T.R. Yuzuncu Yil Universitesi Instutute of Natural and Applied Science Department of Molecular Biology and Genetics

T.R.
YUZUNCU YIL UNIVERSITESI

INSTUTUTE OF NATURAL AND APPLIED SCIENCE
DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS
DETERMINATION OF EXTRACELLULAR HYDROLYTIC ENZYME

PRODUCTION CAPACITY AND 16S rDNA ANALYSIS OF BACILLUS GENUS
BACTERIA ISOLATED FROM ERÇEK LAKE
MASTER THESIS
PREPARED BY: Sardar Hussein RASOOL

SUPERVISOR: Assist. Prof. Dr. Kerem ÖZDEMIR
VAN - 2016
T.R.
YUZUNCU YIL UNIVERSITESI
INSTUTUTE OF NATURAL AND APPLIED SCIENCE
DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS

MASTER THESIS
PREPARED BY: Sardar Hussein RASOOL
VAN - 2016
THESIS STATEMENT ACCEPTANCE and APPROVAL PAGE
This thesis entitled “(Determination of) Extracellular Hydrolytic Enzyme

Production Capacity and 16S rDNA Analysis of Bacillus Genus Bacteria Isolated
from Erçek Lake.” presented by Sardar Hussein RASOOL under supervision of Assit.
Prof. Dr. Kerem ÖZDEMIR in the department of Molecular MicroBiology and Genetics
has been accepted as a M.Sc. thesis according to Legislations of Graduate Higher
Education on …..../......./.......... with unanimity / majority of votes members of jury.
Chair: Signature:
Member: Signature:
Member: Signature:
This thesis has been approved by the committee of The Institute of Natural and
Applied Science on...…../......../...…... with decision number..................
Signature
……………..………..
Director of Institute
THESIS STATEMENT
All information presented in the thesis were obtained according to the ethical
behaviors and academic rules frame. And also, all kinds of statement and source of
information that does not belong to me in this work prepared in accordance with the rules
of theses, were cited to the source of information absolutely.
Signature
RASOOL, Sardar Hussein
ÖZET
ERÇEK GÖLÜ’NDEN İZOLE EDİLEN BACİLLUS CİNSİ

BAKTERİLERIN EKTRASELÜLER HİDROLİTİK ENZİM
ÜRETME KABİLİYETLERİNİN BELİRLENMESİ VE
16S rDNA ANALİZİ

Yüksek Lisans Tezi , Moleküler Biyoloji ve Genetik Anabilim Dalı
Tez Danışmanı: Yrd. Doç. Dr. Kerem ÖZDEMİR
Aralık 2016, 68 Sayfa
Bu çalışmada, Van ilinde bulunan Erçek Gölü’nde belli istasyonlardan alınan su

örneklerinden Bacillus cinsi bakterilerin izolasyonu, ekstraselüler hidrolitik enzim
aktivitelerinin belirlenmesi ve 16S rRNA analizinin yapılması amaçlanmıştır.
Dilusyan plak yöntemi ile 26 Bacillus cinsi bakteri izole edilmiş ve ardından
saflaştırma işlemi yapılmıştır. Bu 26 Bacillus cinsi bakterilerin ekstraselüler hidrolitik
enzim aktivitesi çalışmalarında, 12 izolat ksilanaz aktivitesinde. 7 izolat amilaz
aktivitesinde, 22 izolat proteaz aktivitesinde, 25 izolat katalaz aktivitesinde ve 1 izolat
lipaz aktivitesinde pozitif sonuç vermiştir.
Seçilen 7 izolatın geneomik DNA’sı izole edildikten sonra 16S rRNA gen
bölgesinin PCR amplifikasyonu, 27F ve 1492R evrensel primerleri ile
gerçekleştirilmiştir. Türler arasındaki genetik uzaklığı belirlemek için Maksimum
Likelihood filogenetik ağacı oluşturulmuştur. İzolatların filogenetik ağaçta güçlü bir
homoloji ile kümelendiği gözlemlenmiş olup genetik pozisyonları ortaya konmuştur.
Anahtar Kelimeler: Bacillus, Hidrolitik ektraselüler enzim, 16S rDNA
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ABSTRACT


M.Sc Thesis., Department of Molecular Biology and Genetics
Supervisor: Asst. Prof. Dr.Kerem ÖZDEMİR
December 2016, 68 Page
In this study, from water sample taken from definite stations in Erçek Lake located
in Van district the isolation of Bacillus kind bacteria, the determination of extracellular
hydraulic enzyme activities and doing the analysis of 16S rRNA have been aimed.
With dilution plate method 26 Bacillus type bacteria has been isolated and after
that distillation operation has been done. This, gave positive results in studies of
extracellular hydraulic enzyme activities of 26 Bacillus type bacteria, in 12 isolate
xylanase activity, in 7 isolate amylase activity, in 22 isolate protease activity, in 25 isolate
catalase activity and in 1 isolate lipase activity.
After 7 isolates’ genomic DNA has been isolated 16S rRNA gene region’s PCR
amplification has been realized with 27F and 1492R universal primer. To be able to
determine genetic distance between species Maximum Likelihood phonetic tree has been
formed. It is observed that isolates have been clustered in phylogenic tree with a strong
homology and their genetic positions have been revealed.
Key words: Bacillus, Hydrolytic extracellular enzyme, 16S rDNA
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ACKNOWLEDGMENT
Special thanks to my scientific supervisor Asst. Prof. Dr.Kerem ÖZDEMİR

plantology doctor at YUZUNCU YIL UNIVERSITY Faculty of science biology
Department, and Assoc. Prof. Dr. Musa TURKER Microbiology Department at the same
college, and my special thanks to PhD student, Mr. Metin ERTAS for all his support and
the researches, and
I would like to thank all my brothers (especially Tahsin, Sanger and
Mohammed) and sisters.
Of course at last but not least I wanted to thank my wonderful parents (Hussein,
Asia) that went with me through all the hard times. Of course I would be not able to
manage all this without the love of them.
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TABLE OF CONTENTS
Page
ÖZET …………………………………………………………………………....…i
ABSTRACT……..…………………………………………………..…….………iii
ACKNOWLEDGMENT………………………….……………………..……....…v
TABLE OF CONTENTS……………………............…………………….…..…vii
LIST OF TABLES………………………………...…...………………….…...…..x
LIST OF FIGURES…………………………………………………………...….xii
LIST OF ABBREVATIONS……..……………………………………………...xiv
LIST OF APPENDIX …………………………………………....…………..…xvi
1. INTRODUCTION………………………………………………………………1
2. LITERATURES REVIEW…………..................................................................3
2.1. ENZYMES…………………………….....................................................3
2.1.1. Definition……………………………………………………….....3
2.1.2. History………………………………………………......................3
2.1.3. Nomenclature of Enzyme…………………….…………. ….…….4
2.1.4. The Function of Enzymes in Nature……………………..……..…5
2.2.Factors Affecting Enzyme Activity………..…………………………......6
2.2.1. Enzyme Concentration………………………………………….…6
2.2.2. Substrate Concentration………………………………………..….6
2.2.3. Allostery………………………………..………………………….6
2.2.4. Cofactors…………………………………..……………………….7
2.2.5. Coenzymes……………………………………..…………………..7
2.2.6. Inhibitors…………………………………….……….…………….7
2.3.Applications of Microbial Enzymes……………..…………………….….8
2.3.1. Use of Microbial enzymes in Baking Industry…………...….....….8
2.3.2. Use of Microbial enzymes in Breweries:………… …..…………...9
2.3.3. Use of Microbial enzymes in Leather Industry:……….. ………….9
2.3.4. Use of Microbial enzymes in other industries:……….. ………...…9
2.4.Bacillaceae’………………………..………………………………………9
2.4.1. History of Bacillaceae………………………………………...….10
2.4.2. Bacillus……………………………………..……………..……...10
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2.4.3.Habitat……………………………………………………………..11
2.4.4. General Characteristics…………………………………………....11
2.4.5. Microbiology……………………………………………….……...13
2.4.6. Epidemiology……………………………………..…………...…..13
2.4.7. Taxonomy and Metabolism…………………………………....….13
2.4.8. Bacillus Life Cycle ( Cycle of a Typical Spore forming…………
Bacterium)…………………………………………………….…...14
2.5. Enzymes of Bacillus ………………….……………………………...… 16
2.5.1. Amylases…………………………………………………………..16
2.5.2. Proteases………………………………………………………......16
2.5.3. Catalase…………………………………………………....…........17
2.5.4. Lipases……………………………………………………….....…18
2.5.5. Xylenes……………………………………………………...….…18
2.6. 16S rRNA………………………………………………………………..19
3. MATERIAL AND METHODS…………………………………………….….21
3.1. Materials………………………………………………………………...22
3.2. Method………………………………………………………………......22
3.2.1. Determination and purification of Bacillus bacteria species….….22
3.2.2. Starch hydrolysis test…………...…………………………….......22
3.2.3. Protease hydrolysis test………………………………………..…22
3.2.4. Tween 80 hydrolysis test………………………………………... 23
3.2.5. Xylene hydrolysis test…………………………………………....23
3.2.6. Catalase test……………………..………………………………..23
3.2.7. Solution and dyes used………………………………………...…23
3.2.7.1. Lugol Solution…………………………………………….…………..……23
3.2.7.2. Rhodamine B dye…………………………………………………..………23
3.2.7.3. Kongo red dye………………………………………………..……..………23
3.3. Genomic DNA isolation…………...........................................................24
3.3.1. PCR amplification of 16S rRNA……………….…………......... 26
3.3.2. Analysis of the 16S rRNA Gene Region…………..………..…. 28
4. RESULTS……………………..……………………………………………........29
4.1. Culture for Bacillus…………………………………………………..……....29
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4.2. Xylene Hydrolysis Assay……………………………………………..……... 30
4.3. Amylase hydrolysis assay……………………………………………..…...…32
4.4. Protease hydrolysis assay…………………………………………..…….......34
4.5. Lipase hydrolysis assay……………………………………………..……......36
4.6. Catalase hydrolysis assay…………………………………………..……...…38
4.7. Genomic DNA isolation……………………………………..………….40
4.8. PCR amplification of 16S rRNA…………………….…….....…....40
4.9.Analysis of the 16S rRNA Gene Region……………………….….40
5. DISCUSSION…………………………………………....…………………....43
REFERENCES………………………………………………………….……..…51
APPENDIX……………………………………………………………………....59
CURRICULUM VITAE…………………………………………………………68
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LIST OF TABLES
Table Page
Table 2.1. Origins of isolates of Bacillus species…………….………….. 11

Table 3.1. Collection of Samples .…..……………………………............ 20
Table 3.2 PCR reaction conditions of 16S rRNA gene zone.................... 26
Table 4.1. Results of xylan test for Bacillus bacteria……………. ………....30
Table 4.2. Results of amylase test for Bacillus Bacteria ………………… 32
Table 4.3. Results of Protease test for Bacillus bacteria…………………. 34
Table 4.4. Results of Lipase test for Bacillus Bacteria…………………… 36
Table 4.5. Results of Catalase test for Bacillus Bacteria………………….. 38
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LIST OF FIGURES
Figure Page
Figure 2.1. Cycle of germination, out grouth and sporulation of atypical

sporforming bacterium………………………………..……………15
Figure 3.1. Erçek lake …………………………………….….............................20
Figure 4.1. Bacillus strains on Tryptone Medium soy agar………………….…29
Figure 4.2. The Percentage Results of Xylanase test for Bacillus Bacteria…….30
Figure 4.3. Xylenes positive test(only14)…………………………..……….…30
Figure 4.4. Xylenes positive test(only23)……………………………………...31
Figure 4.5. The Percentage Results of Amylase test for Bacillus Bacteria........32
Figure 4.6. Amylase negative test.(only23)…………………. …….……….…33
Figure 4.7. Amylase positive test(11,12,13)………………………..……….…33
Figure 4.8. The Percentage Results of Protease test for Bacillus Bacteria ……34
Figure 4.9. Positive protease test.……………………………………….……..35
Figure 4.10. Positive protease test ………………………….. …….………...…35
Figure 4.11. The Percentage Results of Lipase test for Bacillus...….………….36
Figure 4.12. Lipase positive test(only) ……………….…………..…….….......37
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LIST OF FIGURES
Figure Page
Figure 4.13. Lipase Negative test………………………………………………..37
Figure 4.14. The Percentage Results of Catalase test for Bacillus Bacteria….....38
Figure 4.15. Catalase positive test………………….……………………….…....39
Figure 4.17. 1% agarose gel image of DNA isolation…..………………….…....40
Figure 4.18. 1.5% agarose gel image of PCR method………………………...…40
Figure 4.19. 16S rRNA analysis as a result of maximum likelihood(ML)

Phylogenetic tree…………………………………………………..42
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LIST OF ABBREVATIONS
Some symbols and abbreviations used in this study are presented below, along
with descriptions.
Symbols Descriptions
Μ Micron
Bp Base pairs
G Gram
M Molar
Mg Miligram
Ml Mililiter
mM Milimolar
Nm Nanometer
Nt Nucleotide
µg Microgram
°C Centigrade degrees
µM Micromolar
µl Microliter
A Adenen
G Guanin
RAPD Randomly enlarged polymorphic DNA
CP Catalase-peroxidases
rRNA Ribosommal RNA
DNA Deoxy ribo nucleotid Asit
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LIST OF ABBREVATIONS
Some symbols and abbreviations used in this study are presented below, along
with descriptions.
Symbols Descriptions
IUB International Union of Biochemistry

NCBI National Center Biotechnology Information
ddH2O Dionize distal water
PCR polimerase chain reaction
TE Tris-EDTA Buffer
TBE Tris-Boric Asit-EDTA Bufer
UV Ultra violet
Nm Nano-meter
ML Maximum Liklihood
µl Micro leter
dNTP Deoxynucleotide Triphosphate
Rpm Revolutions per minute
STE Sodium chloride tris edta
C Cytosine
T Thymine
ß Beta
ATP Adenosine triphosphate
EDTA Ethyeldiamin-tetra-Asitic Asit
H 2 O2 Hydrogen Peroxide
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LIST OF APPENDIX
Appendix page
Ringer solution…………………………………………………………………….59
Glycerol stock solution……………………………………………………………59
Chloroform-iso-amyl alcohol………………………..……………………………61
Phenol-Chloroform-iso-amyl alcohol………………………..………………..….61
Ethidium Bromide…………………………………………………………….…..62
Brom phenol blue……………………………………………………….…….…..62
STE Buffer……………………………………………………….…………….…62
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1. INTRODUCTION
With the exception of a small group of catalytic RNA molecules, all enzymes are
proteins. Their catalytic activity depends on the integrity of their native protein
conformation. If an enzyme is denatured or dissociated into its subunits, catalytic activity
is usually lost. If an enzyme is broken down into its component amino acids, its catalytic
activity is always destroyed. Enzymes, like other proteins, have molecular weights
ranging from about 12,000 to more than 1 million. The action of enzymes depends on
their ability to bind the substrate at a domain of the enzyme molecule called the active
site. Enzymes themselves are not modified (or are only temporarily modified) in the
reaction. The binding site is usually specific for the substrate and depends on the three
dimensional conformation of the enzyme. Common cofactors include metals such as Zn
and Fe and organic molecules such as vitamins. Millions of enzymes, each with a specific
role, are required in nature to break down compounds during the decomposition process
(and make compounds in catabolic processes). When all the benefits of using enzymes
are taken into consideration, it’s not surprising that the number of commercial
applications of enzymes is increasing every year. (Williams, 1904 De Reaumur, 1752;
Smith, 1997; Groves, 1997; Grisham et al,. 1999; Cech, 2000; Bairoch, 2000; Lilley,
2000; )
Commercial enzymes are produced from strains of molds, bacteria, and yeasts.
Up until less than 10 years ago, commercial fungal and bacterial enzymes were produced
by surface culture methods. Within the past few years, however, submerged culture
methods have come into extensive use (Underkofier, 1954; Hoogerheide, 1954; Forbath,
1957).
Microbial enzymes are becoming increasingly important in such diverse fields
as medicine, brewing, and timber preservation. The genus Bacillus has played a major
role in this development as evidenced by the distribution of the papers read at the Fifth
International Fermentation Symposium, 1976, which 23 papers in the session devoted
to "Microbial Enzymes of Industrial Interest" no less than ten were concerned with
enzymes from bacilli. Reasons for the predominance of these bacteria in this area of
study are several , they comprise a group of chemo organotrophs that can be easily
2
maintained and cultivated and yet are markedly heterogeneous in character.

