“EXTRACTION & PURIFICATION of PENICILLIN”: “ EXTRACTION & PURIFICATION of PENICILLIN ” What Is Penicillin?: What Is Penicillin? A class of antibiotics that comes from mold. Discovered by accident in 1928 by Alexander Fleming, is the first anitbiotic. Penicillin antibiotics include ampicillin, phenoxymethylpenicillin, amoxicillin, 1 st -4 th generations. 50 drugs that are now classified as penicillin. Use in WWII and after. How Penicillin Works?: How Penicillin Works? Resembles a protein needed for production of cell wall. Penicillin binds to cell wall of bacteria, prevents peptide chains from linking, and lyses it. PRODUCTION OF PENICILLIN: PRODUCTION OF PENICILLIN During world war II-importance realized, as penicillin had been used to treat many wounded soldiers. A tale by A. Fleming: A tale by A. Fleming The first antibiotic was discovered in 1896 by Ernest Duchesne and "rediscovered" by Alexander Flemming in 1928 from the filamentous fungus Penicilium notatu m . In 1928, Sir Alexander Fleming, a Scottish biologist, observed that Penicillium notatum , a common mold, had destroyed staphylococcus bacteria in culture. A tale by A. Fleming: A tale by A. Fleming He took a sample of the mold from the contaminated plate. He found that it was from the Penicillium family, later specified as Penicillium notatum . Fleming presented his findings in 1929, but they raised little interest. He published a report on penicillin and its potential uses in the British Journal of Experimental Pathology . Slide 7: Thanks to work by Alexander Fleming (1881-1955), Howard Florey ( 1898-1968) and Ernst Chain (1906-1979), penicillin was first produced on a large scale for human use in 1943. At this time, the development of a pill that could reliably kill bacteria was a remarkable development and many lives were saved during World War II because this medication was available . E. Chain H. Florey A. Fleming MOA OF PENICILLIN: MOA OF PENICILLIN All penicillin like antibiotics inhibit synthesis of peptidoglycan, an essential part of the cell wall. They do not interfere with the synthesis of other intracellular components. These antibiotics do not affect human cells because human cells do not have cell walls. Spectrum of Activity : Spectrum of Activity Penicillins are active against Gram positive bacteria Some members (e.g. amoxicillin) are also effective against Gram negative bacteria but not Pseudomonas aeruginosa PRODUCTION OF PENICILLIN: PRODUCTION OF PENICILLIN Penicillin was the first important commercial product produced by an aerobic, submerged fermentation First antibiotic to have been manufacture in bulk. Used as input material for some semi synthetic antibiotics. It is fermented in a batch culture, and a fed batch process is normally used to prolong the stationary period and so increase production. Slide 11: When penicillin was first made at the end of the second world war using the fungus Penicillium notatum , the process made 1 mg dm -3 . Today, using a different species ( P. chrysogenum ) and better extraction procedures the yield is 50 g dm -3 . There is a constant search to improve the yield. The yield of penicillin can be increased by: : The yield of penicillin can be increased by: Improvement in composition of the medium. Isolation of better penicillin producing mold sp. Penicillium chrysogenum which grow better in huge deep fermentation tank. Development of submerged culture technique for cultivation of mold in large volume of liquid medium through which sterile air is forced. Primary and Secondary Metabolites: Primary and Secondary Metabolites Primary metabolites are produced during active cell growth, and secondary metabolites are produced near the onset of stationary phase. Commercial Production Of Penicillin: Commercial Production Of Penicillin Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary phase. INDUSTRIAL PRODUCTION OF ANTIBIOTIC- PENICILLIN : INDUSTRIAL PRODUCTION OF ANTIBIOTIC- PENICILLIN The industrial production of penicillin was broadly classified in to two processes namely, Upstream processing Downstream processing UPSTREAM PROCESSING : UPSTREAM PROCESSING Upstream processing encompasses any technology that leads to the synthesis of a product. Upstream includes the exploration, development and production. DOWNSTREAM PROCESSING: DOWNSTREAM PROCESSING The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing. UPSTREAM PROCESSING INOCULUM PREPARATION: UPSTREAM PROCESSING INOCULUM PREPARATION The medium is designed to provide the organism with all the nutrients that it requires. Inoculation method- submerged technique Spores -major source of inoculum RAW MATERIALS: RAW MATERIALS CARBON SOURCES: Lactose acts as a very satisfactory carbon compound, provided that is used in a concentration of 6%. Others such as