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05/27/13

05/27/13. This presentation is prepared by the coordination of a worthy group. Zahida Yaseen Mamoona Irshad Sahrish Khan. . Health- care products Antibiotics. . Introduction. Antibiotics are prepared by industrial microorganisms.

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05/27/13

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  1. 05/27/13 This presentation is prepared by the coordination of a worthy group • Zahida Yaseen • Mamoona Irshad • Sahrish Khan

  2. Health- care productsAntibiotics

  3. Introduction • Antibiotics are prepared by industrial microorganisms. • Antibiotics are not produced in the greatest quantity nor they are economically valuable. • The other health care products are • Alkaloids • Steroids • Toxins • Vaccines , Vitamins and Enzymes 3

  4. Genetic Engineering Techniques Through genetic engineering we can produced variety of products like • Mammalians proteins and peptides that have therapeutic properties • Medically important and established markets includes • Insulin, Interferon ,Human growth hormones and monoclonal antibodies • All of these are extensively used to test various health and disease states.

  5. Antibiotics • Antibiotics are secondary metabolites produced by • Filamentous fungi and bacteria • Particularly actinomycetes Approximately 4000 antibiotics isolated from various organisms but 50 used as antimicrobial chemotherapy • Medically important antibiotics are • Betalactams,penicilllines,cephloporins,aminoglycosides,(streptomycin), tetracycline 5

  6. Antibiotics(low market value) • Some antibiotics are fail to fulfill certain important criteria particularly • Lack of selectivity • Exhibiting toxicity to humans and animals • High production costs Some antibiotics are acts as antitumor agents e,g; Actinomycin and mytomycin(streptomyces peucetius and S.caeptiosus)

  7. Tools(biochemistry and molecular biology) • Some antibiotics are used to control microbial diseases in plants • Several antibiotics area also added to animal feed as growth promoters With respect to resistance the antibiotics used for humans or may be withdrawn from used in animal feed. For example • The EU Commission voted to ban the application of • Bacitracin,spiromycin,tylomycin and virginomycin as growth promoters after january 1999

  8. Beta-lactams • β-Lactam antibiotics are a broad class of antibiotics • This includes penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems • Most β-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism • more than half of all commercially available antibiotics in use were β-lactam compounds

  9. • Bacteria often develop resistance to β-lactam antibiotics by synthesizing a β-lactamase, an enzyme that attacks the β-lactam ring. • To overcome this resistance, β-lactam antibiotics are often given with β-lactamase inhibitors such as clavulanic acid. • β-lactam antibiotics were mainly active only against Gram-positive bacteria • yet the recent development of broad-spectrum β-lactam antibiotics active against various Gram-negative organisms has increased their usefulness

  10. Mmaoona Irshad • Discovery of Penicllin • Production • Structure of Penicillin • Commercial production • Recovery methods 05/27/13

  11. Penicillin • The first antibiotic • discovered by Flemming • Historically significiant 05/27/13

  12. Flemming 05/27/13

  13. Discovery of Penicillin • Late 1930’s Florey Chain and Heatly characterized the inhibitory compound responsible to produce Penicillin • Developed a protocol to produce it in pure form • Major advancements in both medicine and fermentation technology • Penicillin exhibits the properties of a typical secondary metabolite • P.notatum generated litlle more than 1 mg/L from the surface cultures initially used for penicillin production

  14. Corn steep liquor by product of maize Corn steep liquor 20-25 fold increase in yield • Contains various nitrogen sources, Growth factors and side chain precursors and remains as a major ingredient of penicillin production media.