Psychrophiles, mesophiles, and thermophiles, in addition to alkalophilic, neutrophilic,
and acidophilic species are well represented. Bacteriology secrete a variety of soluble
extracellular enzymes (Priest, 1977).
Bacillus species are gram-positive aerobic or facultative anaerobic, sporulating
rod shaped bacteria that are widely spread in nature, being implicated in food poisoning.
Bacillus species exhibit a wide range of physiologic abilities that allow the organism to
flourish in every environment and compete favorably with other organisms within the
environment, due to its ability to form spores produce metabolites that are heat stable,
cold, radiation, and desiccation disinfect ants and have antagonistic effect on other
microorganisms by Alexander Fleming in 1928.
Antibiotics have been recognized as the only means of effective microbial growth
control, after the discovery of penicillin and other antimicrobial agents (Fleming, 1928).
The increases in antibiotics resistant have been attributed to inappropriate use,
inadequacies on the part of the manufacturers and leads to the steady decline of effective
antibiotics annually worldwide (Kuta, Nimzing and Orka’a: 2009).
Extracellular or exoenzymes are those enzyme that are completely dissociated
from the cell and found free in the surrounding medium.However, the division between
these and cell wall or membrane-bound enzymes is often narrow.Enzymes may be
membrane bound in young cells and released as exoenzymes as the culture enters
stationary phase or solubilized by relatively mild procedures including washing the cells
with water or concentrated salt solution (Cercignani, et al; 1974). Nevertheless, enzymes
from Bacilli are often described as periplasmic in that the procedures adopted in the
laboratory to release them (Birnboim, 1966).
The genus Bacillus comprised a phylogenetically and phenotypically
heterogeneous group of species. Recently, the systematic of the Bacillus group has been
widely modified. On the basis of extensive studies of the small-subunit ribosomal RNA
sequences (Elena, et al; 1999). The aim of the thesis in Bacillus is to determine
Extracellular Hydrolytic Enzyme Production Capacity and analyze its 16S rDNA taken
from Erçek Lake.
2. LITERATURES REVIEW
2.1. Enzymes
Enzymes are proteins that enhance (or accelerate) chemical reactions. Such
process is named catalysis and enzymes accordingly catalyze chemical reactions. In
enzymatic reactions, the molecules existent at the beginning of the reaction are called
substrates. Enzymes change substrates into different molecules, called products. All
processes in nature necessitate enzymes in order to occur at significant rates. Enzymes
are selective for their substrates and hence catalyze only a few reactions from among
many possibilities.Like all catalysts, enzymes work by reducing the activation energy for
a reaction.As with all catalysts, enzymes are not consumed by the reactions they catalyze,
nor do they modify the equilibrium of these reactions. However, enzymes do differ from
most other catalysts by being much more specific. Though almost all enzymes are
proteins, not all biochemical catalysts are enzymes, since some RNA molecules known
as ribozymes also catalyze reactions ( Whitehurst and Oort, 2010).
2.1.1. Definition
“Enzymes are macromolecular biological catalysts. An enzyme is a protein that

catalyzes a specific reaction in the cell” (Stryer, et al; 2002).
2.1.2. History
Biological catalysis was first known and described in the late 1700s, in studies on
the digestion of meat by secretions of the stomach, and research went on in the 1800s
with checkups of the conversion of starch to sugar by saliva and various plant extracts. In
the 1850s, Louis Pasteur, as a result, determined that fermentation of sugar into alcohol
by yeast is catalyzed by “ferments.” He assumed that these ferments were inseparable
from the structure of living yeast cells; this view, called vitalism, prevailed for decades.
Then in 1897 Eduard Buchner discovered that yeast extracts could ferment sugar to
4
alcohol, verifying that fermentation was stimulated by molecules that continued to

function when removed from cells. Frederick W. Kühne called these molecules enzymes.
As vitalistic notions of life were disproved, the isolation of new enzymes and the
investigation of their properties progressed the science of biochemistry (Buchner: 1907;
Manchester,1995; Kuhne, 1877).
Takamine (1894 and 1914) was the first character who recognized the technical
possibility of cultivated enzymes and to introduce them to industry. He was mainly
concerned with fungal enzymes, whereas Boidin and Effront (1917) in France initiated in
the production of bacterial enzymes about 20 years later. Technological progress in this
field during the last decades has been so great that, for many uses, microbial cultivated
enzymes have replaced the animal or plant enzymes. For example, in textile desizing,
bacterial amylase has largely substituted malt or pancreatin. (Underkofler and Hickey,
1954).
The optimum pH and temperature for the activity of crude enzyme were 7.5 and
600C, successively. Chakravarthy et al have isolated a Lipase-producing bacterial strain
Acinetobacter calcoaceticus by using enrichment culture techniques from oil mill. Kumar
et al have described the Production, optimization and purification of lipase from Bacillus
sp. (Duza and Mastan, 2013).
2.1.3. Nomenclature of Enzyme
Enzymes are usually termed according to the reaction they accomplish. Typically,
the suffix ‘ase’ is added to the name of the substrate (e.g. glucose-oxidase, an enzyme
which oxidizes glucose) or the type of reaction (e.g. a polymerase or isomerase for a
polymerization or isomerization reaction). The exclusions to this rule are some of the
enzymes studied originally, such as pepsin, rennin and trypsin. The International Union
of Biochemistry (IUB) initiated standards of enzyme nomenclature which recommend
that enzyme names indicate both the substrate acted upon and the type of reaction
catalyzed (Whitehurst and Oort, 2010).
5
Enzymes can be classified by the kind of chemical reaction catalyzed. Officially,

six groups of enzymes have been categorized:
1. Oxidoreductases.
2. Transferases.
3. Hydrolases
4. Lyases.
5. Isomerases.
6. Ligases.
2.1.4. The Function of Enzymes in Nature
Enzymes have a wide variety of functions inside living organisms. They are
essential for signal transduction and cell regulation, often via kinases and phosphatases.
They also make movement, with myosin hydrolyzing adenosine triphosphate (ATP) to
generate muscle contraction and also moving cargo around the cell as part of the
cytoskeleton. Ion pumps are other ATPs in the cell membrane included active ion
transport. In general, it can be stated that the metabolic pathways in a cell are determined
by the quality and quantity of enzymes existing in that cell. Enzymes have a vital role in
the ‘digestive systems’ of mammals and other animals. Enzymes such as amylases cut
large starch molecules into very small components; proteases break down large protein
molecules.In ruminants, which have herbivorous diets, microorganisms in the gut
produce enzymes like cellulase to break down the cellulose cell walls of plant fibres.
Several enzymes can work together in a specific order, forming metabolic pathways. In a
metabolic pathway, one enzyme takes the product of another enzyme as a substrate.
Glucose, for example, can react directly with ATP to become phosphorylated at one or
more of its carbons. In the nonexistence of enzymes, phosphorylation is trivial.
(Whitehurst and Oort, 2010).
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2.2. Factors Affecting Enzyme Activity
Several factors affect the degree at which enzymatic reactions carry on, such as
temperature, pH, enzyme concentration, substrate concentration and the existence of any
inhibitors or activators. (Whitehurst and Oort, 2010, 9).
2.2.1 Enzyme Concentration
With the intention of studying the effect of increasing the enzyme concentration
upon the reaction rate, the substrate must be existent in an excess amount; that is, the
reaction must be independent of the substrate concentration. Any change in the extent of
product formed over a specified period of time will be dependent upon the level of
enzyme existing. These reactions are said to be ‘zero order’ because the rates are
independent of substrate concentration and are equal to some constant. The relationship
between activity and concentration is influenced by many factors such as temperature,
pH, etc. Highest enzyme activity is generally measured when substrate concentration is
unlimited. (Whitehurst and Oort, 2010, 9).
2.2.2 Substrate Concentration
It has been revealed experimentally that if the amount of the enzyme is kept
constant and the substrate concentration is then gradually increased, the reaction velocity
will increase until it touches a maximum. After this point, increases in substrate
concentration will not increase the velocity. It is hypothesized that when this maximum
velocity is reached, all of the available enzyme is converted to ES, the ES complex
(Whitehurst and Oort, 2010: 9).
2.2.3. Allostery
Allostery or allosteric regulation is the regulation of an enzyme or other protein

by binding an effector molecule at the protein’s allosteric site. Effectors that enhance the
enzyme’s activity are referred to as allosteric activators, whereas those that reduce the
7
protein’s activity are termed allosteric inhibitors. Following this mechanism, allosteric
inhibition is a form of non-competitive inhibition (Whitehurst and Oort, 2010; Changeux
and Edelestein, 2005)
2.2.4. Cofactors
Many enzymes require the existence of other compounds, named cofactors, which
are required so as to demonstrate their catalytic activity. Some enzymes do not require
any additional components to display complete activity. Coenzymes embrace NADH,
NADPH and ATP. These molecules act to transfer chemical groups between enzymes.
(Whitehurst and Oort, 2010; Chapman-Smith and Cronan, 1999).
2.2.5. Coenzymes
Coenzymes are small organic molecules that transport chemical groups from one
enzyme to another. Some of these chemicals for instance riboflavin, thiamine and folic
acid are vitamins. Such compounds cannot be prepared in the body and must be acquired
from the diet. As coenzymes are chemically altered as a result of enzyme action, it is
useful to consider coenzymes to be a special class of substrates, or second substrates,
which are common to many different enzymes. To exemplify, about 700 enzymes are
known to use coenzyme NADH. Coenzymes are usually rejuvenated and their
concentrations preserved at a stable level inside the cell (Whitehurst and Oort:
2010,Wagner, 1975).
2.2.6. Inhibitors
Enzyme inhibitors are substances which change the catalytic action of the enzyme
and thus slow down, or in some cases, stop catalysis. Most theories regarding inhibition
mechanisms are grounded on the presence of the Ezyme Substrate complex. Substrate
inhibition will sometime occur when excessive amounts of substrate are available. There
are three common types of enzyme inhibition – competitive, non-competitive and
8
substrate inhibition. Besides these inhibitor types, a mixed inhibition exists as well.
(Wagner, 1975; Cornish-Bowden,1986).
2.3. Applications of Microbial Enzymes
New and exciting enzyme applications are likely to get benefits in other areas: less
damage to the environment; greater efficiency; lower costs; lower energy consumption;
and the improvement of a product’s properties. Applications of microbial enzymes in
food, pharmaceutical, textile, paper, leather, and other industries are abundant and are
increasing rapidly. Most of the industrially important microbial enzymes, with two major
exclusions at present, are hydrolases, which catalyze the hydrolysis of natural organic
compounds. The enzyme industry is keen to apply this diversity by gathering soil and
water samples from the four corners of the Earth – often at places with extreme physical
and chemical conditions – and testing these samples for the existence of microorganisms
that yield enzymes of particular interest (Duza and Masta, 2013).
2.3.1. Use of Microbial enzymes in Baking Industry
Since 1973, the industry of processing starch has developed to be one of the largest
markets for enzymes. Enzymatic hydrolysis is used to form syrups through liquefaction,
saccharification, and isomerization. Another big market for enzymes is the industry of
baking. Supplementary enzymes are added to the dough to ensure high bread quality in
the form of a uniform crumb structure and bigger size. Special enzymes can also increase
the shelf life of bread by maintaining its freshness longer. A major application in the dairy
industry is to cause the coagulation of milk as the first step in cheese making. At this
point, enzymes from both microbial and animal sources are used. (Duza and Mastan,
2013).
2.3.2. Use of Microbial enzymes in Breweries
In many large breweries, industrial enzymes are added to control the brewing
process and produce consistent, high-quality beer. In food processing, animal or vegetable
9
food proteins with better functional and nutritional properties are obtained by the
enzymatic hydrolysis of proteins. In the juice and wine industries, the extraction of plant
material using enzymes to break down cell walls gives higher juice products, enhanced
color and aroma of extracts, and clearer juice. Enzymes have contributed greatly to the
development and improvement of modern home and industrial detergents which is the
largest application area for enzymes today. (Duza and Mastan, 2013).
2.3.3. Use of Microbial enzymes in Leather Industry
The leather industry is more old-fashioned, and new enzyme applications are
slowly catching on, though bating with enzymes is a long-established application. One
of the prime roles of enzymes is to improve the quality of leather, but they also help to
lessen waste. This industry, like many others, is encountering tougher and tougher
environmental regulations in many districts of the world. The usage of chemicals and
the impact on the environment can be reduced with the use of enzymes. Even chrome
shavings can be dealt with enzymes and recycled (Duza and Mastan,2013).
2.3.4. Use of Microbial enzymes in other industries
As regards pulp and paper, enzymes can minimize the use of bleaching chemicals.
Sticky resins on equipment that cause holes in paper can also be broken down. A
developing area for enzymes is the animal feed industry. In this sector, enzymes are
adopted to yield more nutrients in feedstuffs accessible to animals, which in turn reduce
the production of fertilizer. (Duza and Mastan, 2013).
2.4. Bacillaceae
Members of the family Bacillaceae are among the most robust bacteria on Earth,
which is chiefly because of their capability of forming resistant endospores. This
characteristic is supposed to be the key factor determining the ecology of these bacteria.
Though, they also perform major roles in soil ecology (i.e., the cycling of organic matter)
and in plant health and growth stimulus (e.g., through suppression of plant pathogens and
10
phosphate solubilization). Thus, the researcher describes the high functional and genetic
diversity that is found within the Bacillaceae (a family of low Gram-positive spore-
forming bacteria), their roles in ecology and in applied sciences associated to agriculture
(Mandic-Mulec, et al, 2013).
2.4.1. History of Bacillaceae
One of the most primitive bacteria to be described was “Vibrio subtilis” by

