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Role of Kynureninase in Sporulation of Bacillus Cereus. Chandra Kumar Banerjee Louisiana State University and Agricultural & Mechanical College

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Role of Kynureninase in Sporulation of Bacillus Cereus. Chandra Kumar Banerjee Louisiana State University and Agricultural & Mechanical College Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1974 Role of Kynureninase in Sporulation of Bacillus Cereus. Chandra Kumar Banerjee Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Banerjee, Chandra Kumar, "Role of Kynureninase in Sporulation of Bacillus Cereus." (1974). LSU Historical Dissertations and Theses. 2651. https://digitalcommons.lsu.edu/gradschool_disstheses/2651 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This material was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1.The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. 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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 75-1912 BANERJEE, Chandra Kumar, 1941- ROLE OF KYNURENINASE IN SPORULATION OF BACILLUS CEREUS. The Louisiana State University and Agricultural and Mechanical College, Ph.D., 1974 Microbiology Xerox University Microfilms,Ann Arbor, Michigan 48106 THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. Role of Kynureninase in Sporulation of Bacillus cereus A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Microbiology by Chandra K. Banerjee B.Sc. (Hons.) U. P. Agricultural University, India, 1965 M.Sc. U. P. Agricultural University, India, 1969 August, 1974 ACKNOWLEDGEMENTS The author wishes to express his deep appreciation to Dr. V. R. Srinivasan for his guidance and assistance during the course of this investigation. Special thanks are extended to the staff and faculty members of the Department of Microbiology for their helpful suggestion and cooperation from time to time. Thanks are also extended to his wife Meena, who in various ways contributed to the progress of this work. A sincere appreciation is expressed to Mrs. M. W. Bumm, Dr. M. Fleenor and Mr. Bianchini N. Vittori for their assistance. Thanks are extended to Mrs. Linda McNabb for typing and helping in proof­ reading this dissertation. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . ii LIST OF TABLES.................................................. v LIST OF FIGURES ................... vi ABSTRACT......................................... viii INTRODUCTION.................................................... 1 REVIEW OF LITERATURE..................... 3 A. Heat Resistance of Bacterial Spores................. 3 B. Metabolism of Kynurenine ............................ 6 (i) Biochemical pathways ............................ 6 (1) Niacin pathway .............................. 7 (2) Aromatic pathway ............................ 10 (3) Quinoline pathway..................... 13 (4) Other pathways .............................. 13 (ii) Kynureninase..................................... 20 (ill) Mechanism of kynureninase action ............. 20 (iv) Inhibition of kynureninase by amines........... 22 (v) Importance of kynureninase ...................... 25 MATERIALS AND METHODS ......................................... 28 A. Organism ............................................. 28 B. Media................................................. 28 C. Cultivation of the Organism......................... 28 D. Selection of Heat-Sensitive Mutants................. 29 E. Measurement of Density of Spores ................... 30 F. Germination........................................... 30 G. Preparation of Sepharose-kynurenine Derivative . 30 H. Assay of Kynureninase................ ............... 34 I. Sucrose Density Gradient Centrifugation............. 35 iii Page J. Polyacrylamide Disc Electrophoresis................. 35 K. Analytical Methods .................................. 36 L. Buffers............................................... 37 (i) Phosphate buffer ................................ 37 (ii) Standard buffer ................................ 38 M. Chemicals...................... „ ..................... 38 RESULTS ............. 39 A. Properties of Heat Sensitive Mutants ............... 39 B. Purification of Kynureninase ....................... 52 (i) Preparation of cellular extract................. 52 (ii) Precipitation with PEI 1000 and extraction with ammonium chloride.......................... 52 (iii) Ammonium sulfate precipitation ............... 52 (iv) Affinity chromatography ........................ 53 C. Kinetics of Kynureninase Reaction................... 57 D. Properties of Kynureninase......................... 64 E. Comparison of Kynureninase From the Heat-Resistant Wild Type B. cereus TR-2 and Temperature-Sensitive Sporulation Mutant Ts-17 ............................ 75 DISCUSSION............. 8 6 LITERATURE CITED....................................... 93 VITA............................................................. 103 iv LIST OF TABLES Table Page 1. Growth, sporulation, DPA content and heat resistance of spores of the mutants and the parent strain ......... 40 2. Comparison of chemical constituents of spores of B. cereus T^q -2 and B. cereus N5Y-1..................... 43 3. Comparison of buoyant densities of B. cereus T40"2 B. cereus N5Y-1 spores .................................. 44 4. Comparison of kynureninase activity in the mutant (N5Y-1) and the parent strain (T^q -2) of B. cereus . 49 5. Summary of the purification scheme ...................... 56 6 . Effects of various cations on the activity of kynureninase ............................................. 72 v LIST OF FIGURES Figure Page 1. Niacin pathway of kynurenine metabolism ................ 9 2. Aromatic pathway of kynurenine metabolism .......... 12 3. Quinoline pathway of kynurenine metabolism............. 15 4. Other pathways of kynurenine metabolism ................ 17 5. Anthranilate cycle in Neurospora........................ 19 6 . Mechanism of kynureninase action as proposed by Longenecker and Snell (1955)............................ 24 7. Derivatization of Sepharose-4B.......................... 33 8 . Growth of B. cereus T4 0 - 2 and N5Y-1 in G-medium at 30 and 40 C ........................................... 42 9. Effect of duration of heat-shock at 80 C on germination of B. cereus T4 0 - 2 and N5Y-1 in L-alanine and adenosine ..................... 47 10. Changes in pH absorbance and the -level of kynureninase activity during growth and sporulation of B. cereus T^q -2............ .......................... 51 11. Elution profile of ammonium chloride extract on matrix-B.................................................. 55 12. Polyacrylamide gel electrophoresis of purified kynureninase............................................. 59 13. Hofstee plot of kynureninase activity with L-kynurenine sulfate as the substrate . 61 14. Lineweaver-Burk plot of kynureninase activity with . L-kynurenine sulfate as substrate ................... 63 .15. Sedimentation pattern of kynureninase in a 5-257» sucrose density gradient................................ 6 6 16. Effect of hydrogen ion concentration on the kynureninase activity 6 8 vi Figure Page 17. Variation in kynureninase activity with the amount of pyridoxal-51-phosphate at a substrate concentration used in the routine assay ................................. 71 18. Heat inactivation of kynureninase........................ 74 19. Rate of disappearance of kynurenine in reactions catalyzed by the cellular extracts of the mutant, wild type and wild type following incubation.........
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