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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