Ehrenberg in 1835. In 1872, Cohn gave a new name to the organism: Bacillus subtilis
(Gordon, 1981). That organism was a charter member of a large and different genus,
pioneered by Cohn, that is part of the family Bacillaceae. The endospore, either as the
independent spore or as the structure within the somatic cell, in which case the entire
entity is denoted to as a sporangium, is readily spotted by means of the phase contrast
microscope. This is due to the fact that the spore at a point in the life cycle becomes vastly
refractive. Early workers used stains and special conditions (such as prolonged heating)
to colorize the chemically resistant spore (Doetsch, 1981). However, a Gram-stain is
sufficient to determine the presence of spores because the spore remains unstainable while
the somatic cells or the somatic part of the sporangia will stain. As a result of this ease of
microscopic detection of the spore and its heat resistance, many different
endosporeformers can be easily detected. Using any habitat—soil, water, food, etc.—as
the source, sporeformers can be readily separated by suspending a sample in water and
heating at 80°C for 10 to 30 min. Somatic cells and other resting forms such as cysts and
exospores are usually killed at that temperature. The heat-resistant endospore can then be
plated on appropriate media and isolates recovered in 24 to 48 h. (Mandic-Mulec, et a;
2013).
2.4.2. Bacillus
The Bacillus genus was first discussed and categorized by Ferdinand

Cohn (1872). Thisgenus is the biggest containing gram-positive, endospore-
forming, chemoheterotrophic rods that are usually mobile and peritrichously
flagellated. A lot of species of Bacillus are of great economic importance. e.g
11
Bacillus thuringiensis is an identified insecticide, Bacillus cereus causes food

poisoning and can taint humans, various members of the genus Bacillus are
recognized to produce antibiotics viz. bacitracin, gramicidin, and polymyxin.
Bacillus anthracis similarly, has been reported to be the responsible agent of
the disease anthrax, which has an effect upon farm animals (Singh, et al, 2015).
2.4.3. Habitat
As a consequence of this ease of microscopic detection of the spore and its heat
resistance, many dissimilar endospore formers can be easily acknowledged. Using any
habitat soil, water, food, etc. as the source, spore formers can be easily isolated by
suspending a sample in water and heating at 80 °C for 10 to 30 min. Table 2 displays the
kinds of habitats from which Bacillus species have been isolated (Doetsch, 1981).
2.4.4. General Characteristics
Bacillus species are Gram positive rods often arranged in pairs or chains with
rounded or square ends and usually have a single endospore. The endospores are
commonly oval or sometimes round or cylindrical and are very resistant to contrary
conditions. Sporulation is not repressed by contact to air (Holt, et al., 1994),
(Drobniewski, 1993; )
12
Table 2.1. Origins of isolates of Bacillus species

Name of Bacillus Habitats Name of Bacillus Habitats
species species
B. subtilis Soil, water B. lentimorbus Diseased honeybee
larvae
B. acidocaldarius Thermal acid water B. lentus Soil, foods
and soil
B. alcalophilus pH 10 enrichment B. licheniformis Soil
from soil
B. alvei Soil, diseased bee B. macerans Plant materials, food
larvae
B. amylolyticus Soil B. macquariensis Subantarctic soil
B. anthracis Anthrax-diseased B. marinus Marine sediment
animals
B. azotoformans Soil B. megaterium Soil
B. badius Feces, foods, marine B. mycoides Soil
sources
B. brevis Soil, foods B. pabuli Soil, fodder
B. cereus Soil, foods B. pantothenicus Soil
B. circulans Soil B. pasteurii Soil, water, sewage
B. coagulans Acid foods B. popilliae Diseased scarabid
beetles
B. fastidiosus Soil, poultry litter B. psychrophilus Soil, water
B. firmus Soil, salt marshes B. pumilus Soil
B. globisporus Soil, water B. schlegelii Lake sediment
B. insolitus Soil B. sphaericus Soil, water
sediments, foods
B. larvae Diseased bee larvae B. stearothermophilus Soil, hot spring,
foods
B. laterosporus Soil, water B. thermoglucosidasius Soil
B. lautus Soil, feces B. thuringiensis Soil, foods
B. lentimorbus Diseased honeybee B. validus Soil
larvae
( Slepecky and Hemphill, 2006) cited from Claus and Berkeley (1986).
13
2.4.5. Microbiology
Bacillus spp are aerobic spore forming rods that stain gram positive or gram
variable. With the exception of few species, the large majority have no pathogenic
potential and have never been connected to disease in human beings or animals. Members
of the genus have noteworthy microbiological uses. Plentiful enzymes, antibiotics and
other metabolites have medical, agricultural, pharmaceutical and other industrial
applications. Specimens of antibiotics formed by Bacillus spp include bacitracin by B.
licheniformis or B. subtilis, polymyxin by B. polymyxa and gramicidin by B. brevis.
Certain strains of Bacillus have been exploited as biological controls in antibiotics and
other tests (Bottone, 2010).
2.4.6. Epidemiology
Bacillus organisms are extensively dispersed in the surroundings while the

primary habitat is the soil. These organisms are usually seen in decaying organic matter,
dust, vegetable, water, and some species are part of the normal flora. In the hospital
setting, eruptions and pseudo epidemic have been traced to polluted ventilator equipment,
disinfectant (ethyl alcohol), hospital linen and dialysis equipment. Sources of B. cereus
in food kept outbreaks have been described such as rice, meat loaf, turkey loaf, mashed
potatoes, beef stew, apples and hot chocolate retailed in vending machines.
Epidemiologic studies on the microbiology of street heroin and injection paraphernalia
revealed that Bacillus spp as the predominant isolates from both samples (Curtis, et al:
1967; Dancer, et al., 2002; Turnbull and Kramer,1985).
2.4.7. Taxonomy and Metabolism
Bacillus, established by Chon in 1872 (Cohn: 1872) has undergone significant

taxonomic changes. In the 2nd edition of the Taxonomic Outline of Bergey’s Manual of
Systematic Bacteriology (Ludwig, et al: 2009) phylogenetic classification schemes,
fulfilled mainly by the analysis of 16S rDNA sequence similarities, included in the family
of Bacillaceae the genus Bacillus formed by 94 species.
14
Bacillus species are a vital source of fine biochemicals, antibiotics and

insecticides. Furthermore, the ability of B. subtilis and close relatives to secrete grams per
litre of proteins directly into the growth medium and their well-proven safety have also
made them major candidates for the production of heterologous proteins. In fact, about
two-thirds of the enzyme market (proteases, amylases, rennet substitutes, endonucleases,
glucose-dehydrogenase and pullulanase) for industrial applications are produced by
fermentation from Bacillus species. B. subtilis has been utilized for the production of
nucleotides, traded as food flavour enhancers, amino acids (such as tryptophan, histidine
and phenylalanine) and vitamins such as biotin, folic acid and riboflavin (Queener and
Lively: 1989). Even though δ-endotoxins from B. thuringensis are the most known and
used proteinaceous metabolites come from Bacillus, recently, a large variety of
antimicrobial peptides have been discovered in these bacteria. Some of these peptides can
play a role in competence and in the de-repression of various static-phase genes involved
in sporulation (Sonenshein, 2000).
2.4.8. Bacillus Life Cycle ( Cycle of a Typical Sporeforming Bacterium)
The life cycle of spore-forming Bacillus consists of three dissimilar physiological

processes, somatic growth, sporulation and germination. The alteration from one mode
of evolution to another is motivated by nutrient availability, which is recognized by the
micro organism senses. (Moir 2006; Rosenberg et al. 2012). Multiple signaling pathways
transmit nutritional and evolution rate information immediately to the cell cycle
machinery to permit cells to constantly sample their environments and fine-tune the cell
cycle process (Wang and Levin 2009).
15
Figure.2.1. Cycle of germination, out growth, and sporulation of typical spore forming
bacteria. Also shown are some biochemical and physical events with various
stages. (Slepeccky, 1978).
16
2.5. Enzymes of Bacillus
2.5.1. Amylases
They are glycoside hydrolases, analyze starch into glucose, maltose, maltotriose
and dextrin by hydrolysis of glycosidic bonds. Consequently, they are also called
digestive enzymes. The first enzyme yielded industrially was an amylase from a fungal
basis in 1894, which was exploited as a pharmaceutical assistance for the cure of digestive
disorders (Pandey, 2001). Biodin and Effront were the first to applied B. subtilis and B.
mesentericus for the production of alpha amylase in commercial level using large
fermentors and LSF (Liquid State Fermentation). Truthfully, the employment of bacterial
cultures for the production of commercial enzyme was led the way by them (Underkofler
and Hickey:1954). Amylases contribute as a major class of industrial enzymes comprising
approximately 25% of the enzyme market (Sindhu and Singh, 1997; Rao, 1998). The
most broadly practiced thermostable amylases in the starch industry are formed from B.
licheniformis (Morgan and Priest, 1981).
Microbial production of amylase is more valuable than other sources because it is
economical; production level is high and can be engineered to gain enzymes of desired
characteristics. With the occurrence of biotechnology, the use of amylase has expanded
in clinical research, medical chemistry and starch analytical chemistry. (Lonsane and
Ramesh, 1990).
2.5.2. Proteases
They are a group of enzymes, whose catalytic function is to hydrolyze peptide

bonds of proteins and break them down into polypeptides or free amino acids. They make
up 59% of the global market of industrial enzymes, (Deng et al.,2010). They have got a
great deal of commercial usage in detergents, leather, food and pharmaceutical industries
(Bhaskar et al.,2007 and Jellouli et al.,2009). Sources of proteases embrace all forms of
life, that is, plants, animals and microorganisms. Proteases having pH optima in the range
of 7.0 or around are called neutral proteases.
17
Neutral proteases are primarily of plant origin. Whereas proteases having

optimum activity at pH range of 8 and above are classified as alkaline proteases produced
from microorganisms. Proteases produced from microorganisms play important role in
many industries for instance detergent, tanning, photographic industries, pharmaceutical
and waste treatment etc. (Gupta et al.,2002). Proteases are very common in nature,
microbes serve as a favorite basis of these enzymes because of their very fast growth, the
limited space needed for their cultivation and the ease with which they can be genetically
manipulated to generate new enzymes with reformed properties that are desirable for their
numerous variety of extracellular enzymes, including proteases. Quite a lot of Bacillus
species involved in protease production are e.g. B. cereus, B. sterothermophilus, B.
mojavensis, B. megaterium and B. Subtilis (Shumi et al., 2004). The genus “Bacillus” is
an important source of industrial alkaline proteases and are perhaps the only genera
being commercialized for alkaline protease manufacture (Ferrari et al.,1993). They are
widely spread in soil and water, and certain strains endure extreme environmental
conditions including highly alkaline conditions. Screening of proteases producing
Bacillus sp. from diverse ecological environments can result in segregation of new
alkaline proteases with unique physiochemical features (Singh et al.,1999).
2.5.3. Catalase
It is an enzyme, existing in all aerobic cells, that break down hydrogen peroxide
to molecular oxygen and water. Its chief function is to shield cells from the toxic effect
of hydrogen peroxide. In eukaryotic organisms and in some prokaryotes, catalase is a
molecule incorporates four identical subunits. This enzyme can also act as a peroxidase
for which numerous organic substances can act as a hydrogen donor. Hence, about 20
years ago, a new class of bacterial enzymes, the catalase-peroxidases (CP), was
recognized. These enzymes are regarded to be ancestral forms of catalase or peroxidase
in evolutionary movement. It has been suggested that CPs may act as catalases because
the substrates for peroxidatic activity are not found in cytoplasm.Even though they unveil
high catalase activity, these bifunctional enzymes have little sequence homology with
typical heme-containing monofunctional catalases but have high homology with fungal
cytochrome c peroxidase and plant ascorbate peroxidase. CP is different from typical
18
catalases because it is reduced by dithionite; like peroxidase, it is not inhibited by the

catalase-specific inhibitor 3-amino-1,2,4-triazole but is disabled by hydrogen peroxide,
and it owns a narrow pH range for its maximal activity (Gudelj, et al., 2001).
2.5.4. Lipases
They are a class of enzymes that catalyze the hydrolysis of long chain
triglycerides in the lipids-water interface. Microbial lipases are presently being paid great
attention to the rapid increase of enzyme technology. Lipases have great potential in many
and different industrial uses, chemical, pharmaceutical, medical, cosmetic, leather
industry, paper manufacture, synthesis, biosurfactant, and agrochemicals. In this study,
several bacteria have been tested and selected for their ability to synthesize lipases.
(Laachari, et al: 2014).
Lipases belong to the family of serine hydrolases acting on ester bonds of
triacylglycerols insoluble in water. They are enzymes perfectly soluble in water acting on
insoluble substrates. This heterogeneous biocatalysis has been broken down or
decomposed into two basic steps (Verger and De Haas, 1976). The first step is adsorption
of lipase to the oil / water interface and the second step is the catalysis itself. Grounded
on the concept of heterogeneous catalysis, an interfacial enzymology has been
technologically advanced (Verger and De Haas, 1976; Verger, 1980).
2.5.5. Xylenes
It is an enzyme that catalyzes the hydrolysis of 1,4-beta-D-xylosidic linkages in

xylans that are ingredients or components of hemicellulose, a structural component of
plant cell walls. Arabinoxylans (also known as pentosans) are highly branched xylans
that take place in wheat and rye flour. Numerous xylanases from fungal and bacterial
sources are currently marketed for use in baking (Pariza and Johnson, 2001). Xylanases
designated in this Chemical and Technical Assessment come from genetically engineered
nonpathogenic and nontoxigenic strains of Bacillus subtilis. The information about these
enzymes is given in a dossier submitted to JECFA by the sponsor, Danisco, Inc. (Danisco,
2003).
19
The xylenes-encoding gene was isolated from the wild-type B. subtilis strain 168
and re-introduced into B. subtilis using recombinant DNA technology. Three
recombinant production strains of B. subtilis were created by transformation of the
B.subtilis host strain with an applicable transformation vector. Two of these strains
express xylanases BS1 and BS2 that are identical to the native (wild-type) B. subtilis
xylanase A derived from strain 168. The third strain expresses xylanase BS3 that diverges
from the wild-type enzyme by two amino acids and is endurable or resistant to the
xylanase inhibitor available in flour.
The xylenes production strains contain multiple copies of the corresponding
transformation vectors. All of the DNA introduced into the xylenes production strains is
well-characterized and is not anticipated to give rise to the production of any toxic or
undesirable substances. Xylenes naturally existing in food and xylanases exploited in
food processing as enzymes have not been reported to result in allergic reactions. By
comparison and resemblance, the B. subtilis xylanases debated in this study are not
expected to cause allergic reactions after ingestion of food including the residues of these
enzymes. The recombinant B. subtilis xylenes are unsteady at temperatures above 50o
and are likely to be deactivated during baking or cooking (Ibid).
2.6. 16S rRNA
The most effective approach to Bacillus taxonomy could be analysis of 16S rRNA
molecules by oligonucleotide sequencing (Fox et al., 1977; Stackebrandt and Woese,
1979). That techniqueholds much promise for leading microbial taxonomy into natural
phylogenetic relationships. Nevertheless, traditional taxonomists may be shocked to find
that Bacillus species show affinity with nonsporeforming species. Initial studies with this
powerful tool showed a close relationship among Bacillus, Planococcus, Sporosarcina,
Staphylococcus, and Thermo-action mycetes (Stackebrandt et al., 1987; Stackebrandt and
Woese, 1981). In a latest study 16S rRNA cataloging displayed that B. subtilis and other
ellipsoidal spore forming species, B. cereus, B. megaterium, and B. pumilus, formed a
coherent cluster, while the round-sporeforming species, B. sphaericus, B. globisporus,
and “B. aminovorans” did not cluster. Furthermore, the latter group were closer
phylogenetically to nonsporeforming organisms as follows: B. sphaericus to
20
Caryophanon latum; B. globisporus to Filibacter limicola; B. pasteuri to Sporosarcina

