A Project Report

On
GENETIC ENGNEERING
(Production and purification of antibacterial metabolites from recombinant
strain: Streptomycin)
Submitted to
R.R.Institute of Modern Technology
BKT. Lucknow

Under the guidance of

Mr. Chitranshu Pandey

M.R.D.Lifesciences.Pvt.Ltd.Lucknow
2020

Submitted to Submitted by:

Mr.DHEERENDRA KUMAR Ayushi Katiyar
(Head of Department) Biotechnology(3rd
R.R.I.M.T;B.K.T;Lucknow

ACKNOWLEDGEMENT

1
I owe my soulful thanks to almighty for endowing his immense blessings that

helped me towards the successful completion of my training.I express my deep

sense of gratitude and indebtedness to Mr. Manoj Verma, Director, and Er .D.

K. Verma, Project Manager, MRD Life Sciences, for his kind support and

facilities provided at this well famed Research Institute. I wish to place on record

my indebtedness to Mr. Chitranshu Pandey, Research Scientist, Ms. Pallavi

Sharma, Research Scientist, Ms. Shraddha Prakash, Jr. Research Scientist, Mr.

Raj Shekhar Mishra, Research Assistant, Ms. Pragya Srivastava, Research

Assistant and Ms. Pooja Mishra, Research Assistant, for their sustained co-

operation, interest and encouragement throughout this training work. Sincerely I

express my deep feelings to my friends who rendered a helping hand in the hour of

need. I am indebted to my parents for their moral support and personal sacrifice to

see me through this training work.

Ayushi Katiyar

DECLARATIONS

2
I hereby declare that the present work on “Advanced Biotech Techniques” is a record

of original work done by me under guidance of Mr. Chitranshu Pandey, Research

Scientist, MRD Life Sciences, during 15th July 2020 to 15th August 2020, at MRD

Life Sciences, Lucknow. All the data which were provided in this were through my

own work. I also declare that no part of this thesis has previously been submitted to

any University or any examining body for acquiring any diploma or degree.

Date-

Ayushi Katiyar

LIST OF TABLES

3
SI.No. Name of The Tables Page
No.

1. Serial Dilution of soil sample

2. Identification of isolated microorganism

LIST OF CONTENTS

4
 SI.NO. NAME OF THE CHAPTER PAGENO.
1. INTRODUCTION
2. OBJECTIVE
3. REIVIEW OF LITERATURE
4. METHODOLOGY
5. RESULT
6. DISCUSSION

7. CONCLUSION
8. REFERENCE

INTRODUCTION

The term “antibiotic”, (from the Greek – anti, "against" + biotikos, “fit for life“) was

coined by Selman Waksman in 1942 to describe any substance produced by a

microorganism that is antagonistic to the growth of other microorganism in high

dilution. This original definition excluded naturally occurring substances, such as

gastric juice and hydrogen peroxide (they kill microorganism but are not produced by

microorganism), and also excluded synthetic compounds such as the sulphonamides

(which are antimicrobial agents). Many antibiotics are relatively small molecules with

a molecular weight less than 2000Da. Antibiotic sensitivity is a term used to describe

the susceptibility of bacteria to antibiotic. The word “antibiotics” refers to a metabolic

product of one microorganism in very small amounts which is detrimental or

5
inhibitory to other microorganism. Streptomycin was the first example of an

antibiotic, possessing a broad spectrum of activity, effective against many gram

positive and gram negative bacteria. Other antibiotics with even broader spectra of

activity for example tetracycline have been subsequently discovered. They also

reported that Streptomyces erytheus, S.halstiddi, S.griseus, S.fradiae and

S.aureofaciens produce erythromycin, carbomycin, streptomycin, neomycin and

tetracycline Despite high efficiency of modern antimicrobial preparations, they are the

objects of further improvement. Antibiotics often produce highly selective effects on

biochemical processes and inhibit a single step in the chain of biochemical reactions.

