slide1 n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Primary Metabolites: log phase, use nutrients fast, produce PM Secondary Metabolites: depletion of nutrients, growth r PowerPoint Presentation
Download Presentation
Primary Metabolites: log phase, use nutrients fast, produce PM Secondary Metabolites: depletion of nutrients, growth r

Loading in 2 Seconds...

play fullscreen
1 / 69

Primary Metabolites: log phase, use nutrients fast, produce PM Secondary Metabolites: depletion of nutrients, growth r - PowerPoint PPT Presentation


  • 185 Views
  • Uploaded on

Microbial Products. Primary Metabolites: log phase, use nutrients fast, produce PM Secondary Metabolites: depletion of nutrients, growth retards, produce SM. Primary Metabolites: Vitamins. Vitamins : cannot be synthesized by higher organisms

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Primary Metabolites: log phase, use nutrients fast, produce PM Secondary Metabolites: depletion of nutrients, growth r' - alia


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

Microbial Products

Primary Metabolites: log phase, use nutrients fast, produce PM

Secondary Metabolites: depletion of nutrients, growth retards, produce SM

slide2

Primary Metabolites: Vitamins

Vitamins: cannot be synthesized by higher organisms

But microorganisms are capable of synthesizing (gut)

Thiamine

Riboflavin

Pyridoxine

Folic acid

Pantothenic acid

Biotin

Vitamin B12

Ascorbic acid

b- carotene (provitamin A)

Ergosterol (vitamin D)

  • Studies reveal vitamin deficiencies
  • Reported beneficial health effects
  • Growing vitamin market demand (cost effective)
  • Genetically engineered MO as alternatives to chemical synthesis
slide3

Vitamins

Fat soluble

Water soluble

Carotenoids

b-carotene (provitamin A)

Astaxanthin

Poly unsaturated Fatty acids (PUFA; vitamin F)

Docosahexaenoic acid (DHA)

Arachidonic acid (ARA)

Riboflavin (vitamin B2)

Cobalamin (vitamin B12)

L-Ascorbic acid (Vitamin C)

R-Pantothenic acid (vitamin B5)

D-Biotin (vitamin H or B7)

Vitamin B1 (Thiamine)

Vitamin B6 (pyridoxol)

Folic acid

Ergosterol (vitamin D)

slide4

Vitamin B12 or Cyanocobalamin

  • Water soluble vitamin ; complex sructure
  • Has role in functioning of brain and nervous system, formation of blood
  • Contains rare element cobalt
  • Deficiency causes pernicious anemia which is an causes low Hb, less RBCs
  • Pernicious anemia: autoimmune disorder, parietal cells (stomach) responsible for secreting intrinsic factor are destroyed. Intrinsic factor is crucial for the normal absorption of B12, so a lack of intrinsic factor, as seen in pernicious anemia, causes a deficiency of Vitamin B12
  • dietary reference intake for an adult ranges from 2 to 3 µg per day
  • used in treating cyanide poisoning, prevents brain atrophy in Alzheimer’s patients
  • COMMON INGREDIENT IN ENERGY DRINKS
slide5

Pyrrole nitrogen

4 Pyrrole units

C63 H88 CoN14 O14P

cobinamide

  • Corrin ring
  • Deep red colour due to corrin ring
  • Central Co atom
  • Coordination state 6
  • 4 of 6 coord sites have pyrrole ring
  • 5 has dimethylbenzimidazole group
  • 6 is center of reactivity, variab;e
  • CN, OH, Me, 5-deoxyadenosyl for 4 types of B12

6

2

1

4

3

5

5,6-dimethyl benzimindazole

nucleotide

slide6

Commercial production

Chemical syn not feasible

Genera known to produce vit B12

Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Flavobacterium, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas

20mg/L

  • Most commonly used for industrial production are
      • Streptomyces griesus
      • Pseudomonas denitrificans (aerobic)
      • Salmonella typhimuriu (anaerobic)
      • PropionibacteriumshermaniiGRAS by FDA
      • (anaerobic) (Generally Regarded As Safe)

