penicillin production using rDNA technology

penicillin

1. THE WONDER DRUG PRESENTED BY: Anamika- M.Sc. MB Nayan Dhiman - M.Sc. MB Jyoti Bayalkoti - M.Sc. MB Date- 6/12/2021 1

2. ANTIBIOTICS The term antibiotic has been defined by Selman Waksman as being an organic compound produced by one microorganisms that inhibits the growth of or kills a group of harmful microorganism. These enter and stick to important parts of bacterial cell and interfere with its ability to survive and multiply. 2

3. FIRST NATURALLY OCCURRING ANTIBIOTIC Fleming was already well known upon his return from a long holiday in September 1928,he noted that one petri dish contained colonies of staphylococcus bacteria expect for clear area contaminated with a fungus that appeared to inhibit bacterial growth The mold was found to be Penicillium notatum. 3

4. GENERAL STRUCTURE OF PENICILLIN Penicillin is not a single compound but a group of closely related compounds,all with the same basic ring like structure (a beta lactam) derived from two amino acid(valine and cysteine) via a tripeptide intermediate . The third amino acid of this tripeptide is replaced by an acyl group(R) and the nature of this acyl group produces specific properties on different types of penicillin 4

5. PENICILLIN PRODUCTION Aim: To produce sterile,controlled and homogenous environment in which the fermentation can carry out in a manner that is safe and practical and which optimizes the Penicillin production for ultimate use. 5

6. . INOCULUM PREPARATION Inoculum : A small amount of material containing bacteria,viruses or other microorganisms that is used to start a culture. Out of various species of the fungus Penicillium ,mainly two species used in the fermentation .These are Penicillium notatum and Penicillium chrysogenum WHY Penicillium chrysogenum? Penicillium chrysogenum is high yielding strain and therefore most widely used as production strain. 6

7. MAINTENANCE OF STRAIN The production strain should be carefully maintained because appropriate maintenance and production of reliable pure culture with desirable quality is a key operation. Production strains are stored in dormant form by any means- A spore suspension may be mixed with a sterile, finely divided inert support Spore suspension can be lyophilized in appropriate media Spore suspension can be stored under liquid nitrogen 7

8. INOCULUM BUILD UP The purpose - to develop a pure inoculum in an adequate amount and in the fast growing phase for the production stage fermenter. The starter culture- it is transferred into agar-plate to allow growth. When spores are placed on a vegetative media, the spores start to germinate and form mycelia. After growth on agar plate, cultures were incubated for approx. 70h in a rotary shaker at 200 rpm an 25 to improve oxygen diffusion. Two growth stages ( 2 days incubation) are allowed upon transfer into seed fermenter. 8

9. Initial stages of fermentation are designed for considerable microbial growth and the can be distinguished from main Bioreactors by several factors ,these are : Smaller in size than the main fermenters approx.2 litre capacity. Made of stainless steel. Equipped with agitators which allow continuous mixing of growth medium Provided with pump to deliver sterilized ,filtered air . Must contain all the nutrients including Growth factors . After about 24-28 hrs, the content of the seed tanks is transferred is transferred to fermentation tank. Seed Fermenters 9

10. RAW MATERIAL An abundant growth of mycelium Maximum accumulation of penicillin and Ease of extraction and purification of the antibiotic. Raw materials are primary requirement to design the fermentation broth. CARBON SOURCE NITROGEN SOURCE AMMONIUM SOURCE Lactose acts as a carbon compound. Corn oil supplemented with lactose. Corn steep liquor (CSL) is used as important sources as it results in higher penicillin yields. Cottonseed /soybean meal can be used. Continuous addition of ammonium sulfate is required. Concentration around 250-300 mg/L is required. 10

11. PRECURSORS Certain precursor of the penicillin side chain needed to be added into the fermentation medium. This constitute a major cost item- Penicillin G, requires 0.47g Phenyl acetate. The precursor must be added repeatedly in small amounts during the fermentation process. BUT WHY? PAA can be substituted by other ring system. It is transported across the plasma membrane by free diffusion into mycelium. 11

12. PRODUCTION INSIDE THE BIOREACTOR FIRST PHASE SECOND PHASE THIRD PHASE Growth of mycelium occurs, yield of antibiotic is low. Lactic acid present in corn steep liquor is utilized at the maximum rate by microorganisms Lactose is used slowly Ammonia is liberated into the medium resulting into the rise in pH. There was intense synthesis of penicillin in this phase due to rapid consumption of lactose and ammonia, nitrogen. The mycelia mass increase. The pH remain unchanged. The concentration of antibiotic decreases in the medium. The autolysis of mycelium starts liberation of ammonia and slight rise in pH. 12

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14. RECOVERY Filtration: The penicillin rich filtrate is cooled to 2- 4 to avoid chemical or enzymatic degradation of penicillin. It is usually achieved by using high capacity, rotary vacuum filters for separation of mycelia. Mycelia is filtered and disposed. Extraction: Solvent extraction is accomplished by at low pH such as- 2.5- 3 using amyl acetate as solvent. Continuous, countercurrent, multistage centrifugal extractor are used for this purpose to avoid degradation of penicillin during solvent extraction at low pH, temperature is kept around 2-4 and filtration for 1-2 min. Two extractors used in series result in 99% penicillin recovery. 14

15. Adsorption: Carbon adsorption is used to remove impurities and pigments from penicillin- rich solvent after extraction. Several activated carbon columns in series can be used. Crystallization: Sodium and penicillin concentrations, pH and temperature need to be adjusted for this purpose. Excess amount of sodium are added to the penicillin- rich solvent before crystallization in an agitated vessel. The crystals are separated by a rotary vacuum filter. The crystals are washed and pre-dried with anhydrous butyl alcohol to remove some impurities. Large horizontal belt filters are used for collection and drying of the crystals. PURIFICATION 15

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17. RESULT

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