3.
0 ANTIBIOTICS
❖ Learning objective: Define antibiotics, describe how antibiotics are classified and
synthesised
3.1 Historical background
➢ Microorganisms living in complex environments develop a means by which they
control the growth of competing microorganisms perhaps by producing chemicals -
secondary metabolites.
➢ In early centuries earlier, humans had learned to use crude preparations empirically
for the topical treatment of infections, which we now assume to be effective because
of the antibiotic substances present.
➢ 500 to 600 BC, moulded curd of soybean was used in Chinese folk medicine to treat
boils and carbuncles.
➢ Mouldy cheese had also been used for centuries by Chinese and Ukrainian peasants
to treat infected wounds
➢ Sir Alexander Fleming’s (1929); accidentally discovered the antibacterial properties of
penicillin- largely credited with initiating the modern antibiotic era.
➢ Florey and Chain (1938); introduced penicillin into therapy.
➢ Pasteur and Joubert (1877) that anthrax bacilli were killed when grown in culture in
the presence of certain bacteria, along with similar observations by other
microbiologists.
➢ Vuillemin define antibiosis (literally “against life”) as the biological concept of survival
of the fittest, in which one organism destroys another to preserve itself. The word
antibiotic was derived from this root.
➢ However, the use of the term by the lay public, as well as the medical and scientific
communities, has become so widespread that its original meaning has become
obscured.
3.2 Definitions
➢ Waksman (1942), proposed the definition: “an antibiotic or antibiotic substance is a
substance produced by microorganisms, which has the capacity of inhibiting the
growth and even of destroying other microorganisms.”
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➢ Later proposals both to expanded and restricted the definition to include “any
substance produced by a living organism that is capable of inhibiting the growth or
survival of one or more species of microorganisms in low concentrations”.
Advances made by medicinal chemists to modify naturally occurring antibiotics
and to prepare synthetic analogues necessitated the inclusion of semisynthetic
and synthetic derivatives in the definition.
➢ Other definitions:
Compounds produced naturally by microorganisms and kill or inhibit the growth
of other microorganisms, mainly bacteria
A chemical substance produced by a living organism, generally a microorganism,
that is detrimental to other microorganisms.
Compounds produced by bacteria and fungi which are capable of killing or
inhibiting, competing microbial species.
A type of antimicrobial substance active against bacteria
➢ Generally, for a substance to be classified as an antibiotic it must meet the following
conditions:
1. It is a product of metabolism (although it may be duplicated or even have been
anticipated by chemical synthesis).
2. It is a synthetic product produced as a structural analogue of a naturally occurring
antibiotic.
3. It antagonizes the growth or survival of one or more species of microorganisms.
4. It is effective in low concentrations
➢ Characteristics of an ideal antibiotic:
Kills or inhibits the growth of pathogens
Causes no allergy to host cells
Does not cause damage to host cells
Should be stable when stored in liquid or solid forms.
➢ A limited number of antibiotics have also been found to possess anti-protozoal
activity.
➢ Antibiotics are not effective against the viruses, such as ‘common cold’ or ‘influenza’
➢ Antibiotics may be available in the form of tablets, capsules, oral suspensions and
powder for injection.
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➢ The greatest numbers of antibiotics are derived from soil-dwelling bacteria in the
genera Streptomyces and spore-forming bacteria Bacillus and moulds in the genera
Penicillium and Cephalosporium (renamed to Acremonium).
➢ Of the three, Streptomyces species are the most prolific producers - they are the
source of two-thirds of the antibiotics; about 9,000 different bioactive compounds
have been isolated from them.
➢ There are five major components that are useful drug targets in an actively dividing
cell. These are: (i) the cell wall (ii) cell membrane, (iii) the genetic material, (iv) protein
synthesis, and (v) metabolic pathways.