Sanofi-Aventis (FRENCH) use genetically engineered versions to produce vit B12 under specialized conditions from Propionibacterium since they have no endotoxins or exotoxins

P. denitrificansalso used after strain modification; mutant more efficient than wild type

slide7

Commercial production

  • Produced in continuous culture with 2 fermenters in series
  • Addition of 5,6-dimethylbenzimidazol (0.1%)

Glucose

Corn steep

Betaine (5%)

Cobalt (5ppm)

pH 7.5 +

Anaerobic

70h

Aerobic

50h

Propionibacterium

freudenreichii

Cobinamide production and accumulation

Nucleotide synthesized

Combined with cobinamide

To yield 2ppm of cobalamin

KCN added

Acidification of culture

To 2-3pH/ 100oC

Filter to remove cell debris

CYNACOBALAMIN

80% purity

Used as feed additive

Filtrate

Betaine: sugar beet molasses

slide8

Commercial production

ANAEROBIC PHASE

2-4 DAYS

5-deoxyadenosylcobinamide produced

AEROBIC PHASE

5,6-dimethylbenzimidazole is added and gets incorporated to form 5’-deoxyadenosylcobalamin

During the 7-day fermentation run, adenosylcobalamin is predominantly secreted from the biomass and accumulates in the fermentation broth in milligram amounts.

The down- stream steps comprise filtration, cyanide treatment, chromatography, extraction, and crystallization yielding vitamin B12 in high purity.

If to be used for treatment further purification (95-98% Purity)

slide9

Commercial production

Pseudomonas denitrificans: strain improvements resulted in increase in yeild

From 0.6mg/L to 60mg/L

Glucose : common carbon

Alcohols (methanol, ethanol, isopropanol)

Hydrocarbons(alkanes, decane, hexadecane)

With methanol 42mg/L was obtained using Methanosarcinabarkeri

slide10

Riboflavin (Rf) or Vitamin B2

  • Water soluble
  • Essential for growth and reproduction; key role in energy metabolism, ketone bodies, fats, CHO and protein metabolism
  • Deficiency leads to cheliosis (fissures around mouth), glossitis (purple tounge) and dermatitis
  • Required in coenzymes FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide)
  • Used as an orange-red food colour additive, designated in Europe as E101

7,8-dimethyl-10- (D-19-ribityl) isoalloxazine

Participates in O-R reactions

Flavin is ring moiety with yellow colour to oxidized form

slide11

Isoalloxazine ring

Isoalloxazine ring

H

H

Ribitol

FAD

E101

FMN

E101a

genes encoding the riboflavin biosynthetic enzymes are well conserved among bacteria and fungi

slide12

INDUSTRIAL USE

Processed food is often fortified by the use of riboflavin as a colorant or vitamin supplement.

The main application (70%) of commercial riboflavin is in animal feed, since productive livestock, especially poultry and pigs, show growth retardation and diarrhea in case of riboflavin deficiency.

According to a report by SRIC, a consulting company in Menlo Park (California), in 2005 the need for industrially produced riboflavin was estimated at 6500–7000 tons per year.

slide13

Commercial production

Glucose

50% by biotransformation

usingBacillus pumulis

D-ribose

20% production by Chemical synthesis

Riboflavin

1/3rdproduction by

direct fermentation

Acetone butanol fermentation

Clostridium acetobutylicum

C. butylicum riboflavin as

by product

Ashbyagossypii

Candida famata

Bacillus subtillis (genetically modified)

slide14

Commercial production

Phase I use of glucose, accumulation of pyr, pH acidic, growth stops, no Riboflv

Phase II decrpyr, incr in ammonia, alkalinity incr, prod of Riboflv in form of FAD and FMN

Phase III autolysis, cell disruption, release of free FAD, FMN and riboflv

Carbon sources: glucose, acetate, methanol, aliphatic hydrocarbons

Major riboflavin producers are DSM Nutritional Products (Switzerland) and Hubei Guangji(Hubei Province, China), both using genetically engineered B. subtilisproduction strains, and BASF (first in Germany but now in South Korea), employing genetically engineered A. gossypii.

slide15

Ascorbic acid or Vitamin C

  • Used in collagen biosynthesis, protects against nitrosamines, free radicals
  • Deficiency causes scurvy

Precursor for its chemical synthesis can be obtained by biological methods

feed applications of L-ascorbic acid account for only 10%, whereas the main uses are in the

pharmaceutical industry (50%),

food (25%), and

beverages (15%).

