1 Microbes  Produce Antibiotics Antibiotics  secondary metabolites  inhibit growth at low concentration Alexander Fleming (1929)  Penicillium notatum  inhibit staphlococcus Begining of Antibiotic era  Development :  513 atb  4076 atb an  6000 atb Antibiotics Production  Fermentation Semisynthetic Microbes producing antibiotics:  Bakteria, Actinomycetes, & Fungi Fungi : -Phycomycetes  14 atb -Ascomycetes  299 atb -Aspergillacea  242 atb  Penicillium  123 atb  Aspergillus  115 atb -Basidiomycetes  140 atb -Fungi inperfecti  315 atb -Moniliales  269

-Eubacteriales  274 atb Microbes producing antibiotics: Bakteria : -Pseudomodales  87 atb -Pseudomodaceae  84 -Enterobacterilaceae  36 atb -Micrococcaceae  16 atb -Lactobacillaceae  28 atb -Bacillacea  171 atb  Bacillus  167 atb Microbes producing antibiotics: Bakteria : -Actinomycetales  2078 atb -Mycobacteriaceae  4 atb -Actinoplanaceae  18 atb -Streptomycetaceae  1950 atb - Streptomyces  1922 atb -Micromonosporaceae  41 atb -Thermoactinomycetaceae  17 atb -Nocardiaceae  48 2

3 Classification of Antibiotics: 1. Carbohydrate Atb - pure sugar  nojimisin - aminoglucoside  streptomicin - Ortomisine  Everninomisin - N-glicoside  sterptotrisin - C-glucoside  Vancomisin - glicolipid  Moenomisin 2. Macrocyclic lactone - macrolide Atb  erytromisin - atb polyene  Candisidin - ansamisin  Rifamisin - Mackrotetrolide  tertranamisin 3. quinoneAtb - tetrasiklin  tertrasiklin - anthrasiklin  adriamisin - naftoquinon  actinorhodin - benzoquinon  mitomisin 4. Peptide & amino acid - amino acid derivate  cycloserin - β-laktam  penicillin - peptide  basitrasin - chromopeptide  actinomisin - Chelating peptide  bleomisin Classification of Antibiotics:

4 5. Heterocyclic + nitrogen - nukleoside  polymixinine 6. Heterocyclic + oksigen - polyeter  monensin 7. Alicyclic derivates - cycloalkana derivate  sikloheksimid - steroid  fusidate acid 8. Aromatic - benzene derivate  kloramphenikol - aromatic ether  novobiosin 9. Alifatic Atb - phosphorous compound  phosphomisine Antibiotic Application:  Spektrum atb : - broad  active for many organisms - narrow  active for certain organism 1. Antitumor :  cytostatic agent 2. Plant pathogens:  the first: - streptomisine  Xanthomonas oryzae Pseudomonas 3. Food Preservative :  regulation for their use  piramisin : - food suface - fungiside  tylosin : - effective for bacillus spora  nisin : effective for clostridia Canned food

Germ free Application  regulation !!! Antibiotic Application: 4. Animal Feed :  trigger animal growth 5. Study of biochemistry & molekular biology :  selective inhibitors: - to study cell function -DNA replication - transkription -Translation -Cell wall synthesis Economic value of antibiotics:  Production more than ton per year  1980: selling value + $ 4.2 billion  US + $ 1 billion per year:  1. Cephalosporine 2. Tetracycline  For animal feed: + $ 100 million per year -before 1960 : 5 % new atb isolates were used for terapheutic -then  many isolates were found - % Atb marketable: : 2.6 % –1977 : 1 % High cost of production & clinic assays 5

Hugh number of atb were already found Why exploration still cunducted ???  Natural Atb are not optimal for terapheutic  need to develop: - to increase activity - to reduce side effects - to increase spectrum - to increase selectivity  Resistant development  Chemical Modification  Genetic Modification : - mutasynthesis - DNA rekombinant - fusi protoplasm β-Laktam Antibiotics  Penisilin  Cephalosporin Peptide Antibiotics  Cephamisin Efective Antibiotics Penilisine: -Fleming (1929)  Penicillium notatum -Isolated in Firstly being use in Produced by : - Penicillium - Aspergillus 6