➢ Their mechanism of action of antibiotics can be:
1. Inhibition of cell wall synthesis,
2. Breakdown of the cell membrane structure or function,
3. Inhibition of structures and functions of DNA and RNA
4. Inhibition of protein synthesis, and
5. Blocks on key metabolic pathways.
➢ Antibiotics that produce effects (1) and (2) are considered bactericidal, because the
weakened cell is subject to lysis. It is essential to note that most of these antibiotics
are active only in young, growing cells, because old, inactive, or dormant cells do
not synthesize peptidoglycan.
➢ Penicillin’s and cephalosporins bind and block peptidases that cross-link the glycan
molecules, thereby interrupting the completion of the cell wall. Penicillin’s that do
not penetrate the outer membrane are less effective against Gram-negative
bacteria.
➢ Aminoglycosides (for example streptomycin, gentamicin) insert on sites on the 30S
subunit and cause the misreading of the mRNA, leading to abnormal proteins.
3.3 Resistance
➢ Use, overuse and/or misuse, incorrect usage of many of these agents and the
biochemical fickleness of many bacteria, has led to resistance to antibiotics - a serious
problem in the 21st century.
➢ There is emergence of several resistant strains of bacteria that cannot be managed
with common antibiotics e.g., methicillin-resistant Staphylococcus aureus, ('golden
staph' or MRSA) and Neisseria gonorrhoeae resistant to benzyl penicillin.
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➢ The mechanisms of resistance are:
✓ Enzyme inactivation and modification of the drug.
✓ Modification of the antibiotics target site.
✓ Overproduction of the target.
✓ Replacement of the target site.
✓ Active drug efflux and reduced permeability.
✓ Limiting uptake of a drug
3.4 Classification of Antibiotics
➢ Antibiotics can be classified based on various schemes, such as:
(1) Chemical structure
(2) Mechanisms of action
(3) Spectrum of activity
(4) Mode of action
(5) Absorbability.
The chemical classes of antibiotics are:
(1) Penicillin’s: Antibiotics that possess the β-lactam (a four-membered cyclic amide) ring
structure
(2) Tetracyclines: Tetracycline, rolitetracycline, oxytetracycline, chlortetracycline,
demeclocycline, meclocycline, methacycline, doxycycline, and minocycline. The
tetracyclines are obtained by fermentation procedures from Streptomyces spp. or by
chemical transformations of the natural products.
(3) Cephalosporins: cephalosporins are β-lactam antibiotics isolated from
Cephalosporium spp. or prepared semisynthetically
(4) Quinolones:
(5) Lincomycin’s: sulfur-containing antibiotics isolated from Streptomyces lincolnensis.
Lincomycin is the most active and medically useful of the compounds obtained from
fermentation, Clindamycin
(6) Macrolides: picromycin, (isolated from the actinomycetes), erythromycin and
carbomycin, semisynthetic derivatives oferythromycin (e.g., clarithromycin and
azithromycin). more than 40 such compounds
(7) Sulphonamides
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(8) Glycopeptides: glycopeptide antibiotic vancomycin (Vancocin, Vancoled) isolated
from Streptomyces orientalis (renamed A. orientalis), Teicoplanin (Teichomycin A2,
Targocid) is a mixture of five closely related glycopeptide antibiotics produced by the
actinomycete Actinoplanes teichomyceticus Bacitracin isolated from a strain of B.
subtilis
(9) Aminoglycosides – streptomycin, kanamycin, neomycin, paromomycin, gentamicin,
tobramycin, and netilmicin
(10) Carbapenems
According to Spectrum of Activity
➢ Antibiotics are classified into:
(1) Narrow or short spectrum,
(2) Wide spectrum
➢ The narrow or the short spectrum antibiotics are those antibiotics which target only
specific bacteria and act against narrow range of disease. The examples are: penicillin
G, vancomycin, etc.
➢ The broad-spectrum antibiotics are those antibiotics which work on many different
kinds of bacteria and act against wide range of disease. The examples are: tetracycline,
chloramphenicol, etc.
According to Mode of Action
➢ The antibiotics are again two types:
(1) the bacteriostatic,
(2) the bactericidal.
➢ Bacteriostatic inhibit the growth of bacteria but do not kill them. Example:
tetracycline.