urea and “B. aminovorans” to Planococcus citreus. Cell wall composition agreed except
with the last case ( Slepecky and Hemphill, 2006).
The identification of Bacillus species has been done mainly with morphological
and physiological features, and this method is widely engaged in various fields. Though,
the process adopted needs skillful techniques and is very complex and time-wasting. With
the improvement of genetic engineering, the randomly enlarged polymorphic DNA
(RAPD) method, the hybridization method, or restriction mapping, were altered for the
identification of Bacillus species. These methods are effective for identification or
detection among a small number of species, but they are not appropriate for identification
among a large number of species. Over the years, a good-sized database of 16S rRNA
gene (rDNA) has been built, and this has been successfully practiced or applied in
determining phylogenetic relationships or in pinpointing bacteria. Moreover, it has been
stated that a partial region of 16S rDNA is effective for the classification and
identification of acetic acid bacteria and Streptomyces.Species were subjected to a
comparison of the 16S rDNA sequences. The outcomes showed that the 5 end region was
the hyper-variant region (HV region) and highly specific for each type strain.
Furthermore, sequence analyses of the HV region from 51 strains belonging to four
clusters suggested that the HV region was highly preserved within species. These results
revealed that the HV region is a very efficient index for the rapid identification or
grouping of Bacillus species. (Goto, et al; 1999).
21
3. MATERIAL AND METHODS
Table 3.1 . Collection of Samples

Lab No. Location Coordinate Altittute Date
1 Erçek lake 38380519 East 4278730 North 1820m 20 July 2016

Figure 3.1. Erçek lake.

22
3.1. Materials
The material of this study is Bacillus bacteria species, which are isolated in Erçek
Lake in 2016 years.
3.2. Method
3.2.1. Determination and purification of Bacillus bacteria species
Bacillus bacteria species which were26 isolated in Triptone Soy agar in Erçek
Lake Between 2015-2016 years for this study. Pure cultures were then left in cryogenic
species which had 20% glycerol, and conserved in deep-freezer. The strains, which were
obtained purely, were ready for enzyme tests and molecular studies.
3.2.2. Starch hydrolysis test
Triptone Soy agar isolates, which had 1% soluble starch, was planted in the form
of an intense line with help of loop. Petri dishes were left for 2 days incubation at 37°C.
Following the incubation, lugol solution was added in such a way that it overlaid on the
medium. Clear zones, which were around the colony ,as positive starch digestion and
purple zones as negative starch digestion were assessed in areas where the starch
hydrolysed. (Aygan and et al., 2008).
3.2.3. Protease hydrolysis test
Isolates were planted in Triptone Soy agar which contained 1% Skim Milk
Powder in the form of an intense line with help of loop. Petri dishes were left for 5 days
incubation at 37°C. After incubation, zones which occured around the colonies were
considered as positive for protease activity (Yin and et al., 2010).
23
3.2.4. Tween 80 hydrolysis test
Triptone Soy agar isolates which contained 3% Tween 80 were planted as an

intense line with help of loop. Petri dishes were left for 5 days incubation at 37°C.
Following incubation,at the rate of %0,001 Rhodamin B was added so as to enclose the
surfuce of the petri dishes. Zones which occured around the colonies were considered as
positive for lipase activity. (Karnetova and et al., 1984).
3.2.5. Xylan hydrolysis test
Triptone Soy agar isolates which contained 1% Xylane were planted in an intense
line with the help of loop. Petri dishes were left for 2 days incubation at 37°C. After
incubation, zones which occured around the colonies were considered as positive for
xylene activity. (Karnetova and et al., 1984).
3.2.6. Catalase test
Transfer a small amount of bacterial colony to a surface of clean, dry glass slide
using a loop Place a drop of 3% H2O2 on to the slide and mix. A positive result is the
rapid evolution of oxygen (within 5-10 sec.) as evidenced by bubbling. A negative result
is no bubbles or only a few scattered bubbles.
3.2.7. Solution and dyes used
3.2.7.1. Lugol solution

It was used in amylase test. (Çotuk and Küçüker, 1992).
3.2.7.2. Rhodamine B dye

Rhodamine B (0.001%) dye was used to determine the lipase test.
3.2.7.3. Kongo red dye

Kongo red dye (1%) was used to determine the cellulase test.
24
3.3. Genomic DNA isolation
Ausubel and et al.(1994) method was used by modifying for DNA isolaton from bacteria.
(Ertas, 2009).
The implementation of the method is as follows.

1. 800 µl STE buffer was placed into sterilized, tightly mouth capped eppendorfs
and 4-5 loopfull bacteria biomass was added which was previously improved in
Medium 65 medium.
2. Homogenization was reached by vortex of the tubes at 2700 rpm.
3. After the vortexed tubes were centrifugated at 10000 rpm for 10 minutes, the
supernatant was taken by micropipets and centrifugated again at 10000 rpm
adding 800 buffer on pellet.
4. The supernatant which came into existence after centrifugation was discarded and
400 µl STE buffer was added on it. Buffer was allowed to get mixed with pellet
completely.
5. Tubes were incubated for 30 min in water bath at 75oC adjusted.
6. The tubes, which were removed from the water bath and 50 µl % 10 SDS and 2
µl proteinase K were added in, were waited in at 40oC adjusted water bath for 1
hour.
7. After incubation, 5M NaCl and 0.1 volumes %10 CTAB / 0.7 M NaCl were added
into the tubes to make the salt concentration in the medium between 0.75-0.8.
8. The tubes were incubated in water bath at 65oC adjusted for 10 min.
9. The tubes ,to which an equal volume 24:1 chloroform: isoamyl alcohol was
added, were mixed in hematology shaker at room temperature for 10 min.
10. After the cenrifugation of the tubes at 16000 rpm for 15 min, the supernatant was
transferred to new sterilized eppendorf tubes.
11. The tubes were waited in water bath adjusted at 65 C for 10 min.
12. An equal volume 25:24:1 phenol: chloroform: isoamyl alcohol was added to tubes
and the tubes were mixed in hematology shaker at room temperature for 10-20
min.
25
13. The tubes were centrifuged at 1600 rpm for 15 min.The supernatant which came
into existence after centrifugation was taken to a new sterilized eppendorf and an
equal volume 24:1 chloroform:isoamyl alcohol was added on it.
14. The tubes were mixed gently for 10 min.
15. The supernatant was transferred again to a new sterilized eppendorf tube and 0.8
volume isopropanol(-20 C) was added on it and these tubes were kept for one
night at -20 C.
16. Thereafter, the tubes were centrifuged at 16000 rpm for 15 min and the
supernatant was discarded.
17. For every strain, 100 µl from 70% ethyl alcohol was placed into sterilized
eppendorf tubes. With the help of pipettes, DNA was washed in these tubes.
18. It was centrifuged at 16000 rpm for 5 min and the supernatant was discarded.
19. Until they had a clear apperance,DNAs had been kept at room temperature for
flight of the remained ethyl alcohol and for dry of DNA.
20. According to obtained DNA amount,30 -100 µl TE solution was placed into
sterilized eppendorf tubes.
21. After DNA dissolved thoroughly, 2 µl RNAz was added to the tubes.
22. The for 2-3 sec. centrifuged tubes were kept at 37 oC for 30 min.
23. Afterwards, the tubes were conserved at +4 C˚ thereby being labelled.
DNA samples were checked in agarose electrophoresis gel at the end of the isolation
process. Ethidium bromide dye which has fluorescence feature was added into the gel so
as to make the isolated DNA samples visible in agarose gel. Ethidium bromid has the
feature of 254 or 312 nm wavelength red floresans emission in UV transillüminator
thereby binding to base pair of double chain DNA. This feature allows you to see the
bands in agaroz gel on UV translillüminatör if DNA is isolated. For this, 0.4 gr (%1)
agarose 40 mL 1X TBE buffer with 100 mL erlenmayer flask was added. After being
mixed, with the help of micro-wave oven the agaroz was allowed to dissolve completely
and when it was approximately 60°C ,6 μl etidyum bromid (10mg/ml) was added in it.
The erlenmayer flask was mixed gently so that air bubbles will not be formed.
After electrophoresis plate was placed on a flat surface, the gel solution was poured
without formation of air bubbles. Combs were placed carefully. After the agarose was
frozen, the comb was removed(15-20 min) and placed in elektrophoresis tank which had
26
1X TBe pH 8 buffer. From every DNA sample with together 10 μl and 8 μl loading
dye(Brom phenol blue) was loaded totally 18 μl in the spaces created by comb. Agorose
gel was electrophoresed approximately 20 min at 100 volts and was observed in the UV
transilluminator. It was photographed by using a gel imaging system.
3.3.1. PCR amplification of 16S rRNA
16S rRNA polymerase chain reaction assays were performed in 0.2 ml PCR tubes
in Thermal Cycler (MyGenie-96 Gradient Thermal Cycler, Korea). Two universal
primers (27f : 5’-AGA GTT TGA TCM TGG CTC AG-3’ ve 1492R: 5’
TACGGYTACCTTGTTACGACTT; Lane, 1991) were used for amplification of the
DNA region which coded 16S rRNA gene of DNA samples which were obtained purely
from test organisms. All stock solutions which were prepared for PCR, were prepared
with sterilized ddH2O. Stock solutions were divided small amounts (25-100 µl) into
sterilized eppendorf tubes against the risk of contamination and they were kept - 20 °C
until use.
Preparation:
1. Primer stocks (10 μM)
27f(forward primer: Universal primer binding to starting site of 16S rDNA,5’-
AGAGTTTGATCMTGGCTCAG-3’)
1492R (reverse primer:universal primer binding to last site of 16S rDNA,5’-
TACGGYTACCTTGTTACGACTT-3’)
2. ThermoScientific PCR Master Mix (2X) (Katalog No:0172)
3. DNA (50-100 ng)
4. ddH2O
Thermo Scientific PCR Master Mix (2X) 12.5 µl

27f 3 µl
1492R 3 µl
DNA 3 µl
ddH2O 3.50 µl
Total Volume 25 µl
27
Implementation:
1. The reaction mixture was prepared in 1,5 mL PCR tubes for each sample so that
the total volume was 25 μl and later 22 μl was transferred into 0.2 ml PCR tubes
on ice.
2. Seperately from the reaction mixture, for each sample DNA samples, which were
3μl, were transferred into sterilized 0.2 mL PCR tubes. Total volume of the added
amount of DNA samples was 25 μl.
3. Immediately after the transfer process, PCR reaction (MyGenie-96 Gradient
Thermal Cycler, Korea) was initiated in the conditions of Table 2.
4. 3 µl PCR product was checked on a 1 % agarose gel.
Table 3.2. PCR reaction conditions of 16S rRNA gene zone
Denaturation Amplification End Cooling
Denaturation Bonding Extension Extension

95 °C 95 °C 50 °C 72 °C 72 °C 4 °C
00:25
10 min 00:25 min 1 min 5 min ----
min
1 Cycle 30 Cycle 1 Cycle
Purification process with purification kit after PCR (ROCHE’un High Pure
PCR Product)
1. Put 250 μl.“Binding Buffer” on 50 μl PCR product and mix proper.
2. The filtered tube is placed inside the collection tube.
3. Sample is put into the filtered tube.
4. It is centrifuged at 12.500 rpm for 1.30 min.
5. The downed liquid is discarded.
6. 500 l “Wash Buffer” is added.
7. It is centrifuged at 12.500 rpm for 1:30 min.
8. The downed liquid is discarded.
28

11. The collection tube is discarded and the filtered tube is placed in a 1.5 mL
clean tube.
14. It is our DNA sample which is below.
Sequencing Reaction
Your samples were made thereby using ABI Prısm 310 Genetic Analyzer devices
with ABI PRISM® BigDye Terminator Cycle Sequencing Kit. Thanks to them, sequence
process was made as outsourcing by Soygen Biyoteknoloji firm.
3.3.2. Analysis of the 16S rRNA Gene Region
Taking ab1 files which came after 16S rRNA zone sequencing process,by Codon
Code Aligner V.6.0.2 program the chromatograms were examined for each strain and the
poor quality base sequences (uncertain namely 'N'coded several bases)were removed by
cutting in generally sequence's beginnings and endings, and contiqs were created. 16S
rRNA nucleotide base sequences of all isolates were used for construction of phylogenetic
dendgroms. Blasting in NCBI, access codes of species which are close to every strain
were taken. It was analyzed with both close species in databases and among themselves.
Fasta formats of strains which were sequenced with Mega 7 program and strains in Gene
Banks were asked to program. Thereafter, protected areas were compared by making
Clustal W. After this process, this file in Mega7 program was saved as fasta. This file was
converted to online Phylip format through ALTER (ALignment Transformation
EnviRonment) program. First an account was opened in CIPRES Science Gateway V. 3.3
program and the file which was in Phylip format, was loaded. Then RAxML-HPC
BlackBox file was selected and composed the Maximum Likelihood tree.
4. RESULTS
4.1. Culture for Bacillus
Were transplanted bacteria Bacillus on the medium Tryptone soy agar these
bacteria clearly shows them as in Figure 4.1.
Figure 4.1. Bacillus strains on Tryptone Medium soy agar and Bennett's agar growing
colonies in purification work.
30
4.2. Xylene Hydrolysis Assay
In this study, 12 from 26 Bacillus bacteria, R001, R002, R003, R004, R008,
R009,R010 , R014, R015, R018, R019 and R020, R023 No. isolates showed positive
results, others ngative results.
Table 4.1.Results of xylan test for Bacillus bacteria
Number of bacteria Results of Xylenes Number of bacteria Results of Xylenes
R001 + R014 +
R002 + R015 -
R003 + R016 -
R004 + R017 -
R005 - R018 +
R006 - R019 +
R007 - R020 +
R008 + R021 -
R009 + R022 -
R010 + R023 +
R011 - R024 -
R012 - R025 -
R013 - R026 -
The Results of xylenase Test for Bacilluse Bacteria

70%
54%
60%
46%
50%
40%
Positive Result
30%
Negative Result
20%
10%
0%
Positive Result Negative Result
Figure 4.2. The Percentage Results of xylenase test for Bacillus Bacteria.
31
Figure 4.3.xylenase positive test, (only14).
Figure 4.4. Xylenase positive test, (only 23).