  15. Pencilllin chrysogenum • Isolated from mouldy cantaloupmelon, results greater penicillin yields. • Further increases in yield when production went over to submerged fermentation

  16. Since 1940,s penicillin yield and fermentation has been vastly improved by extensive mutation and selection of producer strains. • Traditional approach involved random mutation and selection of higher producing strains. • Resulting mutants were grown on liquid medium and culture filtrates were assayed fro penicillin • This was a slow and painstaking as large number of strains had to be tested

  17. Such methods were the key to the dramatically increased yields since the discovery of penicillin • Penicillin fermentations now produce yields in excess of 50g/L, • A 50,000 fold increase from the levels first produce by Fleming’s original isolation

  18. Structure of Penicillin • Basic structure if penicillin is 6-aminopenicillanic acid (6-APA) • Composed of thiazolidine ring fused with beta lactam ring whose 6 amino position carries a variety of acyl subtituents • This beta lactam thiazolidine, structure synthesized from

  19. Alpha aminoadipate • L-cystine • L-valine • Side-chain precursors fermentation medium different biosynthetic penicillins • Natural penicillins can be modified chemically to produce compounds with improved characteristics

  20. 05/27/13 General structure of Penicillin

  21. Most penicillins are now semisynthetic obtained by chemical modification of natural penicillin • Modification is achieved by removing their natural acyl group, leaving 6-APA, to which other acyl groups can be added to confer new properties

  22. Semisynthetic penicillins such as Methicillin, carbenicillin and ampicillin various imrovements including • Resistanceto stomach acids to allow oral administration • A degree of resistance to penicillinase • Extended range of activity to Gram negative bacteria

  23. Chemical production of Penicillin • Fed batch process carried out aseptically in stirred tank fermenters of 40,000- 200,000 L capacity • Oxygen level is very important, must be maintained in 25-60mmol/L/h • Oxygen transfer rate viscosity, which increases as fermentation progresses • Maintained at 25-27 c and pH 6.5-7.7 • Specific conditions depends on P.chrysogenum strain used

  24. Carbon sources used • Various carbon sources used for penicillin production, including glucose, lactose, sucrose, ethanol and vegetable oil • 65% is metabolized for cellular maintenance • 25% growth • 10% penicillin production • Mixture of glucose and lactose used in past for penicillin production • Glucose good growth, poor penicillin yield

  25. Lactose poor growth, good yield • Mode of feeding of carbon source is viatlly important • Corn steep liquor still used • Acidic nature creates an environment for calcium carbonate and phosphate buffer to neutralize the medium • Ammonia, mineral salts and specific side cahin precursors, e.g. phenyl acetic acid may also be added

  26. Inoculum development initiated by adding lyophilized spores to a small fermenter • Fungal mycellium may then be grown further to one or two stages • Initially vegetative growth phase devoted to the development of biomass, which doubles every 6h • This high growth phase maintained for the first 2 days

  27. Mycellium must develop as loose pellets rather than compact forms to ensure optimum yield • Carbon source is fed at low rate an dpenicillin production increases • Continues for 6-8 days • Penicillin excreted into the medium is recovered at the end of fermentation

  28. Whole broth extraction can be performed but can be lead to downstream processing problems • Penicillin recovery follows removal mycellium using rotatory vacuum filters • Recovered mycellium is then washed to remove residual penicillin, prior to its use as animal feed or fertilizer

  29. Recovery of antibiotic • Two methods mostly used • Solvent extraction of the cell free medium • ion pair extraction

  30. Solvents extarction • It gives yields of 90% • It involves reducing the Ph of the filtered medium to 2-2.5 by adding sulphuric or phosphoric acid • Followed by a rapid two stage continuous counter current at 03 c using acetone or ketones

  31. Ion pair extraction • It may be used at 5-7 ph, in which range penicillin is stable • Pigments or impurities are removed by treating with activated charcoal • Penicillin is then retrieved from the solvent by adding sodium or potassium acetate • Reduces solubility of penicillin and precipitates as sodium potassium salt • Penicillin crystals are separated by rotary vacuum filtartion

  32. Penicillin crystals are mixed with a volatile solvent as anhydrous ethanol, butanol or iso propanol to remove further impurities • Crystals are collected by filtration and air dried • At this stage penicillin is 99.5% pure • This product may be further processed to form a pharmaceutical grade product

  33. Production of semisynthetic Penicillins Penicillin (sometimes abbreviated PCN or pen) is a group of antibiotics derived from Penicillium fungi. the naturally occurring penicillins (those formed during the process of mold fermentation) and the semisynthetic penicillins (those in which the structure of a chemical substance—6-aminopenicillanic acid—found in all penicillins is altered in various ways)

  34. To generate compounds with improved properties e.g acid stability,resistance to enzymic degradation ,broader spectrum of activity etc. • Remove the side chain of the base Penicillin to form 6-APA by penicillin Acylase from E.Coli • 6-APA then acylated with an appropriate side chain to produce a semisynthetic penicillin.

