PRODUCTION OF ANTIBIOTICS PENICILLIN. STREPTOMYCIN.
- BENITA NANCY RENI.M
What is an Antibiotic.?Greek word Anti against; Bios life.
Microbial products / derivatives kill susceptibleMicrobes / inhibit their growth.
Classification of Antibiotics
Based on , - Mode of Action. - Microbial source.
Mode of ActionCell 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 sourceBacterial 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: -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
D-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
Mutagens used: X-rays, UV rays, Nitrozoguanidine, Alkylating agents,etc,.. Genetic recombination , Protoplast fusion can also be emloyed. P.Chrysogenum NRRL 1951. B25 ( > 200 units/ml) X- Rays U.V
P.Chrysogenum, X-1612 P.Chrysogenum Stanford 25099 ( > 500 units/ml) U.V P.Chrysogenum, Q-176 ( > 761 units/ml)
The processInoculation 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.
PH6.5
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
Penicillin Extracellular present in the medium. Removal of mycelium Rotary vacuum Filtration filter Filtrate subjected to COUNTERCURRENT SOLVENT EXTRACTION. ( PODBIELNIAK EXTRACTOR )
Extraction of the penicillin into an organic solvent.(amyl or butyl acetate / methyl isobutyl ketone). pH 2 - 2.5. Extraction from the organic solvent into an aqueous buffer. pH 7 7.5 Extraction from aqueous buffer into organic solvent.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.
STREPTOMYCINAn 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 Glc1p Glucosamine-6-p dTDPLDihydrostreptose Streptidine-6-p XDP-N-Methyl-L- glucosamine.
Dihydrostreptosyl-streptidine-6-p Dihydrostreptomycin-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 28c; PH range 7.6 8.0
High agitation and aeration are needed.
Process lasts for about 10 days.
The processStrain selection High yielding strains
Strain improvement: Mutation, Protoplast fusion, rDNA tech
Spores are inoculated into a medium - Obtain high mycelial biomass Introduction into an inoculum tank. production tank.
The Phases. 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.
Streptomycin production phase
- Stretomycin production & No new mycelial growth.
- NH3 is utilised.
- PH to 7.0.
- Glc & O2 required in large quantity.
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.
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