7 Natural Penisiline:  effective to many Gram positive bacteria  acid labile  inactivated by penisiline β-laktamase Β-laktam ring inhibit peptidoglikan synthesis  target: transpeptidase & D-alanine carboksipeptidase Polimerase peptidoglican inhibited  Inhibit growing cells  Not inhibit not growing cells Basic structure of penisiline:  6- aminopenisilic acid (6-APA) Compose of thiazolidin ring & β-laktam ring 6-APA containing many acyl groups Fermentation without acyl precursor  produce many natural penisilines Only benzilpenisiline useful for terapeutics + acyl prekursor  produce expected peniiyline

Residu gugus akil6-asam aminopenisilat Cincin β-laktam Cincin thiazolidin Penisiline structure: Commercial production: - natural  Penisilin G,V, & O -semisynthetic:  Penisilin G with chemical or enzimatic Split  6-APA Derivate contruction  + acyl Penisiline acylase 8

Biosynthesis: β-laktam-thiazolidine rings ;  contructed from L-cystein & L-valin  non ribosomal process : tripeptida 2 aa & L-α-asam aminoadipat (L-α-AAA) Product I from cyclication : isopenisilin N (biochemical reaction has not known yet) Benzilpenisiline:  alteration of L-α-AAA with activated penilacetate acid HOOC-CH-CH 2 -CH 2 -CH 2 -COOH Isopenilisin N S N COOH L-α-AAA – N O NH 2 L-α-Asam Aminoadipat (L-α-AAA) Cys L-α-AAA-Cys Val L-α-Aminoadippyl-Cysteinil-D-Valine 2 steps cyclication H Penisilin transakilase fenilasetat L-α-AAA-CoA-SH S N COOH O Benzil-penisilin 9 H - CH 2 -CO – N Biosynthesis of penisiline: Penicillium chrysogenum

Biosynthesis of penisilin:  affected by [fosfat]  repressed by glucose Fermentation use lactose  slow metabolisms of sugar Strain development: Production:  Fleming strain = 2 IU/ml  now = IU/ml Peningkatan dari g/l  50 g/l Strain selection & mutagenesis program Starting in 1943 Method of production: Penisiline G & V  produced by submerged fermentation  capacity – liters  aerobic  O 2 supply limitation 10

11 Growth Curve of penisiline production :  produksi selama 40 jam  Doubling time 6 jam: -pembentukan biomassa  Dg fed-batch: - spi 120 – 160 jam kembali

12 kembali

13 kembali

Cephalosporine  β-laktam + dihidrothiazin rings Cephalosporium acremonium : - isolation of atb in strain isolation in 1945 Acremonium chrysogenum  - Cephalosporin  - Penisilin N  Cephalosporin steroid P1-P5 Fungi: - Emericellopsis - Paecilomyces Streptomyces : S. lipmanii S. clavuligerus S. lactamdurans 14

15 Characteristics: - broad spectrum - low Toxicity - ~ ampisiline - resistant to penisiline beta laktamase - not resistant to cephalosporin beta laktamase

16 Biosynthesis: Low [lisin] Production increased

-Medium :- corn steep liquor Method of Production :  similar with penisiline  Fermentation: - growth phase 90 hour -Up to 90 hours, high consumption of O 2  need high aeration -90 – 160 hours  low consumption of O 2 -pH 7 -Temp 25 – 28 C - meat meal - sucrose, glucose - ammonium acetate  chemically: - from penisiline  price of penisiline  low New β-laktam:  derivated from semisintetic -Nokardisin: - monosiklik β-laktam - efektif pd Gram – - Nocardia uniformis - Streptomyces alcalophilus -Klavulanic acid: - β-laktam-oxazolidin - not efektive - inaktivation by β-laktamase ireversibel - Streptomyces clavuligens - aplikasi kombinasi  aktivitas ningkat -Thienamisin : - β-laktam-pyrrolin - efektive to Gram – & + - inaktivation by β-laktamase - Streptomyces cattleya - not stable  depeloped fro stability 17

