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Production of Avermectins (Avms) from Streptomyces Avermitilis by Fermentation

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Production of Avermectins (Avms) from Streptomyces Avermitilis by Fermentation Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation Production of Avermectins (avms) from Streptomyces avermitilis by Fermentation BY SAMIA SIDDIQUE ROLL NO. 0 5 6 GCU PhD CHEM 2 0 1 0 Session 2010-2013 Department Of Chemistry Government College University Lahore Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation Production of Avermectins (avms) from Streptomyces avermitilis by Fermentation Submitted to GC University, Lahore in partial fulfillment of the requirements for the Award of degree of Ph.D In Chemistry BY SAMIA SIDDIQUE ROLL NO. 0 5 6 GCU PhD CHEM 2 0 1 0 Session 2010-2013 Department Of Chemistry Government College University Lahore Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation RESEARCH COMPLETION CERTIFICATE Certified that the research work contained in this thesis entitled “Production of Avermectins (avms) from Streptomyces avermitilis by Fermentation” has been carried out and completed by MS. SAMIA SIDDIQUE, Registration No.0056-PHD- CHEM-2010 under my supervision during her PhD (CHEMISTRY) in the Department of Chemistry, Government College University, Lahore /PCSIR Laboratories Lahore. Dated: ____________ Supervisor: Co-supervisor: __________________ __________________ Dr. Fahim Ashraf Qureshi Dr. Quratulain Syed General Manager Director General Office of Research, Innovation and PCSIR, Laboratories, Lahore Commercialization, COMSATS Institute of Information and Technology Submitted Through: _______________________ _______________________ Prof. Dr Ahmad Adnan Controller of Examination Chairperson Government College University Department of Chemistry Lahore G. C. University, Lahore Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation D E C L A R A T I O N I, Samia Siddique, Registration No. 0056-PHD-CHEM-2010, student of PhD in the subject of Chemistry session 2010-2013, hereby declared that the matter printed in the thesis entitled “Production of Avermectins (avms) from Streptomyces avermitilis by Fermentation” is my own work and has not been printed, published and submitted as research work, thesis or publication in any form in any University, Research Institution etc. in Pakistan or abroad. Signature of Deponent Dated: __________________________ SAMIA SIDDIQUE Department of Chemistry GC University, Lahore Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation A C K N O W L E D G E M E N T All praises for the most merciful, omnipotent, omnipresent and omniscient Almighty ALLAH, Who enabled me with the blessing of His Prophet Hazrat Muhammad (P.B.U.H), Whose teachings inspired me to wider my thoughts and deliberate the things deeply and to complete this dissertation. I am obliged to pay my heart rendering thanks and gratitude to my respected supervisor Dr. Fahim Ashraf Qureshi, General Manager, Office of Research, Innovation and Commercialization,COMSATS, Institute of Information and Technology, Islamabad. No words can suffice to express my indebtedness to Dr. Quratulain Syed, Director General, Food and BiotechnologyResearch Center, Lahore and Prof. Dr. Ahmad adnan, Department of Chemistry, GC University, Lahore, whose unremitting concentration, determination, hardworking, enthusiastic, highly courteous behavior, scholastic guidance, dedicated attitude, continuous help, congruent encouragement, valuable suggestions, inexhaustible inspiration and kindness in planning and execution of the present research projects and completion of the dissertation. I express my heartiest gratitude and thanks to Prof. Dr. Islam Ullah Khan, Department of Chemistry, G.C. University Lahore, who provided me with all possible facilities of research work. I can never forget the patience and graceful attitude offered to me by by the most compassionate Dr. Nadeem, Dr. Yasir Saleem and Dr. Rubina Nelofer, S.S.O. PCSIR Laboratories, Lahore. I feel pleasure in acknowledging their affectionate guidance, skilled advice and kind co-operation in Practical work. I also feel great pleasure in expressing my heartiest obligation and thanks to whole staff of Food and Biotechnology Research center, PCSIR Laboratories Lahore for their kind cooperation. I express my cordial thanks to Dr. Shahid Baig, Principal Scientists and Dr. Amir Ali, Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation Jr. Scientist at National institute for Biotechnolgy and Genetic Engineering, Faislabad for their kind co-operation and personal interest towards completion of my research work. I am delighted in feeling this occasion for making rightful acknowledgeent of vigorating suggestions blended with incredible patience of my brother Dr. Usman Siddique and my sisters. I owe them most of my life and my love for them will always be closed to my heart. Finaly I would vemture