Pharmaceutical applications include stimulation of collagen synthesis (especially cosmetic products) and high antioxidant capacity, used for the reported health benefits in the prevention of flu, heart diseases, and cancer, as well as an antidote for poisoning.

The food and beverage industry predominantly exploits the antioxidant capacity of L-ascorbic acid to extend durability, prevent discoloration, and to protect flavor and nutrient contents of their products.

slide16

Submerged bioreactor fermentation

Erwiniasp.

Acetobacter sp.

Gluconobacter sp.

2,5-diketogluconic acid

2-keto L-gluconic acid

L-ASCORBIC ACID

D-glucose (200g)

Glucuronic acid

Gluconolactone

L-Gluconolactone

L-ASCORBIC ACID

2,5-diketogluconic acid

reductase

Corynebacterium sp.

D-sorbitol

sorbitol dehydrogenase

L-Sorbose

chemical oxidation

2 keto L gulonic acid

Enol formof

2 keto L gulonic acid

acid treatment

L-ASCORBIC ACID (100g)

Bacillus

megaterium

Acetobacterxylinum,

A,suboxydans

L-Gluconolactone

dehydrogenase

Cloning of gene

2,5-diketogluconic acid

Reductaseof Corynebacterium into Erwiniaherbicola

Reichstein Grussner synthesis

slide17

b- carotene or provitamin A

Provitamin A -----> Vitamin A (intestine)

  • Fat soluble
  • Deficiency leads to night blindness
  • Best source is liver and whole milk also coloured fruits and vegetables
  • Isoprene derivatives
  • Tetraterpenoids with eight isoprene residues
  • 400 naturally occurring carotenoids: b-carotene, a-carotene, d-carotene, lycopene, zeaxanthin

Carotenoids Used as food colorants and animal feed supplements for poultry and aquaculture, carotenoids play an increasing role in cosmetic and pharmaceutical applications due to their antioxidant properties.

The pigments are often regarded as the driving force of the nutraceutical boom, since they not only exhibit significant anticarcinogenic activities but also promote ocular health, can improve immune response, and prevent chronic degenerative diseases.

slide18

Commercial production

  • Microbial fermentation
    • Blakesleatrispora (high yeild; 7g/L)
    • Phycomycesblakesleeanus
    • Choanephoracucurbitarum

Submerged Fermentation process

Corn starch, soyabean meal, b-ionone, antioxidants

Trisporic acid: act as microbial sex hormone, improves yield

b-Ionone: incrb-carotene syn by incr enzyme activity

Purified deodorized kerosene increases solubility of hydrophobic substrates

stimulators

Recovery: b- carotene rich mycelium used as feed additive

Mycelium is dehydrated by methanol, extracted in methylene chloride and crystallized which is 70-85% pure

DSM Nutritional Products (Switzerland) and BASF (Germany) dominate the market with their chemical synthesis processes, but Chinese competitors are catching up.

slide19

Halophilicgreen microalgae Dunaliellasalina. It accumulates the pigments in oil glo- bules in the chloroplast interthylakoid spaces, protecting them against photoinhibition and photodestruction.

Excessive pigment formation in D. salinais achieved by numerous stress factors like high temperature, lack of nitrogen and phosphate but excess of carbon, high light intensity, and high salt concentration, the latter two having the highest impact.

Dried D. salinabiomass for sale contains 10–16% carotenoids, mainly b-carotene. In addition crystalline material obtained after extraction with edible oil is also sold.

slide20

Primary Metabolites: Organic Acids

Organic acids are produced by through metabolisms of carbohydrates. They accumulate in the broth of the fermenter from where they are separated and purified.

