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Penicillin Production.ppt

  1. ◦Industrial ProductionofPenicillin Dr. Muhammad Nawaz
  2. ● First true naturally-occurring antibiotic ever discovered ● Penicillin is a secondary metabolite of certain species of Penicillium Examples: Amoxicillin, ampicillin, phenoxymethylpenicillin. What is Penicillin
  3. History: Discovery & Production ● 1928: Scottish biologist, Alexander Fleming accidently discovered Penicillin. He found that the fungus Penicillium notatum prevented growth of bacteria Staphylococcus spp. ● 1932: Clutterbuck and his colleagues studied the nature of Penicillin and found it as an organic acid
  4. History: Discovery & Production ● 1940; Howard Florey and Ernst Chain managed to purify penicillin in a powdered form. ● 1942:Purified protein was used to treat streptococcal meningitis ● 1943: Penicillin on a large scale. This helped immensely to treat casualties during the WWII that had bacterial infections due to their wounds.
  5. Classes of Penicillins ◦ Natural Penicillins: ◦ Penicillin G (same as Benzylpenicillin) ◦ Penicillin V (same as Phenoxymethylpenicillin) ◦ Aminopenicillins ◦ Ampicillin ◦ Amoxicillin ◦ Hetacillin ◦ Penicillinase-resistant Penicillins (Antistaphylococcal Penicillins) ◦ Methicillin (prototype) ◦ Cloxacillin ◦ Dicloxacillin ◦ Nafcillin ◦ Oxacillin
  6. Classes of Penicillins ◦ Extended Spectrum Penicillins (Antipseudomonal Penicilllins) ◦ Azlocillin ◦ Carbenicillin ◦ Mezlocillin ◦ Piperacillin ◦ Ticarcillin ◦ Beta-lactamase Inhibitors ◦ Clavulanic acid
  7. The basic chemical structure of all penicillin group consists of a beta-lactam ring, a thiazolidine ring, and a side chain (6-aminopenicillanic acid) Structure ofPenicillin:
  8. Penicillin Derivative ●Derivatives produced to deal with the problemof bacterial resistance to penicillin. ●All penicillin or penicillin derivative have a constant core region which is the 6-APA. ●The only region that is different from different types of penicillin derivative is its R group.
  9. Penicillin derivatives:
  10. Biosynthesis of Penicillin: ◦ Three main steps Catalytic step. Oxidative step. Exchange of different chains.
  11. Biosynthesis of Penicillin: ◦ The catalytic step involves an ACV synthetase enzyme that condenses the lateral chain of cysteine, valine, and alpha aminoadipate into tripeptide ACV. ◦ In the second step, tripeptide ACV forms a bicyclic ring by oxidative ring closure. Isopenicillin N synthase is involved resulting in isopenicillin N which is a bioactive intermediate in the pathway. ◦ The third step involves the exchange of L-aminoadipate. Acyl-CoA synthetase and Acyl-CoA racemase, a two enzyme system is involved that helps in converting isopenicillin N into Penicillin G.
  12. Biosynthesis of Penicillin:
  13. Stagesof Production 1. Primary metabolism will be emphasised. Media for this stage will be focussed on achieving maximum growth and biomass production. 2. Once the desired biomass has been achieved, starve (Limiting the amount of C and N available to the culture) the culture and induce the kind of stress conditions that trigger the production of the antibiotic. ★ Use the fed-batch method to feed the culture. As stated above, this allows us to add the substrate to the reactor in small increments and to even change the substrate if we so desire.
  14. Penicillin production process:
  15. Penicillin production steps: 1. Seed Culture development in laboratory. 2. After three days of incubation, the content is used for inoculation and kept in a fermentor that is well equipped with optimum conditions. 3. The content is filtered after six days of incubation which contains penicillin. 4. The penicillin is extracted into amyl or butyl acetate and is transferred into an aqueous solution with phosphate buffer.
  16. Penicillin production steps: 5. Acidify the extract and again re-extract penicillin into butyl acetate 6. In the solvent extract potassium acetated is added to a crystallization tank to crystallize as a potassium salt. 7. Crystals were recovered and further sterilization of salt is done.