32
4.3. Amylase hydrolysis assay
R013, R017 and R020 R023 No. isolates showed positive results, others negative results.
Table 4.2. Results of amylase test for Bacillus Bacteria

Number of bacteria Results of Amylase Number of bacteria Results of Amylase
R001 - R014 -
R002 - R015 -
R003 - R016 -
R004 - R017 +
R005 - R018 -
R006 + R019 -
R007 - R020 +
R008 + R021 -
R009 + R022 -
R010 - R023 +
R011 - R024 -
R012 - R025 -
R013 + R026 -
The Results of Amylase test for Bacillus Bacteria

70% 65%
60%
50%
40% 35%
Positive Result
30%
Negative Result
20%
10%
0%
Figure 4.5. The Percentage Results of Amylase test for Bacillus Bacteria.
33
Figure 4.6. Amylase Positive test, (only23).
Figure 4.7Amylase Positive test,(11,12,13,).

34
4.4. Protease hydrolysis assay
R006, R008, R009, R010, R011, R012, R013, R014, R015, RO16, R017, R018, R019,
R020, R021 and R023 R024 No. isolates showed positive results, others negative results.
Table 4.3. Results of Protease test for Bacillus Bacteria

Number of bacteria Results of Protease Number of bacteria Results of Protease
R001 + R014 +
R002 + R015 +
R003 + R016 +
R004 + R017 +
R005 + R018 +
R006 + R019 +
R007 - R020 +
R008 + R021 +
R009 + R022 -
R010 + R023 +
R011 + R024 +
R012 + R025 -
R013 + R026 -
The Results of Protease Test for Bacillus Bacteria

90% 85%
80%
70%
60%
50%
Positive Result
40%
Negative Result
30%
20% 15%
10%
0%
Figure 4.8. The Percentage Results of Protease test for Bacillus Bacteria.
35
Figure 4.9. Positive protease test.
Figure 4.10. Positive protease test, (23,24).

36
4.5 .Lipase hydrolysis assay
In this study, 1 from 26 Bacillus bacteria, only R007 No. isolates showed
positive results, others negative results.
Table 4.4.Results of Lipase test for Bacillus Bacteria

Number of bacteria results of Lipase Number of bacteria results of Lipase
R001 - R014 -
R002 - R015 -
R003 - R016 -
R004 - R017 -
R005 - R018 -
R006 - R019 -
R007 + R020 -
R008 - R021 -
R009 - R022 -
R010 - R023 -
R011 - R024 -
R012 - R025 -
R013 - R026 -
The Results of Lipase test for Bacillus Bacteria

120%
100% 96%
80%
60% Positive Result

Negative Result
40%
20%
4%
0%
Figure 4.11. The Percentage Results of Lipase test for Bacillus.

37
Figure4.12. Lipase positive test, (only 7).
Figure 4.13. Lipase Negative test.

38
4.6 . Catalase hydrolysis assay
In this study, 26 from 27Bacilluas bacteria,R001,R002, R003, R004, R005,

R006, R007 R008, R009, R010, R011, R012, R013, R014, R015, RO16, R017, R018,
R019, R020, R021, R023 R024, R025 and R026 R027 No. isolates showed positive
results, others negative results.
Table 4.5. Results of Catalase test for Bacillus Bacteria.

Number of bacteria Results of Catalase Number of bacteria Results of Catalase
R001 + R014 +
R002 + R015 +
R003 + R016 +
R004 + R017 +
R005 + R018 +
R006 + R019 +
R007 + R020 +
R008 + R021 +
R009 + R022 -
R010 + R023 +
R011 + R024 +
R012 + R025 +
R013 + R026 +
The Results of Catalase Test for Bacilluse Bacteria

120%
96%
100%
80%
60% Positive Result

40% Negative Result
20%
4%
0%
Figure 4.14.The Percentage Results of Catalase test for Bacillus Bacteria.

39
Figure 4.15. catalase positive test.(R001,R003)
Figure 4.16. Negative Catalase Test.

40
4.7. Genomic DNA isolation
Figure 4.17. 1% agarose gel image of DNA isolation.
4.8. PCR amplification of 16S rRNA
Figure 4.18. 1.5% agarose gel image of PCR method.
4.9. Analysis of the 16S rRNA Gene Region
16S rRNA gene regions of 7 bacteria isolates which have been chosen for
molecular studies have been multiplied with 27F and 1492R primers. For 16S rRNA
strain analysis Bacillus type taken from GenBank and Staphylococcus type’s alignment
have been done and after omission of spaces phylogenic analysis of 1020 nucleotid region
which has been obtained realized. Dendogram bound to test organisms and string analysis
of related type species Maximum Likelihood algorithm has been used.
Bacillus kind bacteria concentrated and clustered in a in a clade different from
Staphylococcus kind which is an outer group. İsolates with number R001, R007, R008,
41
and R022 clustered with a strong homology with members of Bacillus type taken from
GeneBank. On the other hand, isolates with number R011, R009 and R023 showed
clustering in a different clade. Staphylococcus isolates which are outer group showed
clustering in second main clade.
42
Figure 4.19. 16S rRNA analysis as a result of maximum likelihood (ML) phylogenetic
tree.
5. DISCUSSION
Enzymes are substances that have the ability to decompose compound or complex
molecules into smaller units, for example starches are converted into sugars, and these
materials are considered natural materials contribute in all biochemical processes, due to
the specificity of enzymes, each substrate has an enzyme conformity.
The assets include the advantages of lower production costs, and the possibility of
production significantly and on a large scale in the industrial fermentation, and also a
number of physical and chemical properties, and also there is the possibility of gene
manipulation, the absence of any effects caused by the seasons, and the development of
rapid culture and the use by methods are overloaded. These above characteristics that
made enzymes microbial biological catalysts are more suitable for various industrial
applications (Hasan et al., 2006). Therefore, the determination of other new microbial
sources, and especially those that are non-toxic to humans, are of high interest strategy.
In addition to ensuring the supply of the enzyme in various industrial processes, the
development of new enzyme systems that cannot be developed, and obtained from plant
or animal sources make possible progress in the food industry.
Xylenases result from stimulation xylan. This is mainly the yield of these enzymes
by the microorganisms, contributes to the disruption of plant cell walls along with other
enzymes that break down the sugars, as well as digest Xylan through the germination of
some seeds (e.g. in the malting of barley grain).
These can also be obtained from algae, Protozoa, crustaceans, snails, insects, seeds
of land plants. Enzymes are distinct biological polymers that stimulate chemical reactions
and change them into substrates of particular Products. They are specific in function and
accelerating reactions through supplying alternative pathways of lower activation energy
without being consumed.
These are the essential aspects for biochemical processes and exploited in a
number of industries which process food (Haq et al., 2006). They are specific in function
and speeding up reactions by providing alternative ways to decrease the activation energy
without being consumed. This is the task where the key elements in the biochemical
processes take advantage of them in the food processing industries (Haq et al., 2006). The
44
increased demand for the production of different enzymes of microorganisms are in great
quantities.
There was great interest in the production of xylenases and their applications are
important. That is because of the biological conversion of hemicelluloses, which is an
important element of lignocellulosic material.
Enzyme xylenases sparked great interest in the recent period due to the possibility
of their application in many industrial processes. In the past years, and the sustainability
of biotechnology use of xylenes and xylenases grown significantly. xylenases appeared
to be used in the 1980s, at the beginning the preparation of animal feed and then in the
food industry, textiles and paper at the present time xylenases and cellulase along with
protease represents 20% of the world market enzyme (Polizeli et al., 2005). Most
commercial xylenases produced by Trichoderma, Bacillus, Aspergillus, Penicillium,
Aureobasidium, and Talaromyces sp (Godfrey et al., 1996).
In this study, 26 Bacillus bacteria were taken from laboratory of molecular
biology and we had to these bacteria enzymes tests and showed results as follows 46%
serene and transparent areas around the colonies showed and this zone and transparent
areas indicate that the result is positive for Xylenase test.
Amylase is an enzyme that has been obtained from microbial sources which is
used by many industries as a source used in the production of foods and beverages. And
also taking advantage of microorganisms is possible that produces greatly, and can be
manipulated simply desired products too (Sumrin et al., 2011). Generally enzymes
produced from fungal sources and bacterial sources have many applications in various
industries (Aiyer, 2005). In addition, recent advances in biotechnology tool, and also uses
of amylase widened in clinical research, analytical chemistry and medicinal chemistry
and starches. It appeared earlier that the bacterial strains of the genus Bacillus,
Pseudomonas, Clostridium, and the genus Streptomyces are used for making amylase
(Kafilzadeh et al., 2012; Oyeleke et al., 2010). The application of and demand for multi-
enzyme could facilitate the way for increased production of the enzyme amylase and
indigenous search for more efficient procedures (Hmidet et al., 2009).
In this study, 26 Bacillus bacterial were taken from laboratory of molecular
biology and the researcher took these bacteria enzymes tests and showed results as
45
follows: 35% serene and transparent areas around the colonies showed, and this zone as
well as transparent areas indicate that the result is positive for amylase test.
Neutral proteases are primarily of plant origin. Whereas proteases having
optimum activity at pH range of 8 and above are classified as alkaline proteases produced
from microorganisms. Proteases produced from microorganisms play important role in
many industries for instance detergent, tanning, photographic industries, pharmaceutical
and waste treatment etc. (Gupta et al., 2002). Proteases are very common in nature,
microbes serve as a favorite basis of these enzymes because of their very fast growth, the
limited space needed for their cultivation and the ease with which they can be genetically
manipulated to generate new enzymes with reformed properties that are desirable for their
numerous variety of extracellular enzymes, including proteases. Quite a lot of Bacillus
species involved in protease production are e.g. B. cereus, B. sterothermophilus, B.
mojavensis, B. megaterium and B. Subtilis (Shumi et al., 2004). The genus “Bacillus” is
a crucial source of industrial alkaline proteases and are perhaps the only genera being
commercialized for alkaline protease manufacture (Ferrari et al.,1993).
They are widely spread in soil and water, and certain strains endure extreme
environmental conditions like highly alkaline conditions. Screening of proteases
producing Bacillus sp. from diverse ecological environments can cause segregation of
new alkaline proteases with unique physiochemical features (Singh et al.,1999).
In this study, 26 Bacillus bacteria were taken from laboratory of molecular biology
and the researcher took these bacteria enzymes tests showing results as follows: 85%
serene and transparent areas around the colonies showed and this zone and transparent
areas indicate that the result is positive for Protease test.
Enzymes are considered natural catalysts. Fats make up a large part of the Earth's
biomass and enzymes also play a vital role in the decomposition of this fat insoluble in
water. The enzymes involved in the analyzed for fat breakdown and mobilization of fat
into the individual cells of living organisms is also involved in the transfer of fat from the
organism into another organism (Beisson et al., 2000).
Lipase is also considered one of the important catalysts utilized in the field of
biotechnology. Lipase enzyme was isolated from many kinds of plants, animals, bacteria,
fungi and yeasts and is used lipase extracted from microorganisms in different industries,
46
such as dairy products industry, the food industry, the textile industry, the pharmaceutical
industry, the cosmetic industry and manufacturing biodiesel.
That is also used in the manufacture of fine chemicals and chemicals for
agricultural and secondary polymeric materials (Saxena et al., 1999; Jaeger and Eggert
2002).
Microbial Enzymes can often be very useful microbial enzymes extracted from
plants or animals due to the great variety of activities stimulation, high yields, and ease
of genetic manipulation, regular supply due to lack of seasonal fluctuations and rapid
growth on the media are expensive. The microbial enzymes are also more stable than the
corresponding animal and plant enzymes are the output of more convenient and safer
(Wiseman, 1995).
In this study, 26 Bacillus bacteria were taken from laboratory of molecular
biology and the researcher took these bacterial enzymes tests and showed results as
follows: 4 % serene and transparent areas around the colonies showed. Thus, this zone
and transparent areas indicate that the result is positive for Lipase test.
Catalase enzyme is an enzyme, existing in all aerobic cells, that divide hydrogen
peroxide to molecular oxygen and water. Its main function is to shield cells from the toxic
effect of hydrogen peroxide. In eukaryotic organisms. In some prokaryotes, catalase is a
molecule incorporates four identical subunits. This enzyme can also act as a peroxidase
for which numerous organic substances can act as a hydrogen donor. Hence, about 20
years ago, a new class of bacterial enzymes, the catalase-peroxidases (CP), was
recognized. These enzymes are regarded to be ancestral forms of catalase or peroxidase
in evolutionary movement. It has been suggested that CPs may act as catalases because
the substrates for peroxidatic activity are not found in cytoplasm. Even though they unveil
high catalase activity, these bifunctional enzymes have little sequence homology with
typical heme-containing monofunctional catalases but have high homology with fungal
cytochrome c peroxidase and plant ascorbate peroxidase. CP is different from typical
catalases because it is decreased by dithionite; like peroxidase, it is not inhibited by the
catalase-specific inhibitor 3-amino-1,2,4-triazole but is disabled by hydrogen peroxide,
and it owns a narrow pH range for its maximal activity (Gudelj et al,.2001).
In this study, 26 Bacillus bacteria were taken from laboratory of molecular biology
and the researcher took these bacteria enzymes tests which showed results as follows:
47
96% serene and transparent areas around the colonies showed and this bubbles and
transparent areas indicate that the result is positive for catalase test.
Nowadays, studies that have been done with molecular techniques became quite
important in bacteria diagnosis. In species studies, the build-up of findings as a result of
studies have been done with molecular techniques are important for reliability of result.
The diagnosis for the characterization of bacteria, data belong both to phenotypic
and genotypic characters. The result of genotypic and phenotypic data that support each
others is an indication that ideal results have been obtained. First of all phenotypic
observations and later molecular researches are done by means of these data.
These data can be ranged as; microscopic features (form, size, mobility of cell,
whether it forms flagel or not, whether it forms spore or not, cellular inclusions, color,
colony morphology, gram coloration features), chemical composition of cell components
(DNA membrane composition, fatty acids, isoprenoid quinones, cytochromes, cell Wall
structures and components, bacteria antigens, lipopolysaccharides, micholic acids,
peptidoglycan, electrophoretic analysis, polyamines), features related to metabolism
(basic energy metabolism, dietary and metabolic features, special dietary requirements,
enzymes, ecologic parameters) and features defined with relation to nucleic acid probs
(synthesis or isolation of spesific nucleic acids, multiplication and observation of spesific
nucleic acid probs, hybridization of nucleic acid with nucleic acid prob and identification
of hybrids) (Woese et al. 1990; Vandamme et al. 1996; Skirnisdottir 2000; Mora and
Amann 2001; Hugenholtz 2002; Logan et al. 2002; Martin 2002; Bohannan and Hughes
2003; Chelius and Moore 2004; Coenye et al. 2005; Dworkin et al. 2006; Singh et al.
2006).
For the diagnosis and categorization of microorganisms, each of many different
phenotypic and genotypic methods respectively give chance to the phylogenic
categorization from kind to type, subtype and to variety. Therewithal, each method has
some advantages and disadvantages depend on its convenience, applicability, need for
equipment and its dissolubility rate in application. Generally, DNA based methods are
used universally in identification and classification of microorganisms and they are
reliable. These depend on differentiation at the level of kind and type, DNA-DNA
hybridization methods and modern phylogeny that is bound to 16S rRNA alkaline
sequencing on an increasing rate (Vandamme et al. 1996; Rademaker et al. 1997;
48
Daffonchio et al. 1998; Mora and Amann 2001). Molecular techniques have great
importance in identification, characterization, and classification of bacteria in microbial
ecology . (Schloter et al. 2000; Alp 2003; Singh et al. 2006).
Technological developments in Molecular Biology and especially in Polymerase
Chain Reaction (PCR) provides advantages in categorization and classification of
isolates, determination of genetic variation of a population defined with relation to the
analysis of gnomic microorganisms. Especially, thanks to developing PCR based
fingerprint methods like 16S-23S rDNA-PCR, ITS-PCR (Internally Transcribed Spacer),
ARDRA (Amplified Ribosomal DNA Restriction Analysis), T-RFLPs (Terminal
Restriction Fragment Length Polymorphism), tRNA PCR (Transfer RNA), DGGE
(Denaturing Gradient Gel Electrophoresis) and using of specific primers there have been
major improvements in diagnosis and identification of bacteria (Adıgüzel 2006). This
also provides important information about Dynamics of population of bacterial pathogens
and determination of their echologies which use computer supported PCR technology.
The revelation of genome map of microorganisms provide important benefits in
determination of their taxonomic skeleton, their population structure, their disease risks
(Muyzer and Smalla 1998; Louws et al. 1999, 2001).
Recently, by targeting “internal transcribe spacer” gene regions that code 16S-23S
rDNA gene there have been major improvements in studies related to bacterial
identification, scanning applications and differentiation of isolates in one specific type.
In determination and diagnosis of one single microorganism group, instead of techniques
which are highly diseased and have specificity, diagnosis methods bound to ribosomal
genes take part. By definition of nucleotide sequencings of 16S and/or 23S rDNA regions
the classification of bacteria has been done (Ginard et al. 1997; Maiddak et al. 1997, 2001;
Muyzer and Smalla 1998; Woese 2000; Mora and Amann 2001; Martin 2002; Harris and
Hartley 2003; Woese 2004; Adıgüzel 2006; Dworkin et al. 2006; Qiu et al. 2008;
Rusznyak et al. 2008).
In recent years, in identification of family, type and kind of strains at the level of
bacteria genomic profiling method is used prevalently and two of these methods 16S
rRNA and 16S-23S rDNA alkaline sequencing analysis methods provide science World
with important advantages especially in terms of classification of noncultured
microorganisms (Adıgüzel 2006). In this study, for the identification and
49
characterizations of microorganisms by means of cloning of 16S rRNA gene region