to articulate my feelings of profound gratitude for my affectionate father, Dr. Muhammad Siddique and my husband Mr. Tehseen Aslam being a source of continuous encourgament and appreciation for me in completing my research work. SAMIA SIDDIQUE Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation DEDICATED with Love To My affectionate Father Dr. Muhammad Siddique Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation LIST OF CONTENTS CHAPTER 1 Page # INTRODUCTION 01—33 1.1. Actinomycetes 1 1.1.1. Generic classification of Actinomycetes 2 1.1.1.1. Nocardioform Actinomycetes 2 1.1.1.2 Genera with multilocular sporangia 2 1.1.1.3. Actinoplanes 3 1.1.1.4. Streptomyces and other related genera 3 1.1.2. Characteristics of Streptomyces 3 1.1.3. Life cycle of Streptomyces 4 1.1.4. Streptomyces as secondary metabolites producing microorganisms 6 1.2. Streptomyces avermitilis 7 1.2.1. Glucose catabolism 7 1.2.2. Genome structure 8 1.3. Isolation and identification of Streptomyces avermitilis 9 1.4. Strian improvement and mutation 10 1.4.1. Screening of mutants 13 1.5. Fermentation 14 1.5.1. Range of fermentation process 15 1.5.2. Submerged fermentation 15 1.5.3. Components of fermentation 15 1.5.3.1. Medium composition 17 1.5.3.1.1. Carbon source 17 1.5.3.1.2. Nitrogen source 18 1.5.3.1.3. Inorganic phosphate 19 1.5.3.1.4. Inorganic salts 19 1.5.3.1.5 Trace metals 20 1.5.3.1.6. Precursors, co-factors and necleotides 20 1.5.3.1.7. Inhibitors 20 Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation 1.5.3.1.8. Inducers 21 1.5.3.2. Fermentation conditions 21 1.5.3.2.1. Ph 21 1.5.3.2.2. Temperature 22 1.6. Secondary Metabolites 22 1.6.1. Production 23 1.6.2 Functions 24 1.6.2. Resistance mechanisms and secondary metabolite secretion 24 1.7. Avermectins 25 1.7.1. Structure 25 1.7.2. Physical properties 27 1.7.3. Classification 28 1.7.4. Abamectin 29 1.7.5. Biosynthesis of avermectin from Streptomyces avermitilis 29 1.7.6. Spectra of activity 29 1.7.7. Toxicological effects 30 1.7.8. Environmental effects 31 1.8 Application of avermectin B1b 32 OBJECTIVES AND TARGETS 34 CHAPTER 2 LITERATURE REVIEW 35-54 CHAPTER 3 Materials and Methods Medium selection for the production of avermectin from 3.1 Streptomyces avermitilis 41445 55 3.1.1. Microorganism and maintenance 55 3.1,2. Inoculum development 55 3.1.3. Production of avermectin B1b 55 3.1.4. Effect of process parameters 56 3.2. Isolation and identification of microorganism 56 Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation 3.2.1. Isolation of Streptomyces 56 3.2.2. Isolation of Streptomyces avermitilis 57 3.2.2.1. Morphological characterization 57 3.2.2.2. Antimicrobial activities 57 3.2.2.3. Avermectin production 58 3.2.2.4. Agar colonies 58 3.2.2.5. Agar slants 58 3.2.3. Morphological characterization 58 3.2.4. Biochemical characterization 58 3.3. Mutagenesis of Streptomyces avermitilis 41445 59 3.3.1. Mutational analysis 59 3.3.2. Prepration of spore suspension 59 3.3.3. Stock solution of Ethidium bromide 59 3.3.4. Stock solution of ethylmethane sulfonate 59 Physical mutagenesis of Streptomyces avermitilis 41445 by ultra 3.3.5. violet irradiation 60 Physical mutagenesis of Streptomyces avermitilis 41445 by 3.3.6. ethylmethane sulfonate (EMS) 60 Chemical mutagenesis of Streptomyces avermitilis 41445 by 3.3.7. Ethidium bromide (EtBr) 60 Selection and screening of avermectin B1b hyper producing 3.3.8. mutants 61 Defined medium for the growth of mutant strain of Streptomyces 3.3.9. avermitilis 41445 61 3.4. Fermentation technique 61 3.4.1. Submerged fermentation 61 3.4.1.1. Inoculum development for Streptomyces avermitilis 41445 61 Shake flask study for avermectin B1b production from S. avermitilis 3.4.1.2. 41445 62 3.4.1.3. Inoculum development for mutant strain Streptomyces avermitilis 62 Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation 41445 UV 45(m)3 Shake flask study for avermectin B1b production from S.avermitilis 3.4.1.4. 41445 UV 45(m)3 mutant strain 62 3.5. Optimization 63 Optimization of cultural conditions for avermectin B1b production from Streptomyces avermitilis 41445 by Response surface 3.5.1. methodology 63 Single factor optimization methodology for the selection of key 3.5.1.1. factors for avermectin B1b production 63 3.5.2. Statistical experimental designs 63 3.5.2.1. Screening of cultivation variables by Plackett-Burman Design 63 Optimization by Central Composite Design (CDD) and statistical 3.5.2.2. analysis 64 3.5.2.3. Statistical model validation 65 3.6. Optimization 66 Optimization of cultural conditions for avermectin B1b production from Streptomyces avermitilis 41445 by Artificial Neural Network 3.6.1. (ANN) 66 3.6.1.1. Artificial neural network 66 3.6.1.2. Optimization capability of ANN 66 3.6.2. Statistical analysis 67 3.7. Optimization 67 Optimization of cultural conditions for avermectin B1b production from Streptomyces avermitilis 41445 UV 45(m) 3 by Response 3.7.1.
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