I. Terminal end products lactic acid

(pyruvate, alcohol) Propionic acid

II. Incomplete oxidation of sugars citric acid

(glucose) Itaconic acid

Gluconic acid

Glycolysis

Krebs cycle

III. Dehydrogenation of alcohol with O2 acetic acid

Manufactured on large scale as pure products or as salts

slide21

CITRIC ACID: industrial uses

Flavoring agent

In food and beverages

Jams, candies, deserts, frozen fruits, soft drinks, wine

Antioxidants and preservative

Agent for stabilization of

Fats, oil or ascorbic acid

Stabilizer for cheese preparation

Chemical industry

Antifoam

Treatment of textiles

Metal industry, pure metals +citrate (chelating agent)

Pharmaceutical industry

Trisodium citrate (blood preservative)

Preservation of ointments and cosmetics

Source of iron

Acidifyer

Flavoring

Chelating agent

Primary metabolite

Present in all organisms

Detergent cleaning industry

Replace polyphosphates

slide22

Commercial Production

Aspergillusniger

clavatus

Pencilliumluteum

Strains that can tolerate high sugar and low pH with reduced synthesis of undesirable by products (oxalic acid, isocitric acid, gluconic acid)

Glucose

MEDIUM

CYTOPLASM

Pyruvate

Glucose

Pyruvate

Pyruvate

Acetyl CoA

Pyr carboxylase

CO2

OXA

Malate

MITOCHONDRIA

Malate FumarateSuccinyl CoA

OXA

citric acid

a-KG

PyrDehy-

drogenase

CO2

Citrate

synthase

100g sucrose --- 112g any citric acid or 123g citric acid-1hydrate

slide23

Factors for regulation

  • CARBOHYDRATE SOURCE: sugar should be 12-25%
      • Molasses (sugar cane or sugar beet)
      • Starch (potato)
      • Date syrup
      • Cotton waste
      • Banana extract
      • Sweet potato pulp
      • Brewery waste
      • Pineapple waste

High sugar concincr uptake and production of citric acid

  • TRACE METALS:
      • Mn2+, Fe3+, Zn2+ incr yield
      • Mn2+ incr glycolysis
      • Fe3+ is a cofator for enzymes like aconitase
  • pH: incr yield when pH below 2.5, production of oxalic acid and gluconic acid is suppressed and risk of contamination is minimal
  • DISSOLVED O2:high O2, sparging or incr aeration can affect if interrupted
  • NITROGEN SOURCE: addition of ammonium stimulates overproduction, molasses is good source of nitrogen
slide24

Citric acid production

Surface fermentation submerged fermentation

Solid liquid

Stirred Airlift

Bioreactor bioreactor

N alkanes (C9-C23) can also be used to produce citric acid; can result in excess production of isocitric acid

slide26

ACETIC ACID

Incomplete oxidation of ethanol

Vinegar is prepared from alcoholic liquids since ceturies

NAD+ NADH +H+

NADP+NADP +H+

CH3 CH2OH---- CH3CHO-------- CH3CH(OH)2 ------- CH3COOH

Ethanol acetaldehyde acetaldehyde hydrate acetic acid

Alcohol

dehydrogenase

Acetaldehyde dehydrogenase

Gluconobacter, Acetobacterwith acid tolerant A. aceti

One molecule of ethanol one molecule of acetic acid is produced

12% acetic acid from 12% alcohol

It is an obligate anaerobe, Gram-positive, spore-forming, rod-shaped, thermophilic organism with an optimum growth temperature of 55–60 o C and optimum pH of 6.6–6.8.

Clostridium thermoaceticum

slide27

VINEGAR: 4% by volume acetic acid with alcohol, salts, sugars and esters

flauoring agent in sauces and ketchups, preservative also

Wine, malt, whey (surface or submerged fermentation process)

Surface: trickling generator; fermentale material sprayed over surface, trickle thro shavings contaning acetic acid producing bacteria; 30oC (upper) and 35oC (lower). Produced in 3 days.