sequence analysis methods have been used and by means of forming phylogenic tree
evolutionary distance is identified.
In bacteria, 16S rRNA region is accepted as protected region (Lane and oth., 1985)
and it is accepted that in terms of 16S rRNA gene strands, if there is a resemblance
between bacteria more than 95% then they accepted as belongs to the same type (Ludwig
et al. 1998). In this study, as a result of execution of 16S rRNA genes that have been
multiplied in PCR on electrophores gel it has been seen that all strains between 1400-
1500 bp give sole band.
In the year (2008) in a study made by Haghighat and his friends in Tahran
rafineries, they observed morphologic and biochemical characteristics of isolates which
Show gram (+) characteristic and which they got as a result of this study and later they
compared the data they obtained with Bergey’s Manuel’s classification.
Lastly, they supported data on hand with 16S rRNA strand analysis. As a result,
they determined that 99% of these strains are Bacillus licheniformis and Bacillus subtilis.
These bacteria which have been isolated have great importance in industry and
biotechnology and enzymes would be obtained from these isolates can be used in more
different studies and most of enzyme studies. Saving of alkalotolerant bacteria which have
importance in terms of industry to science is important in terms of addition of new types
to bacteria collection have in world literature.
Members of Staphylococcus type and Bacillus type by clustering in a different
clade with test organisms in phylogenic tree shows the accuracy of study and outer group.
In the phylogenic analysis of 16S rRNA gene region of total 7 isolates belong to
Bacillus type;
R023 and R009 isolates with a strong differentiation from other test isolates and
type samples Show clustering in 2nd clade. These two isolates having place in different
clade Show the posibility of them to have monophyletic structure. The isolate with
number R011 forms a different group from other isolates and type samples. Isolates with
number R007, R008, R001 and R022 and ceresus AB506030, Bacillus subtilis NR116017
and Bacillus lichenoformis NR118996 are clustered with a strong homology. İsolates with
number R001 and R022 take place in a different clade and their differentiation is quite
50
strong. Likewise, isolates with number R007 and R008 have had place in a high
differentiation clade.
As a result of this study, molacular characterization of chosen strings has been
done by means of looking at extracellular hidrolithic enzyme activities of Bacillus type
of bacteria isolated from water sample of Erçek Lake. For species identification,
alongside of molacular studies, for this type doing biochemical tests would reveale more
accurate results.
It is foreseen that all this working plannig will provide research material for
attainment of systematic positions of new potential species at the same time and enzyme
activities.
REFRENCES
Aiyer P. V., 2005. Amylases and their applications. Afr. J. Biotechnol. 4(13): 1525-1529.
Alcaraz L. D, Moreno-Hagelsieb G, Eguiarte LE, Souza V, Herrera-Estrella L, & Olmedo
G. 2010. Understanding the evolutionary relationships and major traits of
Bacillus through comparative genomics. BMC Genomics., 11: 311-332.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,
& Struhl, K. (1994): Current protocols in molecular biology. John Wiley and
Sons, New York. 2.0.1–2.14.8
Aygun, A., Arik, B., Korkmaz, H., R., S., Çolak, Ö., 2008. Highly thermostable and
alkaline α -amylas a halotolerant alkaliphilic from Bacillus sp. AB68. Braz. J.
Microbiol., 39: 547-553.
Bairoch A., 2000. The ENZYME database in 2000" (PDF). Nucleic Acids Res 28. (1):
304–305.
Beisson F., Arondel V., Verger R., 2000 . Assaying Arabidopsis lipase activity.
Biochem. Soc. Trans. 28: 773–5.
Berkeley, R.C.W, Logan N.A., 1997., Bacillus, AliscyloBacillus and PaeniBacillus.
edited. Principles and Practice of Clinical Bacteriology. Chichester: John
Wiley & Sons. 185-207.
Bhaskar, N., Sudeepa, E.S., Rashmi, HN., Selvi, AT., 2007. Partial purification and
characterization of protease of Bacillus proteolyticusCFR3001 isolated from fish
processing waste and its antibacterial activities. Bioresour. Technol. 98, 2758-
2764.
Birnboim, H. C., 1966. Cellular site in Bacillus subtilis of a nuclease which preferntially
degrades single-stranded nucleic acids. J. Bacteriol., 91:1004-1011.
Cech, T., 2000. "Structural biology. The ribosome is a ribozyme". Science, 289 (5481):
878–9.
Cercignani, G., M. C. Serra, C. Fini, P. Natalini, C. A. Palmerini, G. Mauni, and P. L.
Ipata. 1974. Properties of a 5'-nucleotidase from Bacillus cereus obtained by
washing intact cells with water. Biochemistry, 13, 3628-3634.
Changeux J,P., Edelstein S.J., 2005. “Allosteric mechanisms of signal transduction”.
Science. 308 (5727), 1424–8.
52
Chapman-Smith A., Cronan J.E., 1999. “The enzymatic biotinylation of proteins: a post-
translational modification of exceptional specificity”. Trends Biochem. Sci.
249: 359–63.
Claus, D. R. Berkeley C. W. 1986. The genus Bacillus.. P. H. A. Sneath (ed.) Bergey’s
manual of systematic bacteriology. Williams and Wilkins. Baltimore. (2),
1105–1139.
Cohn, O. 1872. Untersuchungen über Bakterien. Beitrage zur Biologie der Pflanzen
Heft. 2(1): 127-224.
Cornish-Bowden, A., 1986. “Why is uncompetitive inhibition so rare? A possible
explanation, with implications for the design of drugs and pesticides”. FEBS
Letters. 203 (1): 3–6.
Çotuk, A., Ang Küçüker, M., 1992. Biologists Microbiology Laboratory Manual for the
Nobel Bookstores, Istanbul. 129 pages.
Curtis J.R., Wing A.J., Coleman J.C., 1967. Bacillus cereus bacteraemia-a complication
of intermittent haemodialysis. Lancet. 1:136-8.
Dancer S. J, McNair D, Finn P, & Kolsto AB. 2002. Bacillus cereus cellulitis from
contaminated heroin. J. Med. Microbiol., 51: 278.
Danisco. 2003. Bacillus Xylanase Enzyme. Submission to 63rd JECFA. Danisco A/S
– December 2003.
De Réaumur, RAF. 1752. "Observations sur la digestion des oiseaux". Histoire de
l'academie royale des sciences 266- 461.
Deng A, W.U., J, Zhang., Y, Zhang ,G., Wen, T., 2010. Purification and characterization
of a surfactant-stable high-alkaline protease from Bacillus sp. B001. Bioresour.
Technol., 101: 7100-7116.
Doetsch, R. N. 1981. Determinative methods of light microscopy. 21–33. P. Gerhart
(edited) Manual of methods for general microbiology. American Society for
Microbiology. Washington, D.C.
Doetsch, R. N., 1981. Determinative methods of light microscopy. 21–33. P. Gerhart
(edited) Manual of methods for general microbiology. American Society for
Microbiology. Washington, D.C.
Drobniewski, F. A., 1993. Bacillus cereus and related species. Clin Microbiol Rev. 6:
324-38.
53
Duza, M. B., Mastan S. A., 2013. Microbial enzymes and their applications – a review.
Indo American Journal of Pharm Research. 3(8), 6209.
Eduard Buchner, 1907. Nobel Lecture: Cell-Free Fermentation". Nobelprize.org..
Elena P. Ivanova, Mikhail V., Vysotskii, Vasilii I., Svetashev, Olga I., Nedashkovskaya,
Natalia M. G., Mikhailov V. V., Yumoto N., Shigeri Y., Taguchi T. and
Yoshikawa S. 1999 Characterization of Bacillus strains of marine origin.
Internatl. Microbiol ., 2: 267–271.
Ertas , H.B., 1999. Isolation of Listeria spp. from milk from sheep and caprine in
Elazig region. [PhD thesis.] Firat University, Faculty of Veterinary Medicine,
Elazig, Turkey.
Ferrari, E, Jarnagin , A.S. and Schmidt B.F., 1993. Commercial production of
extracellular enzymes. In: Bacillus subtilis and other Gram positive bacteria.,
American Society for Microbiology Washington. 917-938.
Forbath, T. P., 1957. Flexible processing keys enzymes' future. Chem. Eng. 64: 226-229.
Godfrey, T, and S. West ., 1996. Industrial enzymology: the application of enzymes
industry, MacMillan, New York.
Gordon, R. E. 1981. One hundred and seven years of the genus Bacillus. R. C. Berkeley,
and M. Goodfellow (edited) The aerobic endosporeforming bacteria. Academic
Press. London,
Goto K., Omura T., Hara Y., Sadaie Y., 1999. Application of the partial 16S rDNA
sequence as an index for rapid identification of species in the genus Bacillus.
J. Gen. Appl. Microbiol. 64: 1– 8.
Goto K., Omura T., Hara Y., and Sadaie Y., 1999. Application of the partial 16S rDNA
sequence as an index for rapid identification of species in the genus Bacillus. J.
Gen. Appl. Microbiol. 46: 1–8.
Grisham A., Charles M., Reginald H. & Garrett. 1999. Biochemistry. Philadelphia:
Saunders College Pub. 426– 7.
Groves J. T. 1997. Artificial enzymes. The importance of being selective. Nature. 389
(6649): 329 –30.
Gudelj M., Fruhwirth G. O., Paar A., Lottspeich F., Robra K.-H., A., 2001. Cavaco-Paulo
and G. M. Gübitz. A catalase-peroxidase from a newly isolated thermos
54
alkaliphilic Bacillus sp. with potential for the treatment of textile bleaching
effluents. Extremophiles. 5: 423-429.
Gupta R., Beg Q.,-K., Khan S., Chauhan B., 2002. An overview on fermentation,
downstream processing and properties of microbial alkaline proteases. Appl.
Microbiol. Biotechnol. 60: 381-395.
Haq, I.U., M. H., Javed, T. M., Khan, 2006. An innovative approach for
hyperproduction of cellulolytic and hemicellulolytic enzymes by consortium of
Aspergillus niger MSK-7 and Trichoderma viride MSK- 10. African J. Biotech.
5(8), 609-614.
Hasan, F.; Shah, A. A.; Hameed, A.,2006. “Industrial application of microbial lipase.”
Enzyme and Microbial technology, Vol.39, No.2, pp. 235-251.
Hmidet N., El-Hadj Ali N., Haddar A., Kanoun S., Alya S., Nasri M., 2009. Alkaline
proteases and thermostable alpha-amylase co-produced by Bacillus lichenifor
NH1: Characterization and potential application deterg additive. Biochem.Eng.
J. 47: 71-79.
Holt J. G, Krieg N R, Sneath P. H. A., Staley J. T., Williams S. T., 1994 Bergey's Manual
of Determinative Bacteriology. (edited). 9th ed. Baltimore: Williams and
Wilkins. 559.
Hoogerheide, J. C., 1954. Microbial enzymes other than fungal amylases. In Industrial
fermentations (Edited) by L. A. Underkofler and R. J. Hickey. Chemical
Publishing Co., New York, New York. 2: 122-154.
Isenberg H. D,. 2004. Clinical Microbiology Procedures Handbook. (Edited). American
Society for Microbiology. 13: 332-33
Jaeger K. E, Eggert T., 2002. Lipases for biotechnology, Curr. Opin. Biotechnol., 13:
390 - 397.
Jellouli K, Bougatef A, Manni L, Agrebi R, Siala R., Younes I and Nasri M. 2009.
Molecular and biochemical characterization of an extracellular serine-
protease from Vibrio etschnikovii. Microbiol. Biotechnol., 36: 939-948.
Kafilzadeh, F., F. Dehdari, E. Kadivar and Shiraz, O.B., 2012. Isolation of amylase
producing aquatic Actinomycetes from the sediments of mangrove forests in
south of Iran. Afr .J. Microbiol. Res, 6 (33): 6281-85.
55
Karnetov, J., Matej, J., Rezank, T., Prochazka, P. Nohynek M. Rokos, J., 1984. Estimation
of lipase activity by the diffusion plate method. Folia Microbiol., 29: 346-347.
Kühne W. 1877. "Über das Verhalten verschiedener organisirter und sog. ungeformter
Fermente" [On the behavior of various organized and so-called unformed
ferments]. Verhandlungen des naturhistorisch-medicinischen Vereins zu
Heidelberg. new series (in German). 1 (3): 190–193.
Kuta F. A., Nimzing L. and Orka’A. P. Y., 2009S Screening of Bacillus Species with
Potentials of Antibiotics Production. Applied Medical Information. 24 (1): 42
– 46
Laachari F., Bergadi F., Bahafid W., Sayari A., Elabed S., Mohammed I. & Ibnsouda S.
K. 2014. Biochemical study of lipases from Bacillus subtilis. Moroccan J. Biol.
11: 1-12.
Lane, D.J. 1991. 16/23S rRNA sequencing: In Stackebrandt, E and Goodfellow, M (eds)
Nucelic acid techniques in bacterial systematics. John Wiley and Sons,
Chichester, New York, Brisbane, Toronto and Singapore. 115−175.
Lilley D., 2000. Structure, folding and mechanisms of ribozymes. Curr Opin Struct Biol
15 (3): 313– 23.
Lonsane B. K . Ramesh MV., 1990. Advances in Appl. Microbiol. 35: 54-56.
Ludwig W, Schleifer KH, & Whitman WB. 2009. Revised road map to the phylum
Firmicutes. In: De Vos P, et al., (edited by) Bergey’s Manual of Systematic
Bacteriology, The Firmicutes. New York, NY: Springer-Verlag. 3: 1-17.
MacFaddin JF. 2000. Biochemical Tests for Identification of Medical Bacteria. (edited)
3rd ed. Philadelphia: Lippincott Williams and Wilkins. 363-7
Manchester K. L. 1995. "Louis Pasteur (1822–1895)–chance and the prepared mind".
Trends in Biotechnology. 13 (12): 511–5.
Moir A. 2006. How do spores germinate. J. Appl. Microbiol., 101: 1–5.

