Production, Purification, Characterization of Antibiotic from Locally Isolated of Actinomycetes spp

221 Pages·2016·4.63 MB·English

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 IC about5.24 mg/ml which scavenged 91% of
50
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 R
f
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
that KH14 possesses a powerful effect against used cancerous cell lines,
especially against MCF-7 by inhibiting 75.84% (400 µg concentration) of cancer
cells after 72 hours of incubation, while against the rest of other cell lines,
PC3and A375with moderate activities. High content screen assay was carried out
for six cellular multi-parameters, against MCF-7 cell line. Besides, the
comparison was carried out between the vehicle and extracellular crude extract
of KH14.
Statistical analyses within different (P values) revealed significant
differences between vehicle samples with extracellular crude extract
concentrations exactly in case of cell viability, nuclear morphology, cell
membrane permeability and mitochondrial membrane potential with different
concentrations, while both total nuclear intensity and cytochrome c did not
show any significant differences with the negative control (vehicle).

iii
Table of Contents
Item No. Titles Pages
Summary i
Table of Contents iv
List of Figures x
List of Tables xiv
List of Abbreviations iv
Chapter One: Introduction and Literature Review
1 Introduction and Literature Review 1
1.1 Introduction 1
1.2 Literature review 5
1.2.1 General backgrounds of Actinomycetes 5
1.2.2 Ecology and habitat of Actinomycetes 6
1.2.3 Life cycle of Actinomycetes 8
1.2.4 Structure, Composition and Taxonomy of Actinomycetes 10
1.2.5 Isolation of Actinomycetes 12
1.2.5.1 Choice of substrate 13
1.2.5.2 Selective media 13
1.2.5.3 Incubation 13
1.2.5.4 Colony Selection 14
1.2.6 Identification of Actinomycetes 14
1.2.7 Optimizing Production of Bioactive Compounds from 16
Actinomycetes
1.2.8 Importance and Diversity of Secondary Metabolites Produced 18
by Actinomycetes
1.2.9 Application of Actinomycetes Secondary Metabolite Products 20
1.2.9.1 Antitumor Compounds from Actinomycetes 21
iv
1.2.9.2 Actinomycetes Application in the Environmental and 22
Ecosystem Fields
1.2.9.3 Actinomycetes as Agents of Biodegradation and 23
Bioremediation
1.2.9.4 Potential Enzymes from Actinomycetes 24
1.2.9.5 Immunomodifiers Agents 26
1.2.10 Identification and Profiling of Bioactive Compounds 26
Produced by Actinomycetes
1.2.10.1 Thin Layer Chromatography (TLC) 27
1.2.10.2 Bioautography 29
1.2.10.3 High Performance Liquid Chromatography (HPLC) 30
1.2.10.4 Elucidation of the Structure 30
1.2.11 Future Prospect of the Actinomycetes 31
Chapter Two: Materials and Methods
2.1 Materials 33
2.1.1 Apparatus and equipments 33
2.1.2 Biological Media and Chemicals 34
2.1.2.1 Cultural Media were used in the Study, Lab. prepared 34
2.1.2.2 Biological Media were used in the Study (redy to use) 35
2.1.2.3 Chemical Materials were used in the Study 36
2.1.3 Antibiotic Discs and Kits 37
2.1.3.1 Antibiotic Discs 37
2.1.3.2 The Cellomics Multi-parameter Cytotoxicity 3 kit Composition 37
were used for Cytotoxicity Activity
2.2 Areas of the Study 38
2.3 Methodologies 39
2.3.1 Sample collection 42
v