Amino acid & peptide Atb:  Amino acid derivates : - sikloserin - azoserin  β-laktam  Chromopeptide  Depsipeptide  Linier & cylic peptide D-sikloserin:  D-4-amino-3-isoxazolidone  natural & synthetic - S. orchidoceus - S. lavendulae - S. garyphalus - S. roseochromogenes - Inhibit cell wall synthesis  inhibit alanine racemase - enzyme for alanine production -Efective to mycobacterium  M. tubercolosis -for TBC; combination with isotinate hidrazine & rifampisin or streptomisin Aktinomisin:  atb chromopeptida -fenoksazon-kromofor  unit dg pentapeptida-lakton  as. Amino bervaryasi -hambat RNA polimerase -Sangat toksik  rusak liver & ginjal -Utk tumor/kanker 18

Biosintesis: Atb depsipeptida: Subunit:  as. Amino  as. hidroksil # Valinomisin: - nilai ekonomi rendah - digunakan pd penelitian biokimia  hambat fosforilasi oksidatif  carrier K + - S. fulvissimus 19

20 # Virginiamisin: - efektif Gram + - pemicu pertumbuhan ternak unggas, babi, sapi - S. virginiae Atb peptida linier & siklik: -MO :  Bacillus  sebagian besar atb peptida  Streptomyces -BM : 270 – Sebagian besar siklik - di Bacillus produksi saat sporulasi  berperan dalam proses sporulasi -Aplikasi: terbatas  toksik  obat luar - Gramisidin - Tirosidin - Basitrasin - polimiksin  infeksi Gram – - viomisin - Capreonisin Luka & luka bakar TBC

Basitrasin: A : BM B : BM C: BM nilai ekonomi penting -Produksi th ton -Obat luar dan makanan ternak -Bacillus lincheniformis -Aktivitas  hambat sintesis dinding sel  sintesis peptidoglikan Biosintesis:  tidak melibatkan ribosom, mRNA, & tRNA  komplek multienzim dinamakan: “MESIN THIOTEMPLATE”  ATP & Mg 2+ 21

22 “MESIN THIOTEMPLATE” Metode produksi:

Atb Karbohidrat:  Turunan gula & glikosida Nojirimisin:  efektif thd Sarcina lutea & Xanthomonas oryzae  aktif menghambat α & β glukosidase dan amilase 23

24 Atb Karbohidrat:  Efektif pd Gram –  Utk infeksi akut  Toksik  kerusakan ginjal  Efek samping: kurang pendengaran

25 Streptomisin:  Aktivitas : 1. Hambat sintesis protein  ikat unit 12S dari subunit 30 S  salah kode & baca  12 S situs aktif pengikatan aminoakil-tRNA & tMet-tRNA 2. Rusak membran sel  hambat translokasi peptidil-tRNA  kebocoran molekul BM kecil  Biosintesis:  banyak enzim yg diketahui tapi belum dimengerti benar

26 Metode produksi: -fermentasi :  l  suhu 28 – 30 C  pH 7  waktu 4 – 7 hari  sumber C : pati/dekstrin  sumber N : soy meal

27 Atb makrosiklik lakton:  Efektif pd Gram +  Hambat sintesis protein  ikat subunit ribosom 50 S  S. erythreus -Eritromisin

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Metode produksi: -fermentasi submerged:  glukosa  soy meal  (NH 4 ) 2 SO 4  NaCl  CaCO 3  pH 50 g/l 30 g/l 3 g/l 5 g/l 6 g/l 7 Tetrasiklin:  Struktur dasar cincin naftalena  Pengguanaan luas  Spektrum luas: Gram +, -, riketsia, mycoplasma, spiroket, klamidia  Aktivitas: hambat sintesis protein  subunit 30S: hambat ikatan aminoakil-tRNA ke situs A ribosom -aplikasi : - manusia - veteriner - ternak: unggas, babi - pengawetan : daging, unggas, ikan 29

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31 Pengembangan:  Rekayasa genetik ? -Transformasi  Streptomyces -Antibiotik baru ???