Penicclin production

Penicllin production

1. History: Discovery & Production 1928: Scottish biologist, Alexander Fleming discovered that the Staphylococcus culture he had mistakenly left growing in open was contaminated with a mould which had destroyed the bacteria. After isolating a sample and testing it, he found that it belonged to the Penicillium family. Later the mould was classified as Penicillium notanum. At first, it was difficult to convince people about its potential uses.

2. History: Discovery & Production But later (1939), using Fleming’s work, two medical researchers, Howard Florey and Ernst Chain managed to purify penicillin in a powdered form. 1941: They successfully treated a human. 1943: They produced penicillin on a large scale. This helped immensely to treat casualties during the WWII that had bacterial infections due to their wounds.

3. General Structure of Penicillins

4. General Structure of Penicillins Have β-Lactam functional group, thus they belong to the β-Lactam antibioti c group . They all have a basic ring-like structure (a β-Lactam) derived from two amino acids (valine and cysteine) via a tripeptide intermediate. The third amino acid of this tripeptide is replaced by an acyl group (R ). Th e natur e o f thi s acy l grou p produce s specifi c propertie s on different types of penicillin.

5. Penicillin Derivatives Derivative s produce d to deal with the problem of resistance to 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.

6. How Does Penicillin Work? Inhibits the synthesis of peptidoglycan in cell walls. β-Lactam of penicillin binds to the enzyme - transpeptidase, that is used in the formation of peptidoglycan cross linking. The enzyme is inhibited, thus inability to form cross linking. Cell wall is weakened causing osmotic imbalance in the cell. This leads to cell death. As human cells do not have cell walls, penicillin does not affect them.

7. Penicillin is produced by the fungus Penicilium chrysogenum which requires lactose, other sugars, and a source of nitrogen (in this case a yeast extract) in the medium to grow well. Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary phase.

8. It exhibits the properties of a typical secondary metabolites. I t ac t i v e a g ain s t c e r t a i n G r am- p o siti v e bac t e ria in p r esence of blood , pus and body fluids. It is soluble in water. It is very soluble in acetone, ethyl a lcohol and ether and it is less soluble in benzene, c hloroform . Aqueous solution of penicillin are unstable and must be stored under refrigeration. P eni c illin is mo s t s t a b le in the pH r an g e o f 6 . t o 6 . 5 and reasonably stable over the p H range of 5.5 to 7.5 . Properties of penicillin :

9. Th e Inocu l um : Ma st er s t oc k (spo r es) is the s o u r ce of inoculum. Spores ( P. chrysogenum ) from working stocks cultured are suspended in water or non toxic lauryl sulphonate, then added to the flask containing wheat bran and nutrient soln. A shake flask culture of 4 day old is inoculated into a seed tank for 3 days. The medium : A typical media for penicillin production is given below . The major constituent of typical medium includes, Commercial production of penicillin : 1) Fermentable carbohydrate Potassium dihydrogen phosphate Organic nitrogen source Phenyl acetic acid precursor Edible oil Calcium carbonate (act as buffer) pH after sterilization Corn steep liquor(3.5%), la c t o s e ( 3 . 5%),glu c o s e ( 1% ) . 0.4% 0.25% 1% 5.5 to 6. 5

10. pH 6.5 Temperature 20-24 °C Oxygen Nitrogen: corn steep liquor 8.5 % Glucose 1% 80% ethanol P henylacetic acid Lactose 1% Calcium Carbonate 1% Sodium hydrogen phosphate 0.4% Antifoaming agent: vegetable oil

11. Procedure :

12. ★ Fermentation 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). The fermentation conditions for the Penicillium mold, usually requires temperatures at 20-24°C while pH conditions are kept at 6.5 . 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.

13. 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

14. Aeration (oxygen supply) : supply of oxygen in a bioreactor is the limiting factor in penicillin biosynthesis. Aeration speed is between 150 to 250 rpm . Temperature : Temperature plays an important role in penicillin production it should be m aintained at 2 5 ° C. Biomass production : production of penicillin depends upon biomass production therefore, it is describle to have a high biomass concentration in the vessel. It is achieved by increasing the agitation rate and power. pH : It is maintained early neutrality by calcium and magnesium carbonate in the medium by phosphate buffer. It is also controlled by adding sodium hydroxide or sulphuric acid in the medium.

15. Important Process Parameters Mass Transfer : G ood transfer of oxygen across the liquid interface-the Sparger delivers this oxygen efficiently. Heat Transfer: metabolism as a process tends to give off heat- achieved through cooling jacket whereby cool water is passed through. Bulk Flow and Mixing: Im pellers , bubble columns or loop reactors. Batch, Fed-Batch and Continuous Culture: how nutrients and substrate will be delivered to a culture in a reactor. Steam : Used t o kee p th e reacto r runnin g aseptically. (temperature/pressure of 121°C/15 psi for 15-30min).

16. Batch fermenter

17. Harvested culture broth includes penicillin G along with a variety of other metabolites . Vacuum filter is used for separation of mycelium from the broth on a rotary . C onversion of penicillin to the anionic form occurs at low p H (2.0 to 2.5 ). T he lowering of p H is done by adding phosphoric acid or sulphuric acid.

18. For removal of pigment and other impurities from solvent containing penicillin it is treated with active charcoal. The product is back extracted into water from solvent by adding potassium or sodium hydroxide to form its sail. The product of penicillin is then crystallized into sodium or potassium penicillin.

19. A n tibiotics a r e p r oba bly the m o s t impo r t a n t g r ou p of Conclusion : compounds synthesized by industrial microorganisms. The best known and probably the most medically important antibiotics are the β- lactam, penicillin and cephalosporin's. P eni c illin e xhibit s the p r operti es o f a typi c al se c onda r y metabolites. P eni c illin is c o m me r cially p r oduc e d usuall y v i a a f ed b a t ch process carried out aseptically in stirred tank fermenters.