Study of any effective antimicrobial agent should evaluate not only its effect on

microbial metabolism, but also the mechanism of its selectivity. To this end, the

effects of these compounds on biochemical processes in both microbial and host cells

should be analyzed. Tetracyclines are broad-spectrum agents, exhibiting activity

against a wide range of gram positive and gram negative bacteria, atypical organisms

such as chlamydiae, mycoplasmas, and rickettsiae, and protozoan parasites. The

favourable antimicrobial properties of these agents and the absence of major adverse

side effects has led to their extensive use in the therapy of human and animal

infections. Tetracyclines are also used prophylactically for the prevention of malaria

caused by mefloquine- resistant Plasmodium falciparum. Furthermore, in some

countries, including the United States, tetracyclines are added at subtherapeutic levels

to animal feeds to act as growth promoters. Although the tetracyclines retain

important roles in both human and veterinary medicine, the emergence of microbial

resistance has limited their effectiveness. Undoubtedly the use of tetracyclines in

6
clinical practice has been responsible for the selection of resistant organisms.

Nevertheless, as we enter the new millennium, the use of tetracyclines and other

antibiotics as animal growth promoters is becoming increasingly controversial

because of the concern that this practice may be contributing to the emergence of

resistance in human pathogens.. Tetracycline is an antibiotic and it has high activity

against all Gram positive and Gram negative bacteria.

7
 OBJECTIVES

Antibiotics are a special kind of chemotherapeutic agents usually obtained from

microorganisms. Streptomycin was the first example of an antibiotic, possessing

abroad spectrum of activity, effective against many gram positive and gram negative

bacteria. Other antibiotics with even broader spectra of activity for example

tetracycline have been subsequently discovered. The effectiveness of individual

antibiotics varies with the location of the infection, the ability of the antibiotic to reach

the site of infection, and the ability of the bacteria to resist or inactivate the antibiotic.

Some antibiotics actually kill the bacteria (bactericidal), whereas others merely

prevent the bacteria from multiplying (bacteriostatic) so that the host's immune system

can overcome them. Streptomyces are common soil bacteria, which belong to the

genus Actinomycetes. They grow as mycelia and differentiate to produce spores. They

are important in soil ecology and have gained immense industrial importance by their

production of antibiotics and related commercially important substances. About

60%of known antibiotics are produced by Streptomyces species. The strong selection

for new structures, which is suggested by the vast diversity of antibiotics produced,

makes Streptomyces a particularly interesting group of organism for studying

questions related to genetic variability. Another reason is the large genome size of

about 8 Mb, which is considerably larger than most well-studied metabolically

versatile bacteria such as E. coli and Bacillus subtilis.

8
 REVIEW OF LITERATURE

Studies about antibiotics

1.Patent docs Investigated the method which includes several steps including

obtaining a bacterial sample; identifying the type of bacteria in the bacterial sample;

selecting a set of antibiotics based on the identity of the bacteria in the bacterial

sample; obtaining a control sample from the bacterial sample; placing the bacterial

sample in solutions containing the set of antibiotics; determining concentration of

bacteria in the respective antibiotic solutions; determining growth curves for the

respective antibiotic solutions based on the determined bacterial concentration; and

comparing the growth curves for the respective antibiotic solutions with a growth

curve determined from the control sample.

Studies about Streptomyces

Mikulik K fet al Studied that, Streptomycetes are soil microorganisms exposed to

various stresses that activate specialized responses whose coordinated action promotes

growth under adverse conditions. Ribosomes having a highly cooperative structure are

potential target for control mechanisms that generate signal and activate adaptive

regulons or developmental programs. Streptomyces aureofaciens producing

tetracycline responds to the presence of antibiotics and stresses induced by the

changes in temperature. Tetracycline interacts with 16S RNA and decreases its

thermodynamic stability. The drug also inhibits binding of ternary complex Phe-

tRNA.EF-Tu.GTP to purified ribosomes. They have found that antibiotics that cause

ribosome stall or pause could increase the requirement for tmRNA in the process

9
transtranslation. Increase in tm RNA level was also demonstrated upon downshift in

temperature.

Antibiotic activity of Streptomyces sp

Sejiny MJ et al Concluded that all tested strains showed a progressive increase of

biomass (dry weight) during the first 4-7 days of incubation. On the contrary, the

highest antibiotic activity as recorded in the stationary phase of growth (the last 5 days

of incubation). Although the accumulation of antibiotic started on the 2nd or 3rd day

of incubation, the highest accumulation was observed on 9th and 10th day of

incubation.

