Dr. Michael Benedik, for providing me with invaluable support, guidance and assistance throughout my graduate studies. It has been a blessing and honor to be part of his lab. I also would like to thank my committee members, Dr. Carlos Gonzalez, Dr. Matthew Sachs and Dr. Wayne Versaw, for their constant guidance and evaluation during these years. A special thanks to Dr. Trevor Sewell for his advice and collaboration on the cyanide project and Dr. Bruce Riley for allowing me to rotate in his laboratory during my first semester at Texas A&M University. For their invaluable help, I would like to thank all the undergraduates that I had the opportunity to work with especially Kate Brels, Ilka Maza and Henry Lessen. I would like to thank my labmates and friends, Lacy Basile, Tysheena Charles, Lan Wang, Allyson Martinez, Alvaro Rodriguez and Jason Park. I would like to express my love and gratitude for my family and friends in Lebanon, Australia and USA. Thank you for supporting and encouraging me all these years. v NOMENCLATURE HCN Hydrogen cyanide CN- Cyanide ion NaCN Sodium cyanide KCN Potassium cyanide CynD Cyanide dihydratase CHT Cyanide hydratase CynDpum Cyanide dihydratase from Bacillus pumilus CynDstut Cyanide dihydratase from Pseudomonas stutzeri AK61 CHTcrassa Cyanide hydratase from Neurospora crassa EP-PCR Error-prone PCR vi TABLE OF CONTENTS ABSTRACT .......................................................................................................................ii DEDICATION .................................................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................... v NOMENCLATURE .......................................................................................................... vi LIST OF FIGURES ............................................................................................................ x LIST OF TABLES ...........................................................................................................xii CHAPTER I INTRODUCTION AND LITERATURE REVIEW ................................... 1 Overview ................................................................................................................ 1 Cyanide chemical properties .................................................................................. 2 Cyanide toxicity ..................................................................................................... 3 Cyanide in nature ................................................................................................... 3 Cyanide in industry ................................................................................................ 5 Cyanide detoxification ........................................................................................... 7 Cyanide degrading enzymes ................................................................................. 11 Reduction pathway ......................................................................................... 11 Oxidation pathway ......................................................................................... 13 Hydrolysis pathway ........................................................................................ 15 Substitution/transfer pathway ......................................................................... 16 Nitrilase superfamily ............................................................................................ 21 Nitrilases ......................................................................................................... 23 Nitrilase structure ........................................................................................... 26 Directed evolution and error-prone PCR ............................................................. 31 Summary and purpose .......................................................................................... 32 CHAPTER II RAPID GENERATION OF RANDOM MUTANT LIBRARIES ............ 35 Overview .............................................................................................................. 35 Materials and methods ......................................................................................... 37 DNA fragment preparation ............................................................................. 37 Vector preparation .......................................................................................... 39 Positive selection vector ................................................................................. 39 Transformation ............................................................................................... 39 Results .................................................................................................................. 40 vii Effect of length of homology ......................................................................... 40 Vector preparation .......................................................................................... 43 Effect of induction time .................................................................................. 45 Ratio of fragment to vector ............................................................................ 46 Construction of a positive selection vector .................................................... 47 Discussion ............................................................................................................ 48 CHAPTER III CYANIDE DIHYDRATASE FROM BACILLUS PUMILUS MUTANT WITH HIGHER CATALYTIC ACTIVITY AT pH 7.7 ............................... 50 Overview .............................................................................................................. 50 Materials and methods ......................................................................................... 54 Culture media and reagents ............................................................................ 54 Bacterial strains and plasmids ........................................................................ 54 Error-prone PCR ............................................................................................ 54 PCR fragment cloning ...................................................................................
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