➢ Bactericidal the bacteria. Examples: penicillin, cephalosporin.
According to Mechanism of Action
There are many types of antibiotics, like
(1) Antibiotics that inhibiting the synthesis of bacterial cell wall:
➢ Include penicillins and cephalosporins that are structurally similar and dissimilar
agents, such as cycloserine, vancomycin, bacitracin and the imidazole antifungal
agents.
(2) Antibiotic interfering with cell membrane integrity,
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➢ These agents act directly on the cell membrane of the microorganisms, affecting
permeability, and leading to leakage of intracellular compounds. Examples:
polymyxin, polyene antifungal agents, nystatin, and amphotericin B that bind to cell
wall sterols
(3) Inhibiting nucleic acid synthesis,
➢ Agents that affect the function of 30s and 50s ribosomal subunits to cause reversible
inhibition of protein synthesis: These include tetracyclines, erythromycins,
chloramphenicol, and clindamycin.
(4) Inhibiting protein synthesis
➢ Include agents that bind to the 30s ribosomal subunit and alter protein synthesis.
They include aminoglycosides that leads to cell deaths eventually.
(5) Inhibiting certain metabolic pathway
➢ Agents that affect nucleic acid metabolism: Such as rifamycins, which inhibit DNA
dependent RNA polymerase.
3.5 β-lactam antibiotics – model system
➢ β-lactam antibiotics can be divided into five distinct classes, viz: - penicillin’s,
cephalosporins, cephamycin’s, carbapenems, monolactams.
Figure 3.1: Classes of ß-lactam antibiotics
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➢ Penicillins and cephalosporins are model systems within the antibiotic world because:
(i) They were the first antibiotics produced on a large scale in submerged
fermentations. (Instigated the development of fermentation
technology, and many of the techniques used to produce penicillin’s
have been used as the basis for the production of other microbial
metabolites, especially antibiotics)
(ii) Penicillin was the first antibiotic used worldwide.
Biosynthesis of penicillin’s, cephalosporins, cephamycin’s
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➢ The penam nucleus of penicillin’s and the cephem nucleus of both cephamycin’s and
cephalosporins are formed by the condensation of three precursor amino acids: L-α-
aminoadipic acid, L-cysteine, and L-valine, by a mechanism designated as “non-
ribosomal peptide synthesis”
➢ The mechanism involves activation and condensation of the three component amino
acids and epimerization of the L- to D-valine to form the tripeptide δ-(L-α-
aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV).
➢ Out of the many natural penicillins discovered, only two purified compounds are in
clinical use: penicillin G (intramuscular or intravenous use) and penicillin V (given by
mouth).
➢ Benzylpenicillins (penicillin G) and phenoxymethylpenicillins (penicillin V) are fungal
fermentation products and are precursors to a wide range of semi-synthetic
antibiotics (amoxicillin, ampicillin etc.).
➢ Penicillin G (benzylpenicillin) was first produced from a penicillium fungus that occurs
in nature.
➢ The strain of fungus used today for the manufacture of penicillin G was created by
genetic engineering to improve the yield in the manufacturing process.
Penicillin G
➢ Penicillin G is destroyed by stomach acid, so it cannot be taken by mouth. It is given
by intravenous or intramuscular injection.
➢ It can be formulated as an insoluble salt, and there are two such formulations in
current use: procaine penicillin and benzathine benzylpenicillin, which are used only
in the treatment of syphilis.
1. It is β-lactam antibiotic.
2. Produced by many fungi particularly Penicillium and Aspergillus species.
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3. Natural penicillin’s act on numerous gram +ve bacteria.
4. They are acid labile and can be deactivated by β-lactamase produced by bacteria.
5. Because of low toxicity, large doses could be used. Only 0.5 -2% develop allergies.
6. The basic structure is 6-APA (6-aminopenicillanic acid).
7. If the penicillin fermentation is carried out without addition of side chain precursors,
the natural penicillins are produced.
