ANTIBIOTICS A chemical compound acting against life is known as antibiotics. It can obtain either from natural sources ,microbes or by synthetic methods.
THE MECHANISMOF ANTIBIOTICS
THE BASICS• Used to kill or inhibit the growth of bacteria• Classified as bactericidal or bacteriostatic Kill bacteria directly Prevent cell division• Classified by target specificity:Narrow-spectrum vs Broad range• Most modified chemically from original compounds found in nature, some isolated and produced from living organisms
SITES OF ANTIOBIOTICACTION
MOST COMMONLY USEDANTIBIOTICSPenicillinStreptomycinAmpicillinKanamycinchloramphenicol
PENICILLIN most widely used antibiotic. It is drug of choice when infection is caused by organisms susceptible to it . It is effective drug against gram + bacteriaand also against rickettsia . Mostly penicillin are produced by species of penicillium .
PENICILLIN BIOSYNTHESIS Overall, there are three main and important steps to the biosynthesis of penicillin G (benzylpenicillin).
FIRST STEP is the condensation of three amino acids — L-α- aminoadipic acid, L-cysteine, L-valine into a tripeptide. Before condensing into the tripeptide, the amino acid L-valine must undergo epimerization to become D-valine. The condensed tripeptide is named δ-(L-α-aminoadipyl)-L-cysteine-D-valine (ACV). The condensation reaction and epimerization are both catalyzed by the enzyme δ-(L-α-aminoadipyl)-L- cysteine-D-valine synthetase (ACVS), a nonribosomal peptide synthetase or NRPS.
SECOND STEP The second step in the biosynthesis of penicillin G is the oxidative conversion of linear ACV into the bicyclic intermediate isopenicillin N by isopenicillin N synthase (IPNS), which is encoded by the gene pcbC. Isopenicillin N is a very weak intermediate, because it does not show strong antibiotic activity.
FINAL STEP is an transamidation by isopenicillin N N-acyltransferase, in which the α- aminoadipyl side-chain of isopenicillin N is removed and exchanged for a phenylacetyl side-chain. This reaction is encoded by the gene penDE, which is unique in the process of obtaining penicillins
PRODUCTION Penicillin is a secondary metabolite of certain species of Penicillium and is produced when growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin. α-ketoglutarate + AcCoA → homocitrate → L-α-aminoadipic acid → L-lysine + β- lactam The by-product, L-lysine, inhibits the production of homocitrate, so the presence of exogenous lysine should be avoided in penicillin production. The Penicillium cells are grown using a technique called fed-batch culture, in which the cells are constantly subject to stress, which is required for induction of penicillin production. The available carbon sources are also important: Glucose inhibits penicillin production, whereas lactose does not. The pH and the levels of nitrogen, lysine, phosphate, and oxygen of the batches must also be carefully controlled. The biotechnological method of directed evolution has been applied to produce by mutation a large number of Penicillium strains. These techniques includeerror-prone PCR, DNA shuffling, ITCHY, and strand-overlap PCR. Semisynthetic penicillins are prepared starting from the penicillin nucleus 6-APA.
STREPTOMYCIN It is produced using strains of streptomyces griseus . Basic medium for production of sterptomycin containssoybean meal as nitrogen source ,glucose as carbon source and NaCl .Proteolytic enzymatic activity of S.griseus releases ammonia to the medium from the soybean meal causing rise in pH .during this initial fermentation phase there is a littleproduction of streptomycin.
CLASSICFERMENTATIONPROCESS FOR THEPRODUCTION OFSTREPTOMYCIN
A little additional production of mycelia. The glucose added in the medium & the ammonia released from the soybean meal are consumed during this phase. The Ph remains fairly constant (7.6- 8). It is the final phase of the fermentation, after depletion of carbohydrates from the medium, Streptomycin production ceases & the bacterial cells began to lyse. There is a rapid increase in ph because of the release of ammonia from lysed cells. In the end of fermentation, the mycleium is separated from the broth by filtration & the streptomycin is recovered. The purification consists of adsorbing the streptomycin onto activated charcoal & eluting with acid alcohol.
AMPICILLIN Belongs to β-lactam group of antibiotics – contain β-lactam ring Broad-spectrum Penicillin derivative that inhibits bacterial cell wall synthesis (peptidoglycan cross-linking) Inactivates transpeptidases on the inner surface of the bacterial cell membrane Bactericidal only to growing E. Coli
AMPICILLIN RESISTANCE Cleavage of β-lactam ring by β-lactamase enzyme
β-lactamase is encoded by the plasmid-linked bla (TEM-1) geneHydrolyzes ampicillinAmpicillin levels in culture continually depleted
USE IN SYNTHETIC BIOLOGY• To confirm uptake of gene (eg. of plasmids) by bacteria• Bacterial Transformation: DNA integrates into bacteria’s chromosome and made chemically competent• Exogenous DNA tagged with an antibiotic resistance gene eg. β-lactamase• Grown in medium containing ampicillin• Ampicillin resistance indicates successful bacterial transformation
KANAMYCIN Targets 30s ribosomal subunit, causing a frameshift in every translation Bacteriostatic: bacterium is unable to produce any proteins correctly, leading to a halt in growth and eventually cell death
KANAMYCIN USE/RESISTANCE Over-use of kanamycin has led to many wild bacteria possessing resistance plasmids As a result of this (as well as a lot of side effects in humans), kanamycin is widely used for genetic purposes rather than medicinal purposes, especially in transgenic Plants Resistance is often to a family of related antibiotics, and can include antibiotic-degrading enzymes or proteins protecting the 30s subunit.
CHLORAMPHENICOL Bacteriostatic: functions by halting bacterial growth, which is done by inhibiting the enzyme peptidyl transferase, a protein that assists in the binding of tRNA to the 50s ribosomal subunit Three methods of resistance: reduced membrane permeability, mutation of the 50s subunit, and an enzyme called chloramphenicol acetyltransferase, which inactivates chloramphenicol by covaltly linking groups Easy/cheap to manufacture, but unused in western countries because of possible aplastic anemia as a side effect