STUDIES ON MICROBIAL PRODUCTION OF LIPOXYGENASE INHIBITOR A thesis submitted to the UNIVERSITY OF MYSORE for the award of the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY By CHIDANANDA.C Department of Fermentation Technology and Bioengineering Central Food Technological Research Institute Council of Scientific and Industrial Research Mysore-570020, INDAI June 2008 Chidananda. C, Date: Senior Research Fellow, Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute, Mysore-570 013. DECLARATION I hereby declare that the thesis entitled “STUDIES ON MICROBIAL PRODUCTION OF LIPOXYGENASE INHIBITOR” submitted to the University of Mysore for the award of the degree of DOCTOR OF PHILOSOPHY is the result of the research work carried out by me in the Discipline of Fermentation Technology and Bioengineering, Central Food Technological Research Institute, Mysore, India, under the guidance of Dr Avinash P Sattur during the period April 2005- June 2008. I further declare that the work embodied in this thesis had not been submitted for the award of degree, diploma or any other similar title. (Chidananda. C) Dr. Avinash P. Sattur, Date: Scientist, Fermentation Technology and Bioengineering Department, CERTIFICATE I hereby certify that the thesis entitled “STUDIES ON MICROBIAL PRODUCTION OF LIPOXIGENASE INHIBITOR” submitted to the University of Mysore for the award of the degree of DOCTOR OF PHILOSOPHY by Mr. CHIDANANDA.C, is the result of the research work carried out by him in the discipline of Fermentation Technology and Bioengineering, Central Food Technological Research Institute, Mysore, India, under my guidance during the period April 2005-June 2008. (Avinash P Sattur) Abstract The thesis reports isolation of several fungal cultures from forest soil and screening of the metabolites for their ability to produce inhibitors against lipoxygenase. Of about 74 isolates, one isolate CFTRI-A-24 found to produce consistently high yields of potent inhibitor, was characterized as Penicillium frequentans. The LOX inhibitor was purified using chromatographic techniques and was identified as “Sclerotiorin”. The inhibition kinetics, mode and mechanism of LOX inhibition, its comparative evaluation to standard compounds, in vitro and in vivo (animal) models evaluation as antioxidant additive and other various biological properties of the inhibitors such as antioxidant, antimicrobial, aldose reductase inhibition, platelet aggregation inhibition, antimutagenic and anticancer property are reported. A medium was designed with optimum physical and nutritional parameters for the efficient production of sclerotiorin with better yields in a lab scale process. Effective recovery of the inhibitor in an economical method was also designed. An effort was made to evaluate sclerotiorin for the possible application as an antioxidant additive in commercial oil sample. ACKNOWLEDGEMENTS I take this opportunity to express my sincere gratitude to my guide Dr. Avinash P Sattur, who guided and inspired me throughout the course. It gives me immense pleasure to thank him for his constant support, encouragement, critical suggestions, constructive criticism and for inculcating the right scientific temperament in me. Without his guidance this study would have not been possible. I am grateful to Dr S. Divakar, Head, FTBE department for his constant encouragement and help extended during my course. I also extend my sincere gratitude to Dr. N.G. Karanth and Dr. M.C. Misra, Former heads of the department for their support and encouragement. I thank Dr. V. Prakash, Director, CFTRI, for providing me an excellent infrastructure to carryout my investigation. My sincere thanks to Dr. L. Jagan Mohan Rao, Dr. T.R. Shamala, Dr. K. Srinivasan and Dr. T.P. Krishnakanth for the timely guidance and help rendered during, structure elucidation, culture characterization, animal study experiments and platelet aggregation experiments respectively. I also extend my heartfelt thanks to Dr. S.G. Prapulla, Dr. M.S. Thakur and to the entire staff and students of Fermentation Technology and Bioengineering department who have helped me at various times during this study. It gives me a great feeling to thank and acknowledge all my lab mates, Suresh, Saleem, Chakri, Mamatha Veeresh, Deepak kulkarni, Naveen Babu, Mohan and Sekhar, Without whom the work would have been definitely not interesting. I specially thank Mamatha and Somanna for their timely help rendered during the study. What’s life without friends, my special thanks to all my buddies and pals, Mahesh, Sandesh, Naveen, Tippeswamy, Sharath, Monu, Abhi, Reddy, Girish, Sanchi, Vasu, Sanju, Ashwin, Jyothi, Neeta and Manju, who were with me through