8. From this mixture only benzylpenicillin is therapeutically useful; other compounds
must be removed during product recovery.
Penicillin V
➢ Penicillin V can be taken by mouth because it is relatively resistant to stomach acid.
➢ it has poor absorption. However, it is used for the same bacterial infections as those
of penicillin G and is the most widely used form of penicillin
➢ Some bacteria are able to produce penicillinase that breaks down penicillin and
renders it completely useless and ineffective in clinical treatment.
➢ In order to combat this, scientists have turned to semi-synthetic derivatives of
penicillin in the hope that these will have the properties necessary to beat the
resistance problem.
➢ The fermentation can be better controlled by adding side chain precursor so that only
one desired penicillin is produced. These are biosynthetic penicillins.
➢ In this process Penicillin G, V and O (in limited amount) is produced.
➢ Alternatively, Penicillin G or V is chemically or enzymatically split to form 6-
Aminopenicillanic acid (6-APA), which is chemically recycled to make another Penicillin
derivative. Such are known as semisynthetic Penicillins.
➢ Semi-synthetic penicillins are more frequently used in therapy because they are – acid
stable, resistant to β-lactamases and have expanded antimicrobial spectrum.
➢ Penicillin V (phenoxymethylpenicillin) is produced by adding the precursor
phenoxyacetic acid to the medium in which a genetically modified strain of the
penicillium fungus is being cultured.
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➢ The chemical modification of the fermentation product is initiated by removal of the
natural acyl group leaving the 6-aminopenicillanic acid (6-APA) penicillin nucleus.
➢ Alternative synthetic acyl groups can then be added to confer novel properties to the
antibiotic such as resistance to stomach acid, a certain degree of penicillinase
resistance or an extended range of antibiotic activity.
Penicillin acylase
➢ Industrially, conversion to 6-APA is achieved by use of penicillin acylase.
➢ Penicillin acylases (amidases): are a group of microbial enzymes which can cleave the
acyl chain of penicillins to yield 6-amino penicillanic acid (6-APA) and the
corresponding organic acid.
➢ In a number of cases the same enzyme can be used to direct the synthesis of the new
antibiotic by the addition of the novel acyl group as shown below.
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➢ Penicillin acylases (PAC) are found in a wide range of bacteria, actinomycetes, yeasts
and fungi.
➢ Based on their substrate specificities (side chain preferences), they have been
classified into three groups: penicillin G acylases, penicillin V acylases and ampicillin
acylases.
(i) Penicillin G acylases: are found in species such as Escherichia coli, Alcaligenes
faecalis and Arthrobacter viscosus.
(ii) Penicillin V acylases are found in species such as Bacillus subtilis and Fusarium
oxysporium,
(iii) Ampicillin acylases are found in Streptomyces lavendulae and Pseudomonas
melangenum.
Synthesis of Penicillin V
➢ Penicillin is not a single compound but a group of closely related compounds, all with
the same basic ring-like structure (a β-lactam) derived from two amino acids (valine
and cysteine) via a tripeptide intermediate.
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➢ The third amino acid of this tripeptide is replaced by an acyl group (R in the diagram
below) and the nature of this acyl group produces specific properties on different
types of penicillin.
Synthesis og penicillin G
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Factors effecting synthesis
(i) The amino acid lysine is synthesised from a pathway that involves L-α AAA. Lysine
inhibits penicillin synthesis because it is a feedback inhibitor of homocitrate synthase,
an enzyme involved in synthesis of L-α AAA. If L-α AAA is deficient Penicillin cannot be
synthesised.
(ii) Biosynthesis is affected by glucose repression; thus, sometimes fermentation is done
with slowly metabolizable sugar lactose.
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3.4 Industrial Production of Antibiotics
❖ Learning objective: Describe how antibiotics are produced in industry by fermentation
Key Points
❖ Antibiotics are the secondary metabolites of microorganisms.
❖ During processing, the antibiotic must be extracted and purified to a crystalline
product.
➢ Metabolite: Any substance produced by, or taking part in, a metabolic reaction.
