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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
• Inhibits transpeptidation reaction during peptidoglycan synthesis.
• Has a cidal activity against G+ve.
Structure: β-lactam ring fused with thiazolidine ring.
Produced by,
P.nottatum, P.chrysogenum & some of the Aspergillus sps
6-APA
Discovery…
It was an accident….
By Alexander Fleming in 1928…
Came n handy during world war 11
Types of Penicillin
Natural Penicillin
►No Precursors added during Fermentation;
Eg: Penicillin G, Penicillin V
Biosynthetic Penicillin
► Precursors added during Fermentation.
Eg: Penicillin G, Penicillin V.
Semisynthetic Penicillin
►Prepared from6-Aminopenicillanic acid → Modification by Chemical means. (Eg: acylation)
Eg: Ampicillin, Methicillin, Oxocillin,Propicillin, etc,..
ADVANTAGES….. - can be administerd orally. - Some – Resistant to penicillanase
- Can not be administed Orally.
- Resistant to penicillanase
Chemistry of Penicillin production…
L- Aminoadipic acid.(AAA)
L-Cysteine AAA – Cysteine
L-Valine
L- Aminoadipyl cysteinyl D-Valine
Isopenicillin N Penicillin transacetylase Benzyl Penicillin
Penicillin acylase
6-Aminopenicillanic acid.(6APA) ↓ Semisynthetic penicillns.
Regulation…
- Lysine → -ve regulator.
- sed K, Ammonium ions.
- Catabolic repression by
glucose.
PRODUCTION…
Strain selection… HIGH-YIELDING STRAINS. ↓
Achieved by “Sequential genetic selection”.
P.Chrysogenum NRRL 1951. B25 ( > 200 units/ml)
↓ X- Rays ↓ U.V
P.ChrysogenumP.Chrysogenum, X-1612 P.Chrysogenum P.Chrysogenum Stanford 25099Stanford 25099 ( > 500 units/ml)
↓ U.V
P.ChrysogenumP.Chrysogenum, Q-176 ( > 761 units/ml)
The process…• Inoculation medium…( Moyer & Coghill medium) ► Glycerol, Cane molasses, Corn-steep liquor, MgSO4, 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…
Production strains are genetically unstable.
To store,…at dormancy…
Spore suspension + inert solid support → desiccated.
Spore suspension → lyophilized in app.media.
Spore suspension → stored under liquid nitrogen.
In very strict ASEPTIC CONDITION.
Stored spore suspension. ↓
2 – 3 times culturing n solid media/broth.
↓ Flask culture. ↓ Smaller Fermentor (0.5 – 1 m3) ↓ Little bigger one ( 10 – 20 m3) ↓ Production vessel
Inoculum preparation…
The DSP…. 3 stages…
Penicillin – Extracellular – present in the medium.
Removal of mycelium Rotary vacuum
Filtration filter
Filtrate → subjected to COUNTERCURRENT SOLVENT EXTRACTION.
( PODBIELNIAK EXTRACTOR )
i. Extraction of the penicillin into an organic solvent.(amyl or butyl acetate / methyl isobutyl ketone). pH → 2 - 2.5.
ii. Extraction from the organic solvent into an aqueous buffer. pH → 7 – 7.5
iii. Extraction from aqueous buffer into organic solvent.
iv. Extraction of the solvent to obtain the ↓
Penicillin salt
Obtained penicillin salt solution.
↓Charcoal treatment to remove
Pyrogens
↓ Filter sterilization.
Seitz filter → remove bacteria.
↓ Crystallization.
For Parental use For Oral use
• Powder / • Tabletted.
suspension.
STREPTOMYCIN…An Aminoglycoside…
• Discovered by American
biochemists Selman Waksman,
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.
• Binds to 30S ribosomal subunit & inhibits protein synthesis.
• Cidal against G-ve.
• Treatment of Tuberculosis,
Plague.
• Produced by S.griseus
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
- Rapid growth producing mycelialbiomass.
- Large requirement for O2, Glc, N & P .
- PH up to 8.0 – due to the production of NH3.
- little production of streptomycin.
II. Streptomycin production phase
- Stretomycin production & No new mycelial growth.
- NH3 is utilised.
- PH ↓↓ to 7.0.
- Glc & O2 required in large quantity.
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……..