10
 METHODOLOGY

Isolation of antibiotic producing microorganisms from different soil samples.

Collection of samples:

About 5g of the soil sample was collected from CDRI.

Serial dilution:

Requirements:

 Soil sample

 Test tubes

 Sterilized pipettes

 Distilled water/saline Procedure

 The sterile test tubes were taken (cotton plugged test tubes) and the tubes were

labelled as per dilutions ranging from 10-1 ٫to 10-5 .

 9.0ml of sterile distilled water/saline was added to each test tube aseptically.

 1 g of soil sample was added into the first dilution blank of 9.0ml of distilled

water/saline. The tube was shaken vigorously for few minutes.  Large particles were

allowed to settle.

 1.0ml from the first dilution blank (10-1 ) was added to the second dilution blank

(10-2 ).

 The test tubes were shaken vigorously for two minutes.

 Serial dilution was done till the last tube (10-5 ) dilutions.

Pour plate method: Pour plate was used for quantifying microorganisms that grown

in solid medium and colonies formed within agar matrix. Requirements:

 Serially diluted solutions (10 -2 , 10-4 & 10-5 dilutions)

11
  Petri plates

 Ethanol (70%)

 Bunsen burner

 Incubator

 Starch casein Agar media Composition of media Starch -10g Casein -3g Yeast

extracts -3g Sodium chloride -3g Agar -2g Distilled water -1000ml .

Procedure-

 0.1 ml from the serially diluted samples (10-2 , 10-4 & 10-5 dilutions) was

poured into the sterile petri plates.

 The petri plates were shaken for uniform distribution of the sample.

 After few minutes, the starch casein agar was gently poured into the petri plates.

 After solidification, the plates were incubated for 24hr at 37°C. Identification of

isolated microorganisms:

Preparation of pure culture:

Requirements:

 Pour Plates (10-2 , 10-4 & 10-5 dilutions)

 Test tubes

 Ethanol (70%)

 Inoculation loop.

 Starch casein agar Procedure.

 The starch casein agar was gently poured into the sterile test tubes and was

allowed to solidify as slants.

12
 After agar gets solidified, the inoculation loop was sterilized and a loop full of the

culture was taken from the pour plates and streaked onto the solidified agar.

 After solidification, the tubes were incubated for 24hr at 37°C. 4.2.2. Gram

staining

13
Requirements
Grease free glass slide, Crystal violet, Gram’s iodine, Saffranin, 95% Ethyl alcohol.

Procedure –

 A clean grease free glass slide was taken.

 A thin smear of the isolated organism was made.

 The smear was heat fixed.

 The slide was flooded with crystal violet and allowed to stand for 20-40 seconds.

 The slide was washed with distilled water.

 Gram’s iodine was added to the smear and allowed to stand for 1 minute.

 The slide was flooded with 95% ethyl alcohol for 5-10 seconds.  The slide was

rinsed with distilled water.

 Saffranin was added to the slide and kept for 30 seconds.

 The slide was washed gently for few seconds and air dried.

 The slide was observed under oil immersion

14
Objective-
Gelatin hydrolysis test: Proteins are organic molecules composed of amino acids, in

other words proteins contain carbon, hydrogen, oxygen and nitrogen, and some

proteins contain sulphur too. Amino acids are linked together by peptide bond to form

a small chain or large chain of protein. Gelatin is a protein produced by hydrolysis of

collagen, a major component of connective tissue and tendons in human and other

animals. It dissolves in warm water. Large protein molecules are hydrolyzed by

exoenzymes, and the smaller products of hydrolysis are transported into the cell.

Hydrolysis of gelatin is brought about by microorganisms capable of producing a

proteolytic exoenzyme known as gelatinase, which hydrolyzes this protein to amino

acids. Hydrolysis of gelatin in the laboratory can be demonstrated by growing

microorganisms in nutrient gelatin. Once the degradation occurs, it can be detected by

observing liquefaction or testing with a protein precipitating material because gelatin

is also precipitated by chemicals that coagulate proteins while the end products of

degradation are not precipitated by the same chemicals .

15
Requirements:

 The nutrient agar sample culture.

 Gelatin agar medium.

 Nutrient gelatin deep tubes.

 Inoculating loop.