➢ Secondary metabolite: substa
➢ Antibiotics can be produced in three ways:
✓ Natural microbial production: - uses fermentation technology example
penicillin production.
✓ Semisynthetic production: - post production modification of natural antibiotics
e.g., ampicillin
✓ Synthetic production of antibiotics: made synthetically in the lab e.g., quinoline
➢ Fermentation: Any of many anaerobic biochemical reactions in which an enzyme (or
several enzymes produced by a microorganism) catalyses the conversion of one
substance into another; especially the conversion (using yeast) of sugars to alcohol or
acetic acid with the evolution of carbon dioxide.
➢ Antibiotics are produced industrially by a process of fermentation; the source
microorganism is grown in large containers (100,000 – 150,000 litres) containing a
liquid growth medium.
➢ Oxygen concentration, temperature, pH, and nutrient levels must be optimal and are
closely monitored and adjusted if necessary.
➢ Population size of the microorganism must be controlled very carefully to ensure that
maximum yield is obtained before the cells die.
➢ After the fermentation process is complete, the antibiotic is extracted and purified to
a crystalline product.
➢ Methods of extraction:
✓ Solvent extraction - for those that are soluble in organic solvent
✓ Ion exchange
✓ Adsorption
✓ Chemical precipitation.
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Techniques used to increase yields in fermentation
1. Use of microorganism species that are genetically modified
➢ Mutation is initiated by:
✓ Introducing mutagens such as ultraviolet radiation, x-rays,
✓ Use of chemicals
➢ Selection and further reproduction of the higher yielding strains over many
generations can raise yields by 20-fold or more.
2. Gene amplification: copies of genes coding for enzymes involved in the antibiotic
production can be inserted back into a cell, via vectors such as plasmids.
3.5 The Step-by-Step Manufacturing Process of Penicillin G Antibiotics
1. Start the Culture
➢ The desired microbe is isolated and multiplied and stored.
➢ Usually, microorganism cultures are preserved in the form of inactive spores. This is
because spores are very resistant structures that can survive heat, desiccation, low pH
and mechanical forces that would typically kill a normal vegetative cell.
➢ To increase their longevity, these spores are often kept in a cyropreservative fluid
(e.g., Microbead system) and frozen. In this way the cells can be preserved for many
years.
➢ In this step the microorganism is allowed to multiply many times before fermentation
can begin.
➢ Typically:
o A starter culture is prepared in the lab from a previous cold-stored sample
whereby: (i) A sample of the organism is placed on an agar plate (ii) The initial
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culture is then transferred to shake flasks containing nutrients for growth. This
results in a suspension that can be moved to seed tanks for continued growth.
o The seed tanks are steel tanks designed to provide an ideal environment for
growing microorganisms
o The seed tanks are equipped with mixers, which mix the growth medium with
microbes and a pump to deliver sterilized filtered air
o After 24-28 hours the material in the seed tanks is transferred to the primary
fermentation tank
➢ Source of nutrients added to the seed tank to help the microorganism survive, grow
and thrive:
o Warm water
o Carbon source: Carbohydrate foods e.g., lactose or glucose sugars, acetic acid,
alcohol and hydrocarbons.
o Nitrogen sources: ammonia salts, vitamins, amino acids,
o Minor nutrients: phosphorous, sulphur, magnesium, zinc
o Antifoaming agents: lard oil, octadecanol
➢ The material in the seed tanks is transferred to the primary fermentation tanks after
a few days. Hygiene must be maintained, as contamination can spoil the culture and
destroy the whole batch.
2. Fermentation
➢ Penicillin is an antibiotic thus it is produced in the stationary phase
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➢ A batch fermenter is used
➢ A fed batch process is normally used for growth limiting nutrient to prolong the
stationary period and thus increase production.
➢ The fermentation tank is a larger version of the steel seed tank, made of the same
growth medium.
➢ A fermenter - provides a contained, controlled, homogenous environment in which
the fermentation can proceed in a manner that is both safe and practical and which
optimises the particular objectives of the fermentation. Primary factors to consider
include: cost, reliability and safety Within these parameters, there is a huge range of
variability and flexibility.