Submerged: stainless steel, aerated using suction pump, production is 10X higher

Clostridium thermoaceticum(from horse manure) is also able to utilize five-carbon sugars:

2C5H10O5 --- 5CH3COOH

A variety of substrates, including fructose, xylose, lactate, formate, and pyruvate, have been used as carbon sources in an effort to lower substrate costs. This factor is also important if cellulosic renewable resources are to be used as raw materials.

Typical acidogenic bacteria are Clostridium aceticum, C. thermoaceticum, Clostridium formicoaceticum, and Acetobacteriumwoodii. Many can also reduce carbon dioxide and other one-carbon compounds to acetate.

slide28

1mol

2moles

2moles

1mol

1mol

CODH

These enzymes are metalloproteins; for example, CODH contains nickel, iron, and sulfur; FDH contains iron, selenium, tungsten, and a small

quantity of molybdenum; and the corrinoid enzyme (vitamin B12 compound) contains cobalt. C. thermoaceticum does not have any specific amino acid requirement; nicotinic acid is the sole essential vitamin

slide29

LACTIC ACID: industrial uses

Pharmaceutical grade

>90%

Technical grade

20-50%

Food grade

>80%

Intestinal treatment

(metal ion lactates)

Food additive (sour flour and dough)

Ester manufacture

Textile industry

Glucose

G3P NAD+

NADH +H+

1,3-biphosphoglycerate

Lactic acid

LDH

(Lactate dehydrogenase)

G3P dehy-

drogenase

Pyruvate

slide30

LACTIC ACID

2 isomeric forms L(+) and D(-) and as racemic mixture DL-lactic acid

First isolated from milk

Toady produced microbial

HeterofermentationHomofermentation

Other than lactate products only lactate as product

Lactobacillus

L. delbrueckii Glucose

L. leichmanni

L. bulgaricus

L.helvetii Whey (lactose)

L.lactis ------- Maltose

L.amylophilus -------- Starch

L.pentosus ------ Sulfite waste liquor

Mostly one isomer is produced

slide31

LACTIC ACID: production process

1mol of glucose gives 2 moles of lactic acid; L lactic acid is predominantly produced

Fermentation broth (12-15% glucose, N2, PO4, salts micronutrients)

pH 5.5-6.5/temp 45-50oC/75h

Heat to dissolve Ca lactate

Addition of H2SO4

(removal of Ca SO4)

Filter and concentrate

Addtion of Hexacyanoferrant

(removes heavy metal)

Purification (Ion exchange)

Concentration

Lactic acid

slide32

GLUCONIC ACID: Applications

Used in stainless steel manufacturing, leather (can remove rust and calcareous deposits)