Procedure:

 Gelatin agar medium was taken in test tubes.

 The sample organism was inoculated in these test tubes and one test tube was left

uninoculated and which was taken as a comparative control.  The inoculated tubes

were incubated for 48hr at 37oC.

 All the tubes were kept at 4oC for 15 minutes. Observation: The refrigerated

gelatin tubes were examined to see whether the medium was in a solid or a liquid

form.

Starch hydrolysis test:

Amylase is an exoenzyme that hydrolyses starch, a polysaccharide into maltose, a

disaccharide and some monosaccharides which will enter into the cytoplasm of the

bacterial cell through the semipermiable membrane and there by the endoenzymes.

Starch is a complex carbohydrate composed of two constituents amylase, a straight

chain polymer of 200-300 glucose units, and amylopectin, a larger branched polymer

with phosphate groups. The ability to degrade starch is used as criteria for the

determination of amylase production by microbe. Starch test determines absence and

16
presence of iodine which produces a dark blue coloration of the media and yellow

zone around the growth of the microbe in media.

Requirements:

 Nutrient agar culture sample.

 Starch agar medium.

 Gram’s iodine solution.

 Sterile Petri dishes.

 Inoculating loop.

Procedure:

 The starch agar medium was poured into petri dishes asceptically.

 The tubes were allowed to solidify.

 The organisms were streaked on the plates.

 The tubes were Incubated at 37oC for 24 hr.

 The surface of the plates was flooded with iodine solution with a dropper for 30

sec.

 The excess iodine solution was removed. Observation: The plates were examined

for starch hydrolysis around the line of growth of each organism, i.e. the color change

of the medium.

Catalase test:

During aerobic respiration in the presence of oxygen, microorganisms produce

hydrogen peroxide which is lethal to the cell. The enzyme catalase present in some

microorganism breaks down hydrogen peroxide into water and oxygen.

17
Requirements:

 Culture sample.

 Glass slide.

 Inoculating loop.

 Hydrogen peroxide.

Procedure:

 A clean glass slide was taken.

 Using an inoculation loop a thick smear of the culture was made on the slide.

 A drop of hydrogen peroxide was added on the smear. Observation: Appearance or

absence of gas bubbles was observed.

Screening for tetracycline production from identified organisms: Quadrate

streak - lawn culture plate method:

Requirement

Culture Petri plate LB agar Inoculating loop Bunsen burner Procedure

 LB agar plates were prepared

 Lawn cultures of different organisms were made.

 A loopful of isolated organism was taken and quadrant streaking was done on the

agar plate.

 The plates were incubated at 37°C in inverted position for 24-48 hr. Observation:

 After incubation, the plates were observed for growth. 4.4. Media optimization

studies for the production of antibiotic Different types of media were used for the

18
production of tetracycline. They are Basal solid media, sk2 and pk2 media and starch

media.

Starch media composition

Starch - 45g Calcium carbonates -10.5g NH4 Cl - 1.5g Cotton seed flour - 45g Yeast

- 1.5g Water - 1litter Preparation of media

 A cleaned conical flask was taken.

 4.5g weighed starch was dissolved in 50ml of distilled water and 1.05g of calcium

carbonate was added and stirred well, the remaining chemicals were added like 4.5g

cotton seed flour ,0.15g of ammonium chloride ,0.15g of yeast in conical flask and the

volume was made up to 100ml.

 The conical flask was plugged with cotton wool.

 It was autoclaved at 121°C,15lbs pressure for 15 minutes

 The autoclave was allowed to cool.

 The media was removed and stored at room temperature for the production.

Characterization of purified antibiotic For identification of tetracycline,

the purified sample was run in thin layer chromatography along with standard

tetracycline.

Thin layer chromatography

Materials

Glass rod, Silica gel, Grease free glass slide, Thin Layer Chromatography Chamber,

Chloroform, Acetic acid.

Methods-

19
 Three grease free glass slide were taken.

 The slurry was made with silica gel and distilled water.

 Silica gel was poured onto glass slides and spread.

 Three slides were prepared.

 They were kept in the oven at 42oC for drying.

 The standard and sample were added to the gel, at 1 cm distance from the bottom

of the slide.

 The slides were then kept in the solvent.