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➢ Microorganisms are allowed to grow and multiply in this environment. During this
process, they excrete large amounts of the desired antibiotic.
➢ Important parameters that will greatly affect the reactors process performance:
✓ The medium is typically fed-batch culture. Fed Batch means that the substrate
is added in small increments at various times in the fermentation.
✓ It is constantly agitated and fed a constant flow of sterilised air and anti-
foaming agents
✓ Oxygen and nutrient concentration must be optimum
✓ The tanks are kept at the appropriate temperature
✓ The pH is kept constant at 6.5 Thus the reactor must maintain pH efficiently
for optimal growth (this is frequently done by addition of NaOH).
✓ Phenyl acetic acid or phenoxyaceticacid is fed continously as a precursor (0.5%
- 0.8% of the total)
✓ Glucose concentration of 500 kgm-3 is maintained. About 10% is used for
production, while 25% is used for growth and 65% is used for maintenance
energy.
✓ Population size must be controlled to ensure maximum yield before the cells
die
✓ Steam is used to keep the reactor running aseptically. This is achieved because
the reactor is designed as a pressure vessel and steam is sent through at a
minimum temperature 121°C, pressure of 15 psi for 15-30min.
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Requirements of fermentation:
3. Purification and isolation
➢ Antibiotics are produced in abundance after three to four days. After this, the isolation
process can begin. The fermentation broth is purified by various methods depending
on the antibiotics produced.
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➢ Downstream processing is relatively easy since penicillin is secreted into the medium
– usually there is no need to open the fungal cells
➢ Ion exchange: used to purify water-soluble antibiotic compounds. These methods
separate the compound from the organic waste materials in the broth before passing
it through equipment that separates the other water-soluble compounds from the
desired one.
➢ Solvent extraction method: used to isolate an oil-soluble antibiotic like penicillin. The
broth is treated with organic solvents like butyl acetate or methyl isobutyl ketone,
which can specifically dissolve the antibiotic.
➢ Has relatively high selectivity and yield.
➢ Penicillin is extracted with organic phase several times by shifting the pH to reduce
the impurity in the products.
➢ Other impurities, such as pigments, are simultaneously extracted into the organic
solvent during the extraction of penicillin. Thus, subsequent purification procedures
are necessary, e.g., decolouration and freezing dehydration.
➢ In addition, the formation of rather stable emulsion during the extraction of penicillin
from fermentation broth often causes problems. A demulsifier is used to break
emulsion.
➢ The product is dissolved and then precipitated by a potassium salt to separate it from
other substances in the medium
➢ The separation of antibiotics by ATPS (Aqueous two-phase systems) has been
reported. ATPS can supply a gentle environment for biomaterials over conventional
aqueous-organic solvent systems. It can also alleviate emulsion and enhance the
sedimentation of fine particles.
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4. Refining and Packaging
➢ After isolation, various refining steps are taken depending on the antibiotic's final
form.
➢ Antibiotic products are sold as solutions in intravenous bags or syringes, as pills, gel
capsules, or powders incorporated into topical ointments.
o Intravenous bags: The crystalline antibiotic is dissolved in a solution, placed in
an intravenous bag, and then hermetically sealed.
o Gel capsule: the powdered antibiotic is physically filled into the bottom half of
a gel capsule before the top half is mechanically fitted on top.
o Topical ointments: the antibiotic is mixed into the ointment
➢ Antibiotics are then transported to the final packaging station, stacked and placed in
boxes here, loaded onto trucks and delivered to distributors, hospitals, and
pharmacies. The entire fermentation, recovery, and processing can take five to eight
days.