the ups and downs of my life. I would continue to remain forever grateful to all my fellow colleagues and friends who were associated with me. Last but not the least, I wish to acknowledge the support rendered by Council of Scientific and Industrial Research (CSIR) in the form of Senior Research Fellowship. I owe my gratitude to my parents for their blessings and unconditional love and support. I extend my thanks to my brother Sadu as well for his moral support. Chidananda. C Dedicated to My Beloved Parents … CONTENTS CHAPTER-1. INTRODUCTION Page No 1.1. Enzyme inhibitors 1 1.2. Lipoxygenases 3 1.2.1. Nomenclature of lipoxygenases 3 1.2.2. Classification of lipoxygenases 4 1.2.3. Structure of Lipoxygenases (LOXs) 4 1.2.4. Soybean LOX-1 6 1.2.5. Structure of mammalian lipoxygenase rabbit reticulocyte 15-LOX 8 1.2.6. Assays for lipoxygenases 10 1.2.6.1. Polarographic method 10 1.2.6.2. Spectroscopic method 10 1.2.6.3. Radio labeled assay 10 1.2.7. Catalytic mechanism of lipoxygenases 10 1.2.8. Biological role of lipoxygenases in plants 13 1.2.9. Beneficial attributes of LOXs in Foods 14 1.2.10. Arachidonic acid metabolism in mammals 15 1.2.11. Biological role of lipoxygenases in animals 15 1.3. LOXs as pharmacological target 17 1.3.1. 5-lipoxygenase in inflammatory diseases 17 1.3.1.1. Asthma 18 1.3.1.2. Inflammatory skin disease, psoriasis, bowel disease and rheumatoid arthritis 19 1.3.1.3. Atherosclerosis and cardiovascular disease 19 1.3.2. 5-lipoxygenase in cancer 23 1.3.2.1. Anticancer therapy with LOX inhibitors 24 1.3.2.2. Breast cancer 24 1.3.2.3. Lung cancer 24 1.3.2.4. Prostate cancer 24 1.3.2.5. Colon cancer 24 1.4. Classification of LOX inhibitors 25 1.4.1. Redox inhibitors 26 1.4.2. Non-redox inhibitors 29 1.4.3. FLAP (five lipoxygenase activation protein) inhibitors 30 1.4.4. LTB4 receptor antagonists 31 1.4.5. LTD4 receptor antagonists 32 1.4.6. Antioxidants 32 1.4.7. Substrate analogs 34 1.4.8. Other LOX inhibitors 34 1.4.8.1. Dual inhibitors of COX and LOX 34 1.5. Lipoxygenase inhibitors from various sources 35 1.6. Microbial bioactive metabolites 39 1.6.1. Primary metabolites 40 1.6.2. Secondary metabolites 40 1.7. Production of microbial metabolites through fermentation 41 1.8. Regulation of metabolites production in fermentation 43 1.8.1. Carbon regulation 43 1.8.2. Nitrogen regulation 43 1.8.3. Inorganic salts, trace elements and precursor regulation 44 1.9. Scope of the current investigation 45 CHAPTER-2. MATERIALS AND METHODS 2.1. Screening of fungal cultures 48 2.1.1. Isolation of fungi from forest soil flora 48 2.2. General fermentation conditions 48 2.2.1. Submerged fermentation (SmF) of fungal isolates for the production of lipoxygenase inhibitors 48 2.2.2. Solid State Fermentation (SSF) of fungal isolates for the production of lipoxygenase inhibitors 48 2.2.3. Submerged fermentation of Penicillium frequentans CFTRI A-24 for the production of sclerotiorin 49 2.3. General extraction conditions 49 2.3.1. Extraction of crude lipoxygenase inhibitors from SmF broths 49 2.3.2. Extraction of crude lipoxygenase inhibitors from SSF 49 2.3.3. Extraction of sclerotiorin from Smf broth 50 2.4. Media composition 50 2.4.1. Medium for screening of fungal isolates 50 2.4.2. Medium for maintenance of isolates (PDA) 50 2.4.3. Different production media used for the production of sclerotiorin from Penicillum frequentans CFTRI-A-24 51 2.4.4. Media composition for Ames test 53 2.5. Biological Assays 54 2.5.1. Soybean lipoxygenase assay 54 2.5.1.1. Purification of soybean lipoxygenase 54 2.5.1.2. Extraction of lipoxygenase from Soybean seeds 54 2.5.1.3. Ammonium sulphate precipitation 54 2.5.1.4. Anion exchange chromatography 55 2.5.1.5. Molecular sieve chromatography 55 2.5.1.6. Preparation of linoleic acid substrate 55 2.5.1.7. Soybean lipoxygenase assay 55 2.5.2. 5-Lipoxygenase (5-LO) assay 57 2.5.2.1. Isolation of human polymorphonuclear leukocytes (PMNLs) 57 2.5.2.2. Isolation of 5-Lipoxygenase (5-LO) 57 2.5.2.3. Substrate and reagent preparation 57 2.5.2.4. 5–LO assay 58 2.5.3. Human Platelet Aggregation Assay 58 2.5.3.1. Preparation of platelet rich plasma (PRP) 58 2.5.3.2. Preparation of Epinephrine, Collagen and ADP 58 2.5.3.3. Platelet Aggregation Assay 59 2.5.4. Rat lens aldose reductase assay (ADR) 59 2.5.4.1. Preparation of enzyme, substrate and reagents 59 2.5.4.2. ADR assay 59 2.5.5. Antibacterial assay 60 2.5.6. Antifungal assay 60 2.5.7. Mutagenicity test or Ames test 61 2. 5.7.1. Preparation of S9 fraction 61 2.5.7.2. Tester strain medium 61 2.5.7.4. Mutagenicity Assay 61 II 2.5.8. Human hepatocarcinoma (Hep3B) cell line experiment 61 2.5.8.1. Cytotoxicity assay 62 2.5.8.2. Antiproliferative assay 62 2.5.8.3.