Republic of Iraq Ministry of Higher Education and Scientific Research Al-Nahrain University College of Science Biotechnology Department Production, Purification, Characterization of Antibiotic from Locally Isolated of Actinomycetes spp. and Studying its Cytotoxicity and Antitumor Activity A Dissertation Submitted to the Council of the College of Science, Al-Nahrain University, in Partial Fulfillment of the Requirements for the Degree of Doctorate of Philosophy in Science, Biotechnology By Saman Mohammed Mohammed Amin B.Sc. Biology/ University of Salahaddin- Erbil/ 1999-2000 M.Sc. Biotechnology/ Al -Yarmouk University-Irbid - Jordan/ 2011 Supervised By Dr. Mohsen Hashim Risan Dr. Nidhal Abdulmohaimen (Assistant Professor) (Professor) October- 2016 Al Moharm-1438 Supervisors Certification We, certify that this dissertation entitled “Production, Purification, Characterization of Antibiotic from Locally Isolated of Actinomycetes spp. and Studying its Cytotoxicity and Antitumor Activity” was prepared by "Saman mohammed Mohammed Amin " under our supervision at the College of Science/Al-Nahrain University as a partial fulfillment of the requirements for the Degree of Doctorate of Philosophy in Science (Biotechnology). Signature Signature Name: Dr. Mohsen Hashim Risan Name: Dr. Nidhal Abdulmohaimen Scientific Degree: Assistant Professor Scientific Degree: Professor Date: / / 2016 Date: / / 2016 In view of available recommendations, I forward this dissertation for debate by examining Committee. Signature: Name: Dr. Hameed M. Jasim Scientific Degree: Professor Title: Head of Biotechnology Department Date: / / 2016 Committee Certification We, the examining committee certify that we have read this dissertation entitled " Production, Purification, Characterization of Antibiotic from Locally Isolated of Actinomycetes spp. and Studying its Cytotoxicity and Antitumor activity "and examined the student " Saman Mohammed Mohammed Amin'' in its contents and that in our opinion, it is accepted for the Degree of Doctorate of Philosophy in Science (Biotechnology). Signature: Name: Dr.Jasm Mohammsd Karhoot Scientific Degree: Professor Addres: College of Medicine, Baghdad Unviersity Baghdad Unviersity Date: / / 2016 (Chairman) Signature: Signature Name: Dr. Shatha Ali Shafiq Name: Dr. Shadan Abbas Othman Scientific Degree: Profssor Scientific Degree: Assist.Professor Addres: College of Science, Addres: College of Science, Almostansria Unviersity Baghdad Unviersity Date: / / 2016 Date: / / 2016 (Member) (Member) Signature: Signature: Name: Dr. Abdilwahid Shamkhi Name: Dr. Asma Ali Hassan Scientific Degree: Assist. Professor Scientific Degree: Assist. Professor College of Applied Biotechnology College of Applied Biotechnology Al Nahrian University Al Nahrian University Date: / / 2016 Date: / / 2016 (Member) (Member) Signature: Signature: Name: Dr. Mohsen Hashim Risan Name: Dr. Nidhal Abdulmohaimen Scientific Degree: Assistant Professor Scientific Degree: professor College of Applied Biotechnology College of Applied Biotechnology Al Nahrian University Al Nahrian University Date: / / 2016 Date: / / 2016 (Member/ Supervisor) (Member/ Supervisor) I, here by certify upon the decision of the examining committee. Signature: Name: Dr. Hadi M. A. Abood Scientific Degree: Professor Title: Dean of the Science College Date: / / 2016 Acknowledgments I am extremely thankful to Dr. Mohsen Hashim Risan, my supervisor, who always found time to assist me, and encouraged me to do the best, making continuous and fruitful discussion and directing me to the proper way. My deepest and special thank to my co-supervisor Dr. Nidhal Abdulmohaimen, for her valuable advice and help during this study, also I would like to express my appreciation to University of Al Nahreen, college of science for helping me in many station inorder completing my dissertation. My great thanks are extended to Dr. Dleir Faee‟q Darwesh, Dr. Shwan Kamal Rachid, Dr. Rakhad Adnan, Dr. Mohammad Hassan Fatah, Dr Hassan Mohammad Rostam, Dr. Khalid Palni, Mr. Ismeal Mohammad Rashid, Mr. Hassan Mohammad Tofeeque and Mr. Shwan Ramazan Ali; I learned so much from them, and they spending a lot of time in reading, correcting and analyzing my data. I would like to express my appreciation to University of Garmian, Faculty of Education Biology department, for giving me permission and opening all researches laboratory doors for completing a great part of my work, also for Dr. Kamal Mohammed, head of Biology dept. for always having an open door to me, and for Dr. Ayad Faeq Darwesh and Mr. Karzan Mahmood in chemistry dept. for them help and supporting me with suitable information and materials. My sincere gratitude and deepest appreciation is to my greatest lovely family, particularly my father( Mercy on