PRODUCTION OF ANTIBIOTICS

► PENICILLIN.

► STREPTOMYCIN.

- BENITA NANCY RENI.M

What is an Antibiotic….?

• Greek word… Anti – against; Bios – life.

Microbial products / derivatives – kill susceptible

Microbes / inhibit their growth.
Classification of Antibiotics…

Based on ,

- Mode of Action.

- Microbial source.
Mode of Action……
• Cell wall synthesis inhibitors.
♦ Penicillins - against G+ve.

• Protein synthesis inhibitors.

♦ Aminoglycosides - against G-ve.

• Nucleic acid synthesis inhibitors.

♦ Quinolones - against G-ve > G+ve.

• Cell membrane disruptors.

♦ Polymyxin B - against G-ve only.

• Antimetabolites.
♦ Sulfonamides – broad spectrum.
Microbial source…
• Bacterial origin…

- Bacillin from B.subtilis - against G+ve & G-ve.

• Fungal origin…

- Penicillin from P.nottatum, P.chrysogenum - against G+ve.

• From actinomycetes.

- Streptomycin from S.griseus - against G-ve & some G+ve.

 PENICILLIN
Produced by,

P.nottatum,
P.chrysogenum &
some of the
Aspergillus sps

6-APA

Structure: β-lactam ring fused with

thiazolidine ring.

• Inhibits transpeptidation reaction

during peptidoglycan synthesis.
• Has a cidal activity against G+ve.
Discovery…

It was an accident….

By Alexander Fleming
in 1928…

Came n handy during world

war 11
 Types of Penicillin

Natural Penicillin Semisynthetic Penicillin

►No Precursors added ►Prepared from
during Fermentation; Biosynthetic Penicillin 6-Aminopenicillanic acid →
Modification by Chemical
Eg: Penicillin G, ► Precursors added during means. (Eg: acylation)
Penicillin V Fermentation.

Eg: Penicillin G, Penicillin V . Eg: Ampicillin, Methicillin,

Oxocillin,Propicillin, etc,..

ADVANTAGES…..
- Can not be administed Orally. - can be administerd orally.
- Resistant to penicillanase
- Some – Resistant to
penicillanase
Chemistry of Penicillin production…
L- Aminoadipic acid.(AAA)
L-Cysteine

AAA – Cysteine

L-Valine

L- Aminoadipyl cysteinyl D-Valine

Regulation…

Isopenicillin N - Lysine → -ve regulator.

Penicillin transacetylase
- sed K, Ammonium ions.
Benzyl Penicillin

Penicillin acylase - Catabolic repression by

glucose.
6-Aminopenicillanic acid.(6APA)
↓
Semisynthetic penicillns.
 PRODUCTION…
Strain selection… HIGH-YIELDING STRAINS.
↓
Achieved by “Sequential genetic selection”.

P.Chrysogenum NRRL 1951. B25 ( > 200 units/ml)

↓ X- Rays ↓ U.V

P.Chrysogenum,
P.Chrysogenum X-1612 P.Chrysogenum Stanford 25099
( > 500 units/ml)
↓ U.V

P.Chrysogenum Q-176 ( > 761 units/ml)

P.Chrysogenum,
The process…
• Inoculation medium…( Moyer & Coghill medium)

► Glycerol, Cane molasses, Corn-steep liquor, MgSO 4, KH2PO4,

Peptone, NaCl, CuSo4,Fe-tartarate.

• Fermentation medium…

► Corn-steep liquor, Lactose, Glucose, CaCO3, KH2PO4,

Phenyl acetic acid – precursor.

• PH→6.5

• Submerged production.

• Aerobic process.

• Growth Phase → 40 hrs.

• Doubling time → 6 hrs.

Production strains storage… Inoculum preparation…
Production strains are genetically Stored spore suspension.
unstable.
↓
2 – 3 times culturing n solid
To store,…at dormancy… media/broth.
↓
 Spore suspension + inert solid
support → desiccated. Flask culture.
↓
 Spore suspension → lyophilized in Smaller Fermentor
app.media. (0.5 – 1 m3)
↓
 Spore suspension → stored under
liquid nitrogen. Little bigger one
( 10 – 20 m3)
In very strict ASEPTIC CONDITION. ↓
Production vessel
The DSP…. 3 stages…
Penicillin – Extracellular – present in the medium.

Removal of mycelium Obtained penicillin salt solution.

Rotary vacuum ↓
Filtration filter Charcoal treatment to remove
Pyrogens
Filtrate → subjected to ↓
COUNTERCURRENT SOLVENT EXTRACTION.
( PODBIELNIAK EXTRACTOR )
Filter sterilization.
Seitz filter → remove bacteria.
i. Extraction of the penicillin into an organic solvent.(amyl ↓
or butyl acetate / methyl isobutyl ketone). pH → 2 - 2.5.
Crystallization.
ii. Extraction from the organic solvent into an aqueous
buffer. pH → 7 – 7.5
iii. Extraction from aqueous buffer into organic solvent.
For Parental use For Oral use
iv. Extraction of the solvent to obtain the ↓

Penicillin salt
• Powder / • Tabletted.
suspension.
 STREPTOMYCIN…An Aminoglycoside…
• Binds to 30S ribosomal subunit
& inhibits protein synthesis.

• Cidal against G-ve.

• Treatment of Tuberculosis,
Plague.
• Discovered by American
biochemists Selman Waksman,
• Produced by S.griseus
Albert Schatz, and Elizabeth
Bugie in 1943.

• Consists - an N-methyl-a -L-

glucosamine ring, an a -L-
streptose & a streptidine ring,
linked together by glycosidic
bonds.
Chemistry of Streptomycin production…
D – Glucose

Glc – 6 – p
Glc–1–p Glucosamine-6-p
↓ ↓
dTDP–L–Dihydrostreptose Streptidine-6-p XDP-N-Methyl-L-
glucosamine.

Dihydrostreptosyl-streptidine-6-p

Dihydroxystreptomycine-6-p

↓
Streptomycin-6-p → Streptomycin.
Streptomycin production …
• Submerged culture Method …

• Inoculation medium….Same as penicillin.

• Production medium…

► Glucose / Fructose / Mannitol, Peptone / Meat

extract / Soy extract, Mg++, Ca++, k+, etc,..

• Precursors: Proline, Phenyl acetic acid

• Antioxidant: Sodium sulphite.

• Temperature → 28•c; PH range → 7.6 – 8.0

• High agitation and aeration are needed.

• Process lasts for about 10 days.

The process…
Spores are inoculated into a
medium - Obtain high mycelial
biomass

↓
Introduction into an
inoculum tank.
↓
production tank.
 The Phases….
I. Mycelial growth phase II. Streptomycin production phase
- Rapid growth producing mycelial
biomass. - Stretomycin production & No new mycelial
growth.
- Large requirement for O2, Glc, N
&P. - NH3 is utilised.

- PH up to 8.0 – due to the production - PH ↓↓ to 7.0.

of NH3.
- Glc & O2  required in large quantity.
- little production of streptomycin.

III. Autolysis of mycelium

- Carbohydrates become depleted.

- Streptomycin production ceases .

- Mycelium undergoes Autolysis

- PH ; No O2 requirement; Antibiotic Recovery

The DSP…
Streptomycin – Extracellular – present in the medium.

Culture.
↓ FILTRATION
Filtrate.
↓
Adsorbtion onto activated charcoal.
↓
Elution with acid alcohol.
↓
Precipitation with acetone.
↓
Further purification
by the use of column chromatography.
THANK YOU……..