 The bottom layer of the slide was dipped into the solvent.

 The lid of the chamber was closed.

 It was kept for one and half an hour.

 The bands were observed.

Antimicrobial sensitivity testing

Materials

Muller Hinton agar medium, L-rod, sterile well cutter, Glass wares. Escherichia coli,

Klebsiella pneumonia, Salmonalla typhi.

Methods-

 Muller Hinton agar medium was prepared and sterilized.

 It was poured into sterile petri plates and allowed to solidify.

 A uniform layer of Escherichia coli, Klebsiella pneumonia, and Salmonalla typhi

was applied on appropriately labeled plates by using an L-rod.

 Wells were punched in the plates by used a sterile well cutter.

20
 20, 30, 40, 50μl of the isolated antibiotic were added in to each well.  The plates

were incubated at 37oC for 24-48 hr.

 The formations of zones were observed around the wells.

Quantitative analysis
High Pressure Liquid Chromatography (HPLC) Technically HP stands for high

pressure but it is also known as high performance and high price chromatography.

High performance because this chromatography provides consistent result irrespective

of number of repetitions. High price because the cost of chemicals used as mobile

phase is too high and only HPLC grade solvents are used as mobile phase. HPLC is of

two types, quantitative and qualitative used to determine purity of sample. HPLC is of

two types, preparatory and separating used in downstream processing. Description of

mobile phase Mobile phase is the liquid phase. The solvents are chosen on the basis of

visibility and solubility. The term visibility deals with cut off wavelength for that

particular solvent. The term solubility determines the extent to which the sample is

soluble in solvent system, in case the solubility of sample is found to be less, then the

separation of sample molecules is not achieved. The most frequently used solvents are

acetonitrile (190), methanol (205), and water (205). The number in bracket is cut off

wavelength, which means any wavelength above this number can be used for

detection, hence permits the use of wide range visibility. The other possible solvents

are sodium acetate buffer, chloroform and butanol, but generally not preferred because

of high cut off wavelength.

Preparation of mobile phase:

21
 Single measuring cylinder system should be used. Example, consider 100 ml of

solvent system is required involving aceto nitrile and phosphate buffer in ratio of

60:40, take a cylinder of 100 ml, pour 60 ml of aceto nitrile and in the same cylinder

add 40 ml of phosphate buffer, this defines the single cylinder system.

 pH should be properly calibrated in accordance to nature of sample under

consideration.

 Membrane filtration with vacuum, involving the use of nitrocellulose membrane

should be carried out. The pore size of this membrane ranges from 0.22 micron-0.44

micron. 0.22 micron is generally used because it helps in removal of smallest virus

and bacteriophages.

 Degassing is carried out, to remove air. This can be achieved by heating, sparging,

vacuum desiccators, ultra sonication. Generally heating is avoided because it might

cause damage to mobile phase. The technique of sparging takes more time, hence not

preferred. Ultra sonication based on ultra wavelength is the best way to carry out

degassing.

 It is recommended to store mobile phase in stainless steel cylinders but due to

maintenance problems it’s difficult to use. Plastic and colorless bottles should not be

used for storage purpose. Dark colored bottles like brown are allowed for storage. If

pH is 9 and above, the usage of dark bottles should be strictly avoided.

 Mobile phase is stored at room temperature.

Pump: Pump is one of the components of HPLC, used for maintaining constant flow

rate of liquid through column packed with stationary phase. On the basis of number of

pistons two types of pump systems are available namely single headed pump and dual

22
piston reciprocating pump. Single headed pump has a single piston which sucks the

liquid from reservoir and pushes to column. This system involves back flushing,

which prevents the back movement of liquid. Back flushing provides a kind of storage

for sample. Dual piston reciprocating pump, as name suggests has two pistons. One

piston sucks the liquid while other pushes it into system. Reciprocating term suggests

that work done by a piston is not fixed, which means a piston can either suck or

pushes liquid. On the basis of number of solvents two types of pump are isocratic and

gradient. Isocratic when solvent passing through column is nonvarying in composition

with a single system, whereas gradient involves varying number of solvents with may

be two or three pumps. Gradient system is used to analyze many compounds at a

single time, example analysis of contents of tablets. Solvent flows from reservoir to T

section; from here it goes through two pistons. Each piston possess check valves on

upstream as well as downstream of it, to keep a track on pressure of liquid passing

through it. The downstream check valves open to another T section. From here, it

flows to purger, helps in removal of air bubble if any. Next the liquid travels to

pulsating damper, helps to maintain smooth flow of liquid. This opens to back

pressure sensor, takes the value of pressure limit as a set value for it. If the pressure

increases beyond this set value, the pump switches off automatically. Lastly liquid

passes through filter to remove biological contamination before entering the column.