glucose & sucrose may be used. NITROGEN SOURCES: Corn steep liquor (CSL) Ammonium sulphate and ammonium acetate can be used as nitrogenous sources. MINERAL SOURCES: Elements namely potassium, phosphorus, magnesium, sulphur, zinc and copper are essential for penicillin production. Some of these are applied by corn steep liquor. Calcium can be added in the form of chalk to counter the natural acidity of CSL PAA - precursor FERMENTATION PROCESS: FERMENTATION PROCESS The medium is inoculated with a suspension of conidia of Penicillium chrysogenum. The medium is constantly aerated and agitated, and the mould grows throughout as pellets. After about seven days, growth is complete, the pH rises to 8.0 or above, and penicillin production ceases STAGES IN DOWNSTREAM PROCESSING: STAGES IN DOWNSTREAM PROCESSING Downstream processing is relatively easy since penicillin is secreted into the medium (to kill other cells), so there is no need to break open the fungal cells. However, the product needs to be very pure, since it being used as a therapeutic medical drug, so it is dissolved and then precipitated as a potassium salt to separate it from other substances in the medium. Removal of cells The first step in product recovery is the separation of whole cells and other insoluble ingredients from the culture broth by technique such as filtration and centrifugation. ISOLATION OF BENZYL PENICILLIN : ISOLATION OF BENZYL PENICILLIN The PH is adjusted to 2-2.5 with the help of phosphoric or sulphuric acids. In aqueous solution at low PH values there is a partition coefficient in favor of certain organic solvents such as butyl acetate. This step has to be carried out quickly for penicillin is very unstable at low PH values. Antibiotic is then extracted back into an aqueous buffer at a PH of 7.5, the partition coefficient now being strongly in favor of the aqueous phase. The resulting aqueous solution is again acidified & re-extracted with an organic solvent. These shifts between the water and solvent help in the purification of penicillin. Slide 23: The treatment of the crude penicillin extract varies according to the objective, but involves the formation of an appropriate penicillin salt. The solvent extract recovered in the previous stage is carefully extracted back with aqueous sodium hydroxide. This is followed by charcoal treatment to eliminate pyrogens and by sterilization. Pure metal salts of penicillin can be safely sterilized by dry heat, if desired. Thereafter, the aqueous solution of penicillin is subjected to crystallization. FURTHER PROCESSING: FURTHER PROCESSING For parental use, the antibiotic is packed in sterile vials as a powder or suspension. For oral use, it is tabletted usually now with a film coating. Searching tests (ex: for purity, potency) are performed on the appreciable number of random samples of the finished product. It must satisfy fully all the strict government standards before being marketed The main stages of Penicillin production are:: The main stages of Penicillin production are: PRODUCTS:: PRODUCTS: The resulting penicillin (called penicillin G) can be chemically and enzymatically modified to make a variety of penicillins with slightly different properties. These semi-synthetic penicillins include penicillin V, penicillin O, ampicillin and amoxycillin . PRODUCTION OF PENICILLIN V: PRODUCTION OF PENICILLIN V Phenoxy methyl penicillin Addition of different Acyl groups to the medium. Phenoxyacetic acid as precursor instead of phenyl acetic acid. Slide 31: What is the Carbon source? lactose What is the nitrogen source? yeast What is the energy source? glucose Is the fermentation aerobic or anaerobic? aerobic What is the optimum temperature? 25-27⁰C Is penicillin a primary or secondary metabolite? secondary What volume fermenter is used? 40-200dm^3 Why isn't a larger fermenter used? difficult to aerate When is penicillin produced? 40 hrs-after main increase in fungal mass How long can it be produced for? 140hours(180-40 hours) What was the first fungus known to produce penicillin? Penicillin notatum What species produces about 60mg/dm3 of penicillin? Penicillin crysogenum How did scientists improve the yield still further? Genetic modification What is the substrate? Corn steep liquor Why is batch culture used? secondary metabolite What are the processes involved in down-stream processing? a) filtration of liquid b) extraction from filtrate by counter current butylacetate c) precipitation by potassium salts Why can't penicillin be taken orally? Destroyed by stomach acid Name the form of penicillin which can be taken orally. Penicillin v and ampicillin How does Penicillin kill bacteria? Stops production of cell wall Why are Gram negative bacteria not killed by penicillin? Different cell wall QUERIES ?: QUERIES ?