Oyeleke, S.B., S.H. Auta and Egwim,E.C., 2010. Production and characterization of
amylase produced by Bacillus megaterium isolated from a local yam peel
dumpsite in Minna,Niger State. J. Microbiol. Antimicro. 2(7): 88-92.
Pandey A, Nigam P, Soccol C.R, Soccol V.T, Singh D., Mohan R., 2000. Biotechnol.
Appl. Biochem. 31: 135-152.
56
Pariza, M.W. and Johnson, E.A., 2001. Evaluating the Safety of Microbial Enzyme
Preparations Used in Food Processing: Update for a New Century. Regulatory
Toxicology and Pharmacology. 33: 173-186.
Polizeli M.L.T.M, A.C.S. Rizzatti, R. Monti, H.F. Terenzi, J.A. Jorge, D.S. Amorim .,
2005. Xylanases from fungi:properties and industrial applications. Appl.
Microbiol. Biotechnol . 67: 577-591.
Priest F. G. 1977. Extracellular enzyme synthesis in the genus Bacillus. American Society
for Microbiology. 41 (3): 711-753
Priest F.G., Sonenshein A.L., Hoch J.A., Losick R., 1993. (edited). Bacillus subtilis and
Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular
Genetics. Washington D.C., American Society for Microbiology.
Queener S.W., and Lively D.H., 1989. Screening and selection for strain improvement.
In: Domain AL, Solomon NA, eds. Manual of Industrial Microbiology and
Biotechnology, Washington D.C.: American Society for Microbiology. 3:155–
169.
Ralph A., Slepecky H., Hemphill E., 2006. The Genus Bacillus—Nonmedical.
Prokaryotes. 4: 530–562
Rao M., Tankasale A., Ghatge M., Desphande V., 1998. Microbiol. Mol. Biol. Rev. 62:
597–634.
Saxena R. K, Ghosh P. K, Gupta R, Sheba Dvidson W., Bradoo S., Gulati R., 1999.
Microbial lipases, potential biocatalysts for the future industry. Curr. Sci. 77:
101-115.
Shumi W, Hossain T. Anwar M. N. 2004. Proteolytic Activity of a Bacterial Isolate
Bacillus fastidiosus . J. B. S., 4: 370-374.
Sindhu M. K, Singh B.K , Prased T. 1997. Phytopathol. 34: 269-271.
Singh J., Vohra R., Sahoo D., 1999. Alkaline protease from a new obligate alkalophilic
isolate of Bacillus sphaericus. 21: Biotech. Lett. 921-924.
Singh P., Barolia S.K. & Sharma D.K. Isolation, Characterization and Identification of
Bacillaceae bacterium Spp. from Fecal Contents of Pteropus giganteus in
Udaipur, Rajasthan, India. International Journal of Advanced Research. 3 (8):
331-335.
57
Slepecky, R. A., 1978. Resistant forms. J. R. Norris and M. H. Richmond (edited) Essays
in microbiology. 14: 1–31 John Wiley & Sons. New York.
Smith A.L., 1997. Oxford dictionary of biochemistry and molecular biology. (Edited).
Oxford [Oxfordshire]: Oxford University Press.
Sonenshein AL. 2000. Control of sporulation initiation in Bacillus subtilis. Current
Opinion in Microbiology. 3(6): 561 – 566.
Todar K., 2012. The Genus Bacillus . Todar's Online Textbook of Bacteriology.
Turnbull P.C. and Kramer J.M., 1985. Intestinal carriage of Bacillus cereus: fecal
isolation studies in three population groups. J. Hyg., 9: 629-638.
Underkofler L A & Hickey RJ. 1954. Industrial fermentations. Chemical Publishing Co.,
New York. 2: 325.
Underkofler L. A., Barton R. R., Rennert S. S., 1957. Microbiological Process Report:
Production of Microbial Enzymes and Their Applications. Takamine
Laboratory: New Jersey. Presented at Symposium, Society for Industrial
Microbiology, Storrs, Connecticut.
Underkofler, L. A., 1954. Fungal amylolytic enzymes. In Industrial fermentations.
(Edited by) L. A. Underkofler and R. J. Hickey. Chemical Publishing Co. New
York, New York. 2: 97-121.
Verger R..de Hass G.H. 1976. Interfacial enzyme kinetic of lipolysis. Annuel Review
Biophys. Bioeng. 5: 77-117.
Verger R. 1980. Enzyme kinetic of lipolysis. Methods Enzymol. 64: 341-392.
Wagner AL. 1975. Vitamins and Coenzymes. Krieger Pub Co.
Whitehurst R. J. and Oort M. V., 2010. Enzymes in Food Technology (edited). Sussex:
United Kingdom)
Williams, H. S., 1904. A History of Science: in Five Volumes. Modern Development of
the Chemical and Biological Sciences Harper and Brothers. New York. 2: 231-
239.
Wiseman A ., 1995. Introduction to principles. In: Wiseman A, editor. Handbook of
enzyme biotechnology. 3rd ed. Padstow, Cornwall, UK: Ellis Horwood Ltd.
T.J. Press Ltd.; p. 3–8.
58
Yakoubou S., Xu D., Côté J.C., 2010. Phylogeny of the Order Bacillales inferred from 3’
16S rDNA and 5’ 16S-23S ITS nucleotide sequences. Natural Sciences. 2: 990-
997.
Yin H.-X., Pu J.-N., Wan Y.-T., Xiang B., Bechtel P.J., Sathivel S., 2010. Rheological
and functional poperties of catfish skin protein hydrolysates. Journal of Food
Science, 75: 11–17
ADDS
Ringer's solution,
Ringer's solution, before starting the suspension prior to isolation of the soil in the
stage of obtaining and testing was used to prepare the spore suspension.
59
Ringer solution (Oxoid) 1 tablet

Distilled water 500 ml
1 Ringer one tablet was dissolved in 500 ml of pure water and the mouth of the cap
or cotton sealed glass tube placed in appropriate amounts, were autoclaved for 15
minutes at 121.° C
Glycerol stock solution (Wellington and Williams, 1978)
Glycerol stock at -20 ° C for long Micromonospore spores and mycelia are prepared
for storage.
Glycerol 20 ml
distilled water 80 ml
20% glycerol solution, put into small screw-capped tube in an amount of about 1.5
mL by autoclaving at 1210C for 15 minutes sterile.
0.5 M EDTA, pH 8
EDTA (Merck) 186.1 g

60
ddH20 1000 ml
1 M Tris, pH 8
Tristan 121.1 g
ddH20 1000 ml
TE buffer, pH 8
0.5M EDTA,pH 8 in 2 ml
1M Tris, pH 8, 10 ml
ddH20 1000 ml
10% SDS
SDS (Merck) 10.0 g

ddH20 100 ml
pH 07.Şub
Proteinase K (2 mg / ml)
Proteinase K 2 mg
TE buffer 10ml
Chloroform-iso-amyl alcohol (24: 1 v / v)

61
Chloroform 24ml iso-

amyl alcohol 1ml
70% ethanol
100% alcohol 70 ml
ddH20 (sterile) 30 ml
RNAse (10 mg / ml)
RNAse 10 mg
TE buffer 10 ml
Phenol-chloroform-iso-amyl alcohol (25: 24: 1v / v)
Phenol 25 mL
chloroform 24 ml
iso-amyl alcohol 1 ml
TBE buffer (Tris-boric acid-EDTA; lox, pH 8)
Tristan 121.10 g
Boric acid, 61.83 g
EDTA 5.84 g
ddH20 1000 ml
Ethidium Bromide (10 mg / ml stock)

62
Ethidium bromide 100 mg

ddH20 10 ml
Brom phenol blue (Loading Buffer)
Brom phenol blue 40 mg

Glycerol 5 ml
0.5 M EDTA 1.5 ml
ddH20 3.5 ml
STE Buffer
5 M NaCl 10 ml
1 M Tris pH 8.0 25ml
0.5 M EDTA 100 ml
ddH20 365ml
APPENDIX
Ringer's solution,
Ringer's solution, before starting the suspension prior to isolation of the soil in the
stage of obtaining and testing was used to prepare the spore suspension.
Ringer solution (Oxoid) 1 tablet

Distilled water 500 ml
1 Ringer one tablet was dissolved in 500 ml of pure water and the mouth of the cap
or cotton sealed glass tube placed in appropriate amounts, were autoclaved for 15
minutes at 121.° C
Glycerol stock solution (Wellington and Williams, 1978)
Glycerol stock at -20 ° C for long Micromonospore spores and mycelia are prepared
for storage.
Glycerol 20 ml
distilled water 80 ml
20% glycerol solution, put into small screw-capped tube in an amount of about 1.5
mL by autoclaving at 1210C for 15 minutes sterile.
60
0.5 M EDTA, pH 8
EDTA (Merck) 186.1 g

ddH20 1000 ml
1 M Tris, pH 8
Tristan 121.1 g
ddH20 1000 ml
TE buffer, pH 8
0.5M EDTA,pH 8 in 2 ml
1M Tris, pH 8, 10 ml
ddH20 1000 ml
10% SDS
SDS (Merck) 10.0 g

ddH20 100 ml
pH 07.Şub
Proteinase K (2 mg / ml)
Proteinase K 2 mg
TE buffer 10ml
61
Chloroform-iso-amyl alcohol (24: 1 v / v)
Chloroform 24ml iso-

amyl alcohol 1ml
70% ethanol
100% alcohol 70 ml
ddH20 (sterile) 30 ml
RNAse (10 mg / ml)
RNAse 10 mg
TE buffer 10 ml
Phenol-chloroform-iso-amyl alcohol (25: 24: 1v / v)
Phenol 25 mL
chloroform 24 ml
iso-amyl alcohol 1 ml
TBE buffer (Tris-boric acid-EDTA; lox, pH 8)
Tristan 121.10 g
Boric acid, 61.83 g
EDTA 5.84 g
62
ddH20 1000 ml
Ethidium Bromide (10 mg / ml stock)
Ethidium bromide 100 mg

ddH20 10 ml
Brom phenol blue (Loading Buffer)
Brom phenol blue 40 mg

Glycerol 5 ml
0.5 M EDTA 1.5 ml
ddH20 3.5 ml
STE Buffer
5 M NaCl 10 ml
1 M Tris pH 8.0 25ml
0.5 M EDTA 100 ml
ddH20 365ml
63
GENİŞLETİLMİŞ TÜRKÇE ÖZET (EXPANDED TURKISH SUMMURY )
Tezin Adı: Erçek Gölü’nden İzole Edilen Bacillus cinsi Bakterilerin Ektraselüler
Hidrolitik Enzim Üretme Kabiliyetlerinin Belirlenmesi ve 16S rDNA Analizi
Yüksek Lisans Öğrencisi: Serdar Hussein RASOOL
Danışman: Yrd. Doç. Dr. Kerem ÖZDEMİR
Erçek Gölü , Doğu Anadolu Bölgesi’nin Van ilinde yer almaktadır. Sahanın yüz
ölçümü 1526 km² dir. Erçek Gölü, orijinal bir ekolojik ve limnolojik yapıya sahip
olmasına rağmen üzerinde çok fazla çalışılmamıştır. Yapılan sınırlı sayıdaki çalışma daha
çok gölün batımetrik özellikleri, inci kefalının biyolojik özellikleri, balık dağılımı,
zooplankton türlerinin belirlenmesi gibi konulara ilişkindir. Erçek Gölü’nün en derin
noktası 40 m ve ortalama derinliği 18.45 m olarak tespit edilmiştir. Yapılan çalışmada
gölün yüzey sıcaklığı hava sıcaklığına bağlı olarak yıl içersinde 2-23 oC arasında değiştiği
ve göl suyunun alkali özellikte olup en yüksek pH değerinin 10.75, en düşük değerin ise
9.40 olduğunu kaydedilmiştir (Sarı ve İpek, 1998).
Enzimler, canlı organizmalar tarafından üretilen özelleşmiş katalitik
fonksiyonlara sahip protein molekülleridir ve canlı organizmaların hayatsal faaliyetlerini
gerçekleştirmeleri için gerekli pek çok biyokimyasal reaksiyonlardan sorumludurlar. İlk
zamanlar, enzimatik reaksiyonlar nitelikleri bilinmemesine rağmen yazının
kullanılmasından çok yıllar önce gözlemlenmiş ve kullanılmıştır. Sütün ekşimesi, şekerin
fermentasyon ile alkol oluşturması, şarap, sirke ve peynirin yapılması, ekmeğin
mayalanması gibi enzimatik reaksiyonlar çok eskiden beri bilinmekte ve
kullanılmaktaydı.
Enzim kaynağı olarak mikroorganizmaların tercih edilmesinin diğer nedenleri ise,
oluşturdukları yan ürünlerin az olması, aktivitelerinin yüksek ve daha stabil olması,
ekonomik ve yüksek oranlarda saf olarak üretilebilmeleridir. Örneğin, mikrobiyal
enzimlerin ekstrem sıcaklık ve pH değerlerinde, çok yüksek düzeyde aktivite
göstermeleri endüstri açısından oldukça önemlidir (Horikoshi, 1999, Kirk ve ark. 2002).
64
MATERYAL ve YÖNTEM
Materyaller
Bu araştırmanın materyalini; Erçek Gölü’den 20.07.2016 tarihinde farklı

noktalardan alınan su örnekleri oluştuçmaktadir.
Yöntem
Su örneklerinin toplanması
Erçek Gölü’nden alınan su örnekleri 1000 ml’lik steril çam şişelerle toplandı ve
5 saat içerisinde laboratuvara ulaştırılmıştır.
Su örneklerinden Bacillus bakterilerinin izolasyonu
Bacillus bakterilerin sayımında kullanılan su örneklerinden dilüsyon hazırlandı.