The above description of pump comes under operating system of pump. The

functional system of pump involves pressure limit (set value for back pressure sensor),

current limit (the actual or working pressure of liquid), flow rate (ml/min of liquid

flowing), on/off switch. Injector Injector is installed with either 6 ports or 7 ports

23
binary valve system. In case of 7 ports, first port is found to be nonfunctional. The

term binary is used because two ports are connected to each other. The function of

ports is as follows:

 injection port

 sample loop, for storage of sample

 pump port

 column port

There are five types of column.

 Guard- used in HPLC

 Capillary- used in GC

 Derivatizing- it can resist high temperature

 Fast-used in downstream processing for purification of compound

 Preparatory Two types of column are used, namely normal and reverse.

Normal involves polar stationary phase and nonpolar mobile phase, while reverse has

nonpolar stationary phase and polar mobile phase. Reverse type of column is used in

HPLC because it exhibits more adsorbent properties. The stationary phase can range

from C1 to C18, C-diol, C-NH2, C-phenol, C-CN. C1- silane is used in normal type of

column. For everse phase C2 to C18 can be used. Most frequently used are C5 and

C18 that is pentasilane and octadecasilane. For isolation of protein C-phenol, C-18 are

mostly used. Column is made up of large number of plates, the number of which is

proportional to resolution obtained in separation.

Column goes through three types of analysis: installation analysis, running analysis

and certification analysis. During installation of column, working of it is thoroughly

24
checked by following the protocol given by manufacturer is strictly followed. The

protocol results are compared to once which are obtained through analysis, if found to

be same then only running analysis is carried out. During this, sample analysis is

carried out, if results obtained are found to be satisfactory, it approaches final stage of

certification analysis. After removal of sample from column, it is washed with 70%

ethanol, even washing should be done before putting it to use. The column can be

washed with different solvents to remove clog by proteins. Column forms the heart of

HPLC; hence its maintenance is must.

Detector Seven types of detector are available

 Visible

 UV

 UV visible

 Refractory Index

 Infrared

 radioactive elements UV visible type of detector is commonly used. It involves two

lamps, tungsten is used for visible range and deuterium/mercury/ hydrogen are used in

UV lamps. To increase the intensity of light coming from lamp, it is passed to silver

mirror. The light is incident on lens system, which helps to focus light on small slit.

From slit, it travels to monochromator, to select a light of particular wavelength. The

monochromator can be prism type or it can involve grating (small edges) system. A

light absorber of monochromator absorbs light of all wavelengths except one which is

required for analysis of sample. The transmitted light from absorber passes through

the cuvette.

25
Objective:

To estimate tetracycline content in the purified sample Protocol: Test mix: tetracycline

Mobile phase: acetonitrile: phosphate buffer (pH 2.5), (60:40) Flow rate: 1ml/min

Temperature: ambient Sample Volume: 20 μl Column: C18 Detection: UV (268 nm) .

Preparation of sample:

 1ml of purified sample was taken in to 25ml volumetric flask

 The volume was made up to 25ml by adding the mobile phase

 It was membrane filtered using 0.22 micron membrane.

 Then the samples were ready for injection.

Preparation of standard:

10mg tetracycline was taken in 25ml volumetric flask and volume was made up to

25ml by adding the mobile phase, then membrane filtered. Calculation: Average

Amount of tetracycline= Sample area X Standard amount X Dilution X weight

Standard area X Dilution X Sample amount Retention Standard Deviation= (Higher

area- Lower area) X 100 Higher area Within 0.5% results are accepted.