Quality control
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➢ Steps to ensure no contamination is introduced at any point the production process
➢ During manufacturing, the quality of all products must be checked regularly
➢ Frequent checks on the conditions of the microorganism culture during fermentation
➢ Various physico-chemical properties of the product must be checked
Product
Penicillin G which can be chemically modified to make a variety of penicllin with different
properties
3.6 Aminoglycoside antibiotics:
➢ Antibiotics composed of one or more amino sugars and an aminocyclitol (6-carbon)
ring are referred to as aminoglycosides. These complex compounds are exclusively the
products of various species of soil actinomycetes in the genera Streptomyces and
Micromonospora.
➢ Streptomycin is obtained from, Streptomyces griseus and dihydrostreptomycin can be
isolated from S. humidus.
Streptomycin
➢ Streptomycin is an antibiotic drug, the first of a class of drugs called aminoglycosides
to be discovered, and was the first antibiotic remedy for tuberculosis. It is derived
from the actinomycetes, Streptomyces griseus. It is an aminoglycoside antibiotic. It
causes kidney damage and deafness as side effects. Streptomycin is active against a
large number of bacteria found among the gram-negative, gram-positive, and acid-
fast groups and among the spirochaetes; it has relatively little activity against
anaerobic bacteria, fungi, protozoa, and viruses.
➢ Streptomycin was found to be soluble in water and insoluble in organic solvents, such
as ether, chloroform, and acetone. The chloride was completely soluble in methanol,
less soluble in ethanol, and virtually insoluble in butyl alcohol, acetic acid, and
pyridine. The sulfate was only slightly soluble in methanol and virtually insoluble in
the other solvents.
Parameters for Production
✓ Medium contains soybean meal which is the nitrogen source, glucose as the carbon
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✓ source and NaCl.
✓ Fermentation is carried out at 28°C.
✓ The pH should be in the range of 7.6 to 8.0
✓ High agitation and aeration are needed.
✓ It takes 10 days to complete the process before streptomycin can be extracted from
the broth.
Biosynthesis of aminoglycoside
✓ Glucose is the precursor.
✓ Over 30 separate enzymatic steps are known. One of the principal components of
streptomycin, streptidine is synthesized from glucose-6-Phosphate via myo-inositol.
✓ After a number of enzymatic steps, there is formation of streptidine-6-phosphate.
✓ After the final, dephosphorylation step, there is formation of streptomycin from
streptomycin-6-P
✓ Yield have increased from 100 - 200µg/ml to 15mg/ml by strain development process.
✓ Factor A (iso-capryloyl-3-hydroxymethyl-γ-butyrolactone) controls the biosynthesis. It
is active at extreme low concentration of 10M. Factor A- mutants that do not
synthesize streptomycin, can do so by addition of Factor A.
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Figure: Flow chart showing biosynthesis steps of streptomycin production
Figure: Role of A-factor in production
Production Process
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➢ The production of streptomycin can be explained in three different phases. The
changes that occur is these stages are as follows:
Phase I:
✓ Rapid growth occurs resulting in production of mycelia biomass.
✓ Little production of Streptomycin is obtained.
➢ Proteolytic activity of the microbe releases ammonia to the medium from the soybean
meal causing a rise in pH.
Phase II:
✓ Additional production of mycelium takes place.
✓ Streptomycin starts accumulating in the medium.
➢ The glucose and ammonia released are consumed during this phase. The pH remains
fairly constant between 7.6 and 8.0
Phase III:
➢ The process has completed.
➢ Finally, the mycelium is separated by filtration and antibiotic starts after this stage of
production.
Extraction and Recovery
(1) Adsorption of the Streptomycin from the culture filtrate on 1 % (w/v) activated
charcoala t pH 6-8.
(2) Elution of the streptomycin from the charcoal with 1.2 % (v/v) aqueous phosphoric
acid at pH 1-0-2.0.
(3) Adsorption of the streptomycin on 3-4% (w/v) activated charcoal from the neutralized
phosphate eluate at pH 7.
(4) Elution of the streptomycin under anhydrous conditions with acidified methanol.
(5) Evaporation of the neutralized methanol eluate to one-eighth of its original volume.
(6) Precipitation of the streptomycin by dilution of the concentrate with acetone, or other
precipitant of higher boiling-point, e.g., amyl acetate.
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