Metabolism, enzymology, and genetic characterization of caffeine degradation by pseudomonas putida CBB5

Abstract

To my girls, Emmie, Jane, and most especially Rachel, for their support throughout this entire process and for the joy they bring to my life. iii ACKNOWLEDGMENTS Obviously, a work of this magnitude requires assistance from many people. I am deeply grateful to all of those listed here and to anybody that I might have forgotten. First and foremost, I would like to thank my thesis advisor, Dr. Mani Subramanian, for accepting me into his lab. His industry-style approach and constant lab meetings helped me to focus, stay on track, and not get distracted as I worked on this research. I always wanted to have some good news whenever he asked the question, "Anything interesting?" His knowledge of microbiology, biochemistry, and molecular biology was a vast resource from which I was able to draw ideas countless times over the last four years-from his suggestion to add iron to the N-demethylase activity assay to his plan to clone the ndmA and ndmB genes separately, and everything in between. There are many talented people in our research group who contributed greatly to this project. Dr. Chi Li Yu isolated CBB5 from the soil and performed many of the studies to determine its metabolism of caffeine and theophylline. She was also my mentor on enzyme purification, teaching me how to equilibrate columns, load and elute proteins, assay for activity, and many other procedures. Dr. Michael Louie both performed research and served as my mentor for all of the molecular biology work performed here. He constructed the gDNA libraries, suggested and performed many of the techniques used to sequence and clone the ndmA and ndmB genes, and answered more questions in four years than anybody should have to answer. I've received a lot of credit for the research described herein, but these two people have also put a lot of work and brainpower into the project. Katelynn Louie also helped with the molecular biology work and continuously kept me supplied with HPLC vials, filters, and inserts in order to get all of the kinetic assays done. Bhavita Bhatt has continued to keep the supplies in stock. Dr. Shuvendu Das has served as a resource for questions regarding HPLC and LCMS. Fellow graduate v TABLE OF CONTENTS LIST OF TABLES .

A. Genetic characterization of the caffeine C-8 oxidation pathway in Pseudomonas Sp. CBB1 B. Validation of caffeine dehydrogenase as a suitable enzyme for a rapid caffeine diagnostic test

“…However, unlike higher organisms, caffeine metabolism in microbes did not receive much attention until the 1970s [Gopishetty et al, 2011]. Within the microbial kingdom, although biochemistry and enzymology of caffeine degradation in bacterial systems is well studied [Dash et al, 2006;Yu et al, 2008Gopishetty et al, 2011;Summers et al, 2011;Summers et al, 2012], little is known about caffeine utilization by yeast and fungi. Caffeine metabolism in fungi like Aspergillus niger [Ina et al, 1971] and Penicillium roqueforti [Schwimmer et al, 1971] occurs via a similar N-demethylation pathway as found in higher organisms, where N-7 demethylation of caffeine yields theophylline [Hakil et al, 1998].…”