1/10’luk ve 1/10.000’lik seyreltmelerden Triptic Soy agar plakların yüzeyine 100µl ilave
edilerek iyice yayılacaktır. Petri kapları 37C’de 24-48 saat inkübasyona bırakılmıştır.
İnkübasyonu müteakip üreyen koloniler, muhtemel Bacillus bakteri kolonileri olarak
değerlendirilmiştir.
Bacillus bakterilerinin saf kültürlerinin elde edilmesi
Triptic Soy agar besiyerinde gelişen karışık kültürden streril Triptic Soy agar
besiyerlerine öze yardımıyla çizgi plak yöntemi ile ekimler yapılarak saf kültürler elde
edilecektir. Saf kültürler % 20’lik gliserol içeren kriyojenik tüplere aktarılıp derin
dondurucuda muhafaza edilmiştir.
 Katalaz testi
 Nişasta hidroliz testi
 Kazein hidroliz testi
 Tween 80 hidroliz testi
 Xylane hidrolizi testi
65
 16S rDNA Geninin Polimeraz Zincir Reaksiyonu (PCR) İle Çoğaltılması

 16S rRNA Gen Bölgesinin Analizi
Mega7 programı ile bu dizilemesi yapılan suşlar ve Gen Banktaki suşların fasta
formatları programa çağrıldı. Daha sonra Clustal W yapılarak korunmuş bölgeler
kıyaslandı. Bu işlemin ardından Mega7 programındaki bu dosya fasta şeklinde
kaydedildi. Bu dosya ALTER (ALignment Transformation EnviRonment) programı ile
online olarak Phylip formatına çevirildi. CIPRES Science Gateway V. 3.3 programında
hesap açılarak Phylip formatındaki dosya yüklenerek RAxML-HPC BlackBox dosyası
seçilerek Maksimum Likelihood ağaçı oluşturuldu.
BULGULAR
Erçek Gölü’nden alınan numunelerden toplamda 26 Bacillus cinsi bakteri
dilüsyon plak yöntemi ile izole edilerek saflaştırılmıştır.
Ekstraselüler hidrolitik enzim çalışmalarında 26 izolattan 12’si ksilanaz, 7’si
amilaz, 22’si proteaz, 1’i lipaz ve 25’i katalaz aktivitesi göstermiştir.
Şekil Ekstraselüler hidrolitik enzim aktivisei çalışmasından görüntüler.

Moleküler çlışmalar için seçilen 7 bakteri izolatının 16S rRNA gen bölgeleri 27F
ve 1492R primerleri ile çoğaltılmıştır. 16S rRNA dizi analizi için 7 izolatın ve
GenBanktan alınan Bacillus cinsi ve Staphylococcus tip türlerinin toplu hizalaması
yapılmış ve boşluklar çıkarıldıktan sonra elde edilen 1020 nükleotidlik bölgenin
filogenetik analizleri gerçekleştirilmiştir. Test organizmaları ve ilgili tip türlerinin dizi
analizine bağlı dendogramı Maximum Likelihood algoritması kullanıldı.
Bacillus cinsi bakteriler dış grup olan Staphylococcus cinsinden ayrı bir kladda
yoğunlaşarak kümelenmiştir. R001, R007, R008, ve R022 nolu izolatlar GenBank’tan
alınan Bacillus cinsi üyeleri ile güçlü bir homoloji ile kümelenmiştir. R011, R009 ve
R023 nolu izolatlar ise farklı kladlarda kümelenme göstermiştir. Dış grup olan
Staphylococcus izolatları ile ikinci ana kladda kümelenme göstermiştir.
66
Şekil. 16S rDNA gen bölgesinin ML filogenetik ağacı.
TARTIŞMA VE SONUÇ
Enzim üretme yeteneğine sahip olan bakteriler, doğada organik madde

ayrışmasında ve besin döngüsünde önemli rol oynarlar. Mikrobiyal enzimler yenilenebilir
kaynaklardan üretilebilme ve biyolojik olarak bozulabilme potansiyeline sahiplerdir.
Enzimlerin üretiminden elde edilen atıklar, toprak verimini arttırmada gübre olarak
kullanılabilir. Özellikle fazla miktarda ekstrasellüler enzim üreten ve salgılayan Bacillus
suşları en önemli endüstriyel enzim üreticileri arasında yer alır. Bacillus cinsi bakteriler
toprak, hayvan dışkıları ve bitkisel atıklar üzerinde yaygın olarak bulunurlar. Bu cinsin
bireylerinin çoğu zararsız, izolasyonu ve teşhisi kolay, hızlı büyüme oranı ile
fermentasyon süresi kısadır. Yapılan bu çalışmada 26 Bacillus Trypton Soy agar besi
yerinde izole edilmiştir ve enzim testleri ve 16S rDNA analizleri için hazır hale
getirilmiştir.
Mikrobiyal yolla enzim üretiminin ilk aşaması uygun mikroorganizmanın
seçimidir. Kültür ortamı ve fermantasyon koşulları da enzim üretimini etkileyen önemli
67
parametrelerdir. Ortam içeriğinin optimize edilmesi amacıyla farklı kaynaklı karbon, azot
ve metal iyon kaynakları kullanılmaktadır.
Çalışmamızda 26 izolatın %46’sı Ksilinaz, % 26’sı Amilaz, % 84’ü Proteaz, %
4’ü lipaz ve % 96’sı katalaz aktivitesi göstermiştir. Enzimler, endüstride hemen her
alanda kullanılabilmekte ve bu alanların sayısı gün geçtikçe artmaktadır. Bu nedenle
birçok bilim adamı doğal kaynaklardan bakteri izolasyonuna gitmekte, böylelikle yeni
türlerin ortaya çıkması sağlanmaktadır.
Bacillus canine ait toplam 7 izolatın 16S rRNA gen bölgesi filogenetik
analizinde;
R023 ve R009 izolatı diğer test izolatları ve tip örneklerinden güçlü bir ayrımla 2.
Kladda kümelenme göstermiştir. Bu iki izolatın ayrı bir kladda yer alması monofiletik
yapıya sahip olduğu ihtimali olduğunu göstermektedir. R011 nolu izolat diğer izolat ve
tip örneklerinden farklı bir grup oluşturmuştur. R007, R008, R001 ve R022 nolu izolatlar
ve Bacillus ceresus AB506030, Bacillus subtilis NR116017 ve Bacillus lichenoformis
NR118996 tip örnekleri ile güçlü bir homoloji ile kümelenmiştir. R001 ve R022 nolu
izolatlar ayrı birkladda yer alarak ayrımları oldukça güçlüdür. Aynı şekilde R007 ve R008
nolu izolatlarda ayrım gücü yüksek bir kladda yer almıştır.
Bu çalışmanın sonucunda Erçek Gölünden alınan su numunelerinden izole edilen
Bacillus cinsine ait bakterilerin ektraselüler hidrolitik enzim aktivitelerine bakılarak
seçlen suşlarının moleküler karakterizasyonu yapılmıştır. Tür teşhisi için moleküler
çalışmaların yanında bu cins için morfolojik ve biyokimyasal testler yapılması daha doğru
bir teşhis ortaya koyacaktır.
CURRICULUM VITAE
Sardar Hussein Rasool born at 18/February/1985 in Erbil, Iraq .After completing

Primary, secondary and high education school in Erbil, he graduated from the Biology
department of Science College at Salahaddin University in 2011. The main language is
Kurdish and fluent speak in Arabic, English and intermediate in Turkish. Currently
enrolled in University Institute of Natural and Applied Science Department of Molecular
Biology and Genetic.
YÜZÜNCÜ Y1L ÜNIVERSITESI
FEN BILIMLER ENSTITÜSÜ
LİSANSİSTÜ TEZ ORIJINAL LIK RAPORU
Tarih: 15/12/2016
Tez Başlığı / Konusu:

Determination of Ertracellular Hydrolytic Enzyme Production Capacity and 16S rDNA Analysis of
Bacillus Genus Bacteria Lsolated from Erçek Lake.
Yukarıda başlığı/konusu belirlenen tez çalışma= Kapak sayfası, Giriş, Ana bölümler ve Sonuç bölümlerinden
oluşan toplam 66 sayfalık kısa= ilişkin, 15/12/2016 tarihinde şahsımıtez damşmanun tarafından Turnitin
intikal tespit programından aşağıda belirtilen filtrelenae uygulanarak alınmış olan orijinallik raporuna göre,
teziııiin benzerlik oranı % 11 (onbir)
Uygulanan filtreler aşağıda
- Kabul ve onay sayfası hariç,
- Teşekkür hariç,
- içindekiler hariç,
- Simge ve kısaltmalar hariç,
- Gereç ve yöntemler hariç,
- Kaynakça hariç,
- Almanlar hariç,
Tezden çıkan yayınlar hariç,
7 kelimeden daha az &itiş' me içeren metin kısımları hariç (Limit inatrh size to 7 words)
Yüzüncü Yıl Üniversitesi Lisansüstü Tez Orijinallik Raporu Alınması ve Kullanılmasına ilişkin Yönergeyi
incelediın ve bu yönergede belirtilen Warni benzerlik oranlarına göre tez çalışma= herhangi bir intilıal
içermediğini; aksinin tespit edileceği muhtemel dunurıda doğabikcek her türlü hukuki sorumluluğu kabul
ettiğimi ve yukarıda vermiş olduğum bilgilerin dogru olduğunu beyan ederim.
Gereğini bilgilerinize arz ederim.

Tarih ve İmza
Adı Soyadı: Sardar Hıı çein RASOOL

Öğrenci No:I49102162
Anabilim Dalı: Moleküler Biyoloji ve Genetik
Programı:
Statüsü: Y. Lisans X Doktora O
DANIŞMAN ONAY1 ENSTITÜ ONAYİ

UYGUNDUR UYGUNDUR
Yrd. .1<ertrın
ia., elZDEMİEİL------
n re
Doç. Dr. erem ÖZDEMIR

(Unvan, Ad Soyad, İmza)

T.R. Yuzuncu Yil Universitesi Instutute of Natural and Applied Science Department of Molecular Biology and Genetics

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T.R. Yuzuncu Yil Universitesi Instutute of Natural and Applied Science Department of Molecular Biology and Genetics

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YUZUNCU YIL UNIVERSITESI

DETERMINATION OF EXTRACELLULAR HYDROLYTIC ENZYME

PREPARED BY: Sardar Hussein RASOOL

DETERMINATION OF EXTRACELLULAR HYDROLYTIC ENZYME

PREPARED BY: Sardar Hussein RASOOL

This thesis entitled “(Determination of) Extracellular Hydrolytic Enzyme

ERÇEK GÖLÜ’NDEN İZOLE EDİLEN BACİLLUS CİNSİ

RASOOL, Sardar Hussein

Bu çalışmada, Van ilinde bulunan Erçek Gölü’nde belli istasyonlardan alınan su

Anahtar Kelimeler: Bacillus, Hidrolitik ektraselüler enzim, 16S rDNA

DETERMINATION OF EXTRACELLULAR HYDROLYTIC ENZYME

RASOOL, Sardar Hussein

Key words: Bacillus, Hydrolytic extracellular enzyme, 16S rDNA

Special thanks to my scientific supervisor Asst. Prof. Dr.Kerem ÖZDEMİR

RASOOL, Sardar Hussein

Table 2.1. Origins of isolates of Bacillus species…………….………….. 11

Figure 2.1. Cycle of germination, out grouth and sporulation of atypical

Figure 3.1. Erçek lake …………………………………….….............................20

Figure 4.1. Bacillus strains on Tryptone Medium soy agar………………….…29

Figure 4.3. Xylenes positive test(only14)…………………………..……….…30

Figure 4.4. Xylenes positive test(only23)……………………………………...31

Figure 4.6. Amylase negative test.(only23)…………………. …….……….…33

Figure 4.7. Amylase positive test(11,12,13)………………………..……….…33

Figure 4.9. Positive protease test.……………………………………….……..35

Figure 4.10. Positive protease test ………………………….. …….………...…35

Figure 4.11. The Percentage Results of Lipase test for Bacillus...….………….36

Figure 4.12. Lipase positive test(only) ……………….…………..…….….......37

Figure 4.13. Lipase Negative test………………………………………………..37

Figure 4.15. Catalase positive test………………….……………………….…....39

Figure 4.17. 1% agarose gel image of DNA isolation…..………………….…....40

Figure 4.18. 1.5% agarose gel image of PCR method………………………...…40

Figure 4.19. 16S rRNA analysis as a result of maximum likelihood(ML)

IUB International Union of Biochemistry

maintained and cultivated and yet are markedly heterogeneous in character.

“Enzymes are macromolecular biological catalysts. An enzyme is a protein that

alcohol, verifying that fermentation was stimulated by molecules that continued to

2.1.3. Nomenclature of Enzyme

Enzymes can be classified by the kind of chemical reaction catalyzed. Officially,

2.1.4. The Function of Enzymes in Nature

2.2. Factors Affecting Enzyme Activity

2.2.1 Enzyme Concentration

2.2.2 Substrate Concentration

Allostery or allosteric regulation is the regulation of an enzyme or other protein

2.3. Applications of Microbial Enzymes

2.3.1. Use of Microbial enzymes in Baking Industry

2.3.2. Use of Microbial enzymes in Breweries

2.3.3. Use of Microbial enzymes in Leather Industry

2.3.4. Use of Microbial enzymes in other industries

2.4.1. History of Bacillaceae

One of the most primitive bacteria to be described was “Vibrio subtilis” by

The Bacillus genus was first discussed and categorized by Ferdinand

Bacillus thuringiensis is an identified insecticide, Bacillus cereus causes food

2.4.4. General Characteristics

Table 2.1. Origins of isolates of Bacillus species

Bacillus organisms are extensively dispersed in the surroundings while the

2.4.7. Taxonomy and Metabolism

Bacillus, established by Chon in 1872 (Cohn: 1872) has undergone significant

Bacillus species are a vital source of fine biochemicals, antibiotics and

2.4.8. Bacillus Life Cycle ( Cycle of a Typical Sporeforming Bacterium)

The life cycle of spore-forming Bacillus consists of three dissimilar physiological

2.5. Enzymes of Bacillus

They are a group of enzymes, whose catalytic function is to hydrolyze peptide