  35. Production of Cephalosporin Yields of Cephalosporins from fermentation are much lower than those for penicillins 6-APA is used as a starting material Penicillin is converted by ring expansion to 6-APA & then to 7-ADCA A suitable side-Chain can also be attached

  36. Emergence of Antibiotic resistance Antibiotic resistance was recognized soon after the natural penicillins were introduced The use of antibiotics creats selection pressure favouring the growth of antibiotic resistant mutants, by the misuse and overuse of drugs The emergence of pathogenic bacterial strains, show multiple resistance to a broad range of antibiotics

  37. Many of the antibiotic-resistance genes of staphylococci carried on plasmids that can be exchanged with species of Bacillus and Streptococcus, providing the means for additional genes and gene combinations. Some resistance genes are carried on transposons,that are segments of DNA that can exist either in the chromosomes or within plasmids

  38. Uses of Antibiotics in Medicine

  39. Cell wall synthesis inhibitors usually stop bacteria from forming their cell walls. They kill bacteria and not human cells because human cells do not form cell walls,e.g beta lactums, semisynthetic penicillins Cell membrane inhibitors kill bacterial cells by disorganizing the outer membranes of bacteria,e.g polymyxin. Protein synthesis inhibitors interfere with the process of translation in protein synthesis,e.g tetracyclines, chloramphenicol, macrolides,

  40. Chemotheraputic agents affecting the synthesis of nucleic acids block the division and growth of cells by inhibiting synthesis of DNA and RNA. Most of these agents affect both animal and bacteria cells, so they cannot be used as an antibiotic. Competitive inhibitors are mostly synthetic. These drugs work by disrupting the metabolic rate of bacteria. Some examples include sulfonamides, isoniazid, paraaminosalicylic acid, and ethambutol

  41. Production of semisynthetic Penicillins Penicillin (sometimes abbreviated PCN or pen) is a group of antibiotics derived from Penicillium fungi. the naturally occurring penicillins (those formed during the process of mold fermentation) and the semisynthetic penicillins (those in which the structure of a chemical substance—6-aminopenicillanic acid—found in all penicillins is altered in various ways)

  42. To generate compounds with improved properties e.g acid stability,resistance to enzymic degradation ,broader spectrum of activity etc. • Remove the side chain of the base Penicillin to form 6-APA by penicillin Acylase from E.Coli • 6-APA then acylated with an appropriate side chain to produce a semisynthetic penicillin.

  43. Production of Cephalosporin Yields of Cephalosporins from fermentation are much lower than those for penicillins 6-APA is used as a starting material Penicillin is converted by ring expansion to 6-APA & then to 7-ADCA A suitable side-Chain can also be attached

  44. Emergence of Antibiotic resistance Antibiotic resistance was recognized soon after the natural penicillins were introduced The use of antibiotics creats selection pressure favouring the growth of antibiotic resistant mutants, by the misuse and overuse of drugs The emergence of pathogenic bacterial strains, show multiple resistance to a broad range of antibiotics

  45. Many of the antibiotic-resistance genes of staphylococci carried on plasmids that can be exchanged with species of Bacillus and Streptococcus, providing the means for additional genes and gene combinations. Some resistance genes are carried on transposons,that are segments of DNA that can exist either in the chromosomes or within plasmids

  46. Uses of Antibiotics in Medicine

  47. Cell wall synthesis inhibitors usually stop bacteria from forming their cell walls. They kill bacteria and not human cells because human cells do not form cell walls,e.g beta lactums, semisynthetic penicillins Cell membrane inhibitors kill bacterial cells by disorganizing the outer membranes of bacteria,e.g polymyxin. Protein synthesis inhibitors interfere with the process of translation in protein synthesis,e.g tetracyclines, chloramphenicol, macrolides,

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