Food additive for breverages

Used in Ca and Fe therapy

Na gluconate used in sequestering agent in detergets

Desizing polyester or polyamide fabric

Manufacture of frost and cracking resistant concrete

Bacteria: Gluconobacter, Acetobacter, Pseudomonas, Vibrio

Fungi: Aspergillus, Penicillium, Gliocladium

slide33

intracellular

extracellular

PQQH2

Bacteria

PQQ

Glucose

dehydrogenase

H2O

D-gluconolactone

Gluconic Acid

D-Glucose

Lactonase

Glucose

oxidase

fungi

Extracellular

Inducible

FAD

FADH2

Fungi

Catalase

PQQ: pyrroliquinolinequinone

coenzyme

H2O2

O2

High conc of glucose and pH above 4

H2O2 antagonist for other micro-organisms

Submerged fermentation process

Use glucose from corn

H 4.5-6.5

28-30oC for 24h

Incr supply of O2 enhances yield

slide34

ITACNIC ACID: Applications

Aspergillusitoconicusand A.terreus

Used in plastic industry, paper industry

Manufacturing of adhesives

Cis-aconitic acid undergoes decarboxylation

Itaconic acid Oxidase

Itaconic acid

Itatartaric acid

(-) By Ca to incr yield

slide35

SECONDARY METABOLITES

ANTIBIOTICS

BROAD SPECTRUM

NARROW SPECTRUM

Control growth of wide range of unrelated organisms

Tet, Cm

Control growth of selected number of organisms

Pen, Str

Streptomyces,eg. Tetracyclin, actinomycin D,

slide36

ANTIBIOTICS: applications

Antimicrobial agents for chemotherapy

Antitumour antibioticseg. Actinomycin D and mitomycin D

Food preservative antibiotics eg in canning (chlortetracycline) or fish or meat preservation (pimarcin, nisin)

Antibiotics in animal feed and veterinary medicine egenduracidin, tylosin and hygromycinB, theostrepton, salinomycin

Control of plant diseases egblasticidin, teranactin, polyoxin

Molecular biology

slide37

MODE OF ACTION OF ANTIBIOTICS

RNA ELONGATION

DNA GYRASE

CELL WALL SYNTHESIS

DNA DIRECTED RNA POLYMERASE

DNA

PROTEIN SYNTHESIS

(50S INHIBITORS)

PROTEIN SYNTHESIS

(30S INHIBITORS)

PROTEIN SYNTHESIS

(tRNA)

THF

RNA

RIBOSOMES

DHF

CYTOPLASMIC

MEMBRANE STRUCTURE

AND FUNCTION

LIPID BIOSYNTHESIS

PABA

slide38

SYTHETIC ANTIBIOTICS

Selective toxicity: concept, Paul Ehrlich

  • GROWTH FACTOR ANALOGS:
    • structurally similar to a growth factor required in a micro-organism; small differences of analogs in authentic growth factor prevent analog to function in the cell.
    • SULFA DRUGS: specifically inhibit bacteria (streptococcal infections) eg. SULFANILAMIDE: is an analog of PABA (p-aminobenzoic acid) which is part of folic acid and nucleic acid precursor. Combination: sulfamethoxazole and trimethoprim; disadvantages and advantages
    • ISONIAZID: important growth factor with narrow spectrum only against Mycobacterium. It interferes with synthesis of mycolic acids, a cell wall component. It is an analog of nicotinamide (vitamin). Single most effective drug against tuberculosis.
slide39

NUCLEIC ACID BASE ANALOGS

URACIL 5-FLOUROURACIL (Uracil analog)

PHENYLALANINE p-FLOUROPHENYLALANINE

THYMINE 5-BROMOURACIL (thymine analog)

Addition of F or Br does not alter the shape but changes chemical properties such that the compound does not function in the cell metabolism, thereby blocking the nucleic acid synthesis.

These analogs are used in treatment of viral and fungal infections and many of these occur as mutagens.

  • QUINOLONES:
    • Antibacterial compounds interfere with bacterial DNA gyrase, prevent supercoiling (packaging of DNA) egFlouroquinolones like ciprofloxin (UTI, anthrax). B. anthracis maybe resistant to pencillin. These are effective in both G+ve and G-ve bacteria since DNA gyrase is present in all.
    • Also used in beef and poultry for prevention and treatment of respiratory diseases.
slide41

NATURALLY OCCURING ANTIBIOTICS

FROM BACTERIA, FUNGI

LESS THAN 1% OF 1000S OF ANTIBIOTICS ARE USEFUL BECAUSE OF TOXICITY OR LACK OF UPTAKE BY HOST CELLS

Natural antibiotics can be artificially modified to enhance their efficacy then they are semi-synthetic antibiotics

Broad spectrum antibiotics: effective against both gram +ve and gram-ve

Narrow may also be beneficial to target specific group of bacteria eg. Vancomycin: narrow spectrum effective for gram positive pencillin resistant Staphylococcus, Bacillus, Clostridium

Targets for antibiotics maybe

ribosomes (Cm and Str for Bacteria and Cyclohexamide for eukarya), Cell wall, cytoplasmic membrane, lipid biosynthesis, enzymes, DNA replication and transcription elements