Observation: The peaks were observed

26
 RESULT

Isolation of antibiotic producing microorganism from different soil sample: More

than 5 soil samples were collected from CDRI. and isolation of organisms was done

by serial dilution using starch casein agar media. After 72 hr incubation, according to

the colony morphology, pure cultures were prepared. This culture was subjected to

staining and biochemicals test to confirm the isolate as Streptomyces aureofaciens

Collection of sample:

Soil sample was taken and used further for serial dilution.

Serial dilution of soil sample:

Preparation of pure culture:

Pure culture was prepared on the slant and kept in refrigerator.

27
Table No.2

 Gram Staining-

 Gelatin Hydrolysis Test

 The test was positive due to the formation of solids.

 Starch Hydrolysis Test

28
  The test was positive due to the colour change of the medium.
 Catalase Test

 Formation of gas bubble indicated that test was negative.

 Quadrate streak- plate method

 Characterization of purified antibiotic:

 The purification was confirmed by running the purified tetracycline on the TLC
plate along with standard tetracycline obtained from sigma Aldrich.
Characterization of microbial study was proved by using different pathogens
like Klebsiella pneumonia and Salmonella typhi on observing the zone of
inhibition.
 Thin layer chromatography:

 Antimicrobial sensitivity testing:

 Antimicrobial activity by using K. pneumonia in basal solid

media pellet (acet)

29
 20μl was sufficient for obtaining the zone of inhibition

 Antimicrobial activity by using K. pneumonia in pk2 media

liquid (acet)

 30μl was sufficient for obtaining the zone of inhibition

 Antimicrobial activity by using S.typhi in basal solid media pellet

(acet)

50μl was sufficient for obtaining the zone of inhibition

 Antimicrobial activity by using K. pneumonia in basal solid

media pellet (met)

30
 20μl was sufficient for obtaining the zone of inhibition

 Antimicrobial activity by using S.typhi in basal solid media

liquid(met)

50μl was sufficient for obtaining the zone of inhibition

 Antimicrobial activity by using S.typhi in basal solid media

pellet (met)

20μl was sufficient for obtaining the zone of inhibition

 Quantitative analysis
 High Pressure Liquid Chromatography (HPLC)

31
 DISCUSSION

The tetracyclines, which were discovered in the 1940s, are a family of antibiotics that

inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the

ribosomal acceptor (A) site. Tetracyclines are broad spectrum agents, exhibiting

activity against a wide range of gram positive and gram negative bacteria, atypical

organisms such as chlamydiae, mycoplasmas, and rickettsiae, and protozoan parasites.

The favourable antimicrobial properties of these agents and the absence of major

adverse side effects has led to their extensive use in the therapy of human and animal

infections.

Chlortetracycline and oxytetracycline, both discovered in the late 1940s, were the first

members of the tetracycline group to be described. These molecules were products of

Streptomyces aureofaciens and S. rimosus, respectively. Other tetracyclines were

identified later, either as naturally occurring molecules, e.g., tetracycline from S.

aureofaciens, S. rimosus, and S. viridofaciens and demethylchlortetracycline from S.

aureofaciens, or as products of semisynthetic approaches, e.g., methacycline,

doxycycline, and minocycline.

32
 CONCLUSION

Streptomyces are common soil bacteria, which belong to the genus Actinomycetes.

They grow as mycelia and differentiate to produce spores. They are important in soil

ecology and have gained immense industrial importance by their production of

antibiotics and related commercially important substances. About 60% of known

antibiotics are produced by Streptomyces species.. Another reason is the large genome

size of about 8 Mb, which is considerably larger than most well studied metabolically

versatile bacteria such as E. coli and Bacillus subtilis.

Streptomyces are Gram positive, mycelial organisms and they produce several

extracellular hydrolases. Streptomyces proteases came into focus as a byproduct of

antibiotic fermentation. Protease production seems to be interlinked with the complex

regulation of secondary metabolite biosynthesis and sporulation.. The study of the

genetics of Streptomyces is important not only because of many antibiotics it

produces, but also because its differentiation and its regulation of secondary

metabolism are of basic interest.

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3712.

3. Nadir babay. Smear layer remove from root dentin using tetracycline

hydrochloride concentrations, An SEM study Saudi dental journal. 2003;15(1).

4. Dawson CR, Daghfous T and Whitcher J. Intermittent trachoma

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Bulletin of world health organization.

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