  17. Strains for Penicillin Production ◦ Penicillium notatum ◦ Penicillium chrysogenem
  18. Strain Improvement • At the end of the WWII, penicillin was first made using the fungus Penicillium notatum, which produced a yield of 1mg/dm3 ● Today, using a different species known as Penicillium chrysogenum, and better extraction procedures, the yield is 50 mg/dm3
  19. Media Formulation ● pH 6.5 ● Temperature 20-24 °C ● Oxygen ● Nitrogen: corn steep liquor 8.5 % ● Glucose 1% ● 80% ethanol ● phenylacetic acid ● Probenecid ● Lactose 1% ● Calcium Carbonate 1% ● Sodium hydrogen phosphate 0.4% ● Antifoaming agent: vegetable oil
  20. ★ Medium for penicillin 1. The Penicillium chrysogenum usually contain its carbon source which is found in corn steep liquor and glucose. 2. A medium of corn steep liquor and glucose are added to the fermenter. Medium also consists of salts such as MgSO4, K3PO4 and sodium nitrates. They provide the essential ions required for the fungus metabolic activity.
  21. Production Method ● Secondary metabolites are only produced in times of stress when resources are low and the organism must produce these compounds to kill off its competitors to allow it to survive. ● It is these conditions that we wish to duplicate in order to achieve the maximum amount of product from our fermentation.
  22. Heat sterilization 3.Medium is sterilized at high heat and high pressure, usually through a holding tube or sterilized together with the fermenter. 4.The pressurized steam is used and the medium is heated to 121°C at 30 psi or twice the atm. pressure
  23. Seed Culture 9. The seed culture is developed first in the lab by the addition of Penicillium chrysogenum spores into a liquid medium. When it has grown to the acceptable amount, it is inoculated into the fermenter. 10. The medium is constantly aerated and agitated. Carbon and nitrogen are added sparingly alongside precursor molecules for penicillin fed-batch style. Typical parameters such as pH, temperature, stirrer speed and dissolved oxygen concentration, are observed.
  24. Seed Culture 11. After about 40 hours, penicillin begins to be secreted by the fungus. 12.After about 7 days, growth is completed, the pH rises to 8.0 or above and penicillin production ceases. The Penicillium fungus
  25. Fermentation: 5.It is done in a fed-batch mode as glucose must not be added in high amounts at the beginning of growth (which will result in low yield of penicillin production as excessive glucose inhibit penicillin production). 6.The fermentation conditions for the Penicillium mold, usually requires temperatures at 20-24°C while pH conditions are kept at 6.5 7.The pressure in the bioreactor is much higher than the atmospheric pressure (1.02atm). This is to prevent contamination from occurring as it prevents external contaminants from entering.
  26. Fermentation It is necessary to mix the culture evenly throughout the culture medium. Fungal cells are able to handle rotation speed of around 200 rpm. Fermentors
  27. Product Recovery • Harvest broth from fermented tank by filtration (rotary vaccum filtration) chill to 5-10 C (because penicillin is highly reactive and destroyed by alkali and enzyme) • Acidify filtrate to pH 2.0-2.5 with H2SO4 ( to convert penicillin to its anionic form) • Extract penicillin from aqueous filtrate into butyl acetate or amyl acetate (at this very low pH as soon as possible in centrifugal counter current extractor) • Discard aqueous fraction Rotary vacuum filter
  28. ◦ Allow the organic solvent to pass through charcoal to remove impurities and extract penicillin from butylacetate to 2% aqueous phosphate buffer at pH 7.5 ◦ Acidify the aq. Fraction to pH 2-2.5 and re-extract penicillin into fresh butylacetate ( it concentrated up to 80-100 times) ◦ Add potassium acetate to the solvent extract in a crystallization tank to crystalize as potassium salt ◦ Recover crystal in filter centrifuge ◦ Sterilization ◦ Further processing ◦ Packaging
  29. Storage Penicillin is stored in containers and kept in a dried environment. The White Penicillin-Sodium salt The resulting penicillin (called Penicillin G) can be chemically and enzymatically modified to make a variety of penicillins with slightly different properties. These can be semi-synthetic penicillins, such as; Penicillin V, Penicillin O, ampicillin and amoxycillin.
  30. SpecificConditions forPenicillin Production ● Most penicillins form filamentous broths. This means they can be difficult to mix due to their high viscosity. Also the increasing viscosity of the broth can hinder oxygen transfer.
  31. A solution for the viscosity and the filamentous growth of penicillium species could be bubble columns (air lift reactors) which would distribute the oxygen equally and also to agitate the medium. SpecificConditions forPenicillin Production
  32. ● Penicillin is an aerobic organism; oxygen supply is critical: reactor must have an efficient oxygen supply system. ● The optimum pH for penicillin efficiently (pH controller growth is 6.5: maintain pH and acid-base reservoir). ● Strain Stability problems (mutations): careful strain maintenance is required. ● Biomass doubling is about 6h: provisions must be made. SpecificConditions forPenicillin Production
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Notes de l'éditeur

  1. Alpha-aminoadipyl-cysteinyl-valine synthetases Isopenicillin N synthase Acyl-CoA synthetase and Acyl-CoA racemase