Protein synthesis, Transcription (RNA poly, RNA elongation etc)

slide42

Produced By Fungi

B-LACTAMS (b-lactam ring)

Penicillin

Cephalosporins

Produced by Prokaryotes

AMINOGLYCOSIDES (amino sugars with glycosidic linkage)

MACROLIDES (lactone ring bonded to sugars)

TETRACYLINES (Streptomyces)

PEPTIDE ANTIBIOTICS (Daptomycin, (Streptomyces)

PLATENSIMYSIN (Streptomyces)

slide46

Beta Lactam Antibiotics

PENICILLINS,

CEPHALOSPORINS,

MONOBACTAMS AND

CARBAPENEMS

slide47

PENCILLIN--------b-LACTAM ANTIBIOTIC

Pencillin G and V (natural)

Penicilliumchrysogenum

Alexander Fleming

Pencillin G first clinically useful antibiotic

For Gram positive bacteria

Used for

Pneumococcal

Streptococcal infections

6-AMINOPENICILLIANIC ACID

slide48

Ampicillin, carbencillin

Slight modification in N-acyl groups results in semi synthetic penicillin which is able to act on gram negative bacteria (goes past outer membrane) to act on cell wall

MANY BACTERIA HAVE BETA LACTAMASE HENCE THOSE BACTERIA ARE PENCILLIN RESISTANT

EG.Oxacillin and Methicillin beta lactamase resistant semi synthetic antibiotics

MECHANISM OF ACTION

  • Pencillins block cell wall synthesis: transpeptidation (cross linking 2 glycan peptide chains)
  • Transpeptidases bind to pencillin hence they are called PENCILLIN BINDING PROTEINS (PBP)
  • Newly synthesized bacterial wall is no longer cross linked and has poor strength
  • PBP also stimulates release of AUTOLYSINS (ENZYMES TO DIGEST CELL WALL)
  • Osmotic pressure differences cause lysis
  • VANCOMYCIN: does not bind PBPs but D-alanyl- Dalanine peptide to block transpeptidation
  • BECAUSE OF SELECTIVE PROCESS B-LACTAMS DO NOT AFFTECT HOST CELLS AND MECHANISM IS UNIQUE TO BACTERIA
slide49

MECHANISM OF ACTION

Natural penicillin: i.e. V and G are effective against several gram positive bacteria

They are effective against b-lactamase producing MO (enz which can hydrolyze penicillins)

Eg. Staphylococcus aureus

Production of penicillin is used: 45% (human), 15% (animal health) and 45% for production of semi synthetic penicillin

P. notatum, P.chrysogenum and its mutant strain which is a high yeilding strain (Q176)

Genetically engineered strains for improved pencillin production are being used now

slide50

UDP tansfers NAG to bactoprenol-NAM peptapeptide. For pentaglycine use special glycyl-tRNA moc but not ribosomes

UDP deriv of NAM and NAG are synthesized

Transport of completed NAM-NAG-pepntapeptide across membrane

Sequentially aa are added to UDP-NAM to form NAM -pentapeptide

ATP is used, no tRNA or ribosomes involved in peptide bond formation

Transfer of UDP-NAM-pentapeptideto bactoprenol PO4

LIPID I

LIPID II

Bactoprenol carrier moves back across membrane by losing one PO4 for a new cycle

Attached to growing end of PG chain and incr by one repeat unit

slide51

Bactoprenol is a 55 carbon alcohol and linked to NAM by pyrophosphate

In S. aureus pepntapeptide has L-lys and in

E. coli DAP

UDP glucose

slide52

Final step is TRANSPEPTIDATION which creates peptide cross links between PG chains. The enzyme removes terminal D-alanine as cross link is formed

slide53

The b-lactam group of antibiotics includes an enormous diversity of natural and semi-synthetic compounds that inhibit several enzymes associated with the final step of peptidoglycan synthesis.

All of this enormous family are derived from a b-lactam structure: a four-membered ring in which the b-lactam bond resembles