him ) ,My mom and My lovely wife , all time they gave me encouragement and support. Without their prayers and guidance this project would not have been completed. Thanks to all my brother and sisters whom always I find them when I need. For all of them, I said “Thank you all” ……. Saman Amin Dedications I dedicate the outcome of my efforts to Who always encouraged me to do the best, My supervisions Who I never forget him, the soul of my father Who their words of encouragement and push for tenacity ring in my ears. My mother The most greatest and patient wife, My lovely wife The fruit of my life and most loved ones, My sons Dwarozh and Bawk, My beloved and dear in my life, my brothers and sisters. Everyone who contributed to the progress of science and service to the humanity Saman Amin Summary Recently, the increase of the pathogens resistant to recent antimicrobial agents has become a health problem. Accordingly, the current study aimed at the isolation of local actinomycetes spp. that is capable of producing novel bioactive compounds, as well as investigating their biological (antimicrobial and anticancer) activities, in addition to, characterizing and partial purifiying the antimicrobials metabolites. Forty soil samples were collected in Garmian area, during the period from 2nd to 15th Feb. 2015. Out of the forty soil sources were screened, only (65%) of them harbored potential actinomycetes, and 55 suspected colonies with different morphotypes were isolated. All isolates were tested for antimicrobial activities by primary screening against reference strains. Among all suspected actinomycetes candidates, only six isolates KH14, KH16, KH18, KA19, KA38 and KA39 were able to inhibit the growth at least two of tested microorganisms. Optimizing media composition for enhancing the production of antimicrobial metabolites, five different broth media ISP1, ISP2, ISP4, ISP5 and Glycerol yeast extract broth were used and ISP2 revealed the best results (inhibition zone) among all media used. Furthermore, five different organic solvents were used to obtain the maximum extraction of extracellular antimicrobial metabolites. Results exhibited the extraction by n- butanol, methanol, ethyl acetate, n- hexane and chloroform, for recording zones of inhibition as 46.6%, 33.3%, 20%, 13.3% and 6.6% respectively against the tested microorganisms. Comparisons of antimicrobial activities between extra and intracellular crude extracts were carried out. In general, the extracellular exerted more potent antimicrobial activity than the intracellular. i Ten different synthetic antibiotic discs were used to compare their activities with the extracellular crude extract. Interestingly, the extract, when compared their activities, show a higher inhibitory activity against the pathogens than synthetics discs activity. The results of the optimization experiments, such as growth kinetic, pH and salt tolerance NaCl revealed that the 3 days of incubation at pH 8 and 5% of NaCl tolerance especially for KH14 isolate. Minimum inhibitory concentration against Staphylococcus aureus, Escherichia coli and Candida albicans shows that 64, 500 and 500µl/ml respectively. The extracellular crude extract exhibited a strong inhibitory activity against Gram- positive bacteria and a moderate inhibitory activity against both Gram-negative bacteria and yeast. In addition to the antimicrobial actions, DPPH assay has been performed to determine the antioxidant activity. The extracellular crude extract from KH14 isolates showed IC50 about5.24 mg/ml which scavenged 91% of formed DPPH free radicals. Determining the nature of the antimicrobial compounds was analyzed by TLC, which indicated the presence of four separate spots, only one of them revealed the antimicrobial activities, as well as their position was determined by bioautography, with different solvents (mobile phase) exhibited different Rf values. HPLC-MS analysis of the scraped active spot and ethyl acetate extract indicated the presence of many compounds. In addition, the results showed that most of them have a similar mass, which becomes noticeable in chromatogram. UV absorbance designated the presences of only two peaks at(221.85)and (264.85)nm from the scraped spot, while three distinguished peaks were shown at 222.85, 264.85 and 442.85 nm from ethyl acetate extract. Furthermore, the similarity between the two extracts on the basis of UV absorbance was achieved by the second peak of both extracts which is 264.85nm, and this was considered ii as an antimicrobial absorbency. GC-MS analysis results for ethyl acetate extract of KH14 displayed 12 peaks, their molecular weight and molecular formulas were determined depending on NIST library. The antitumor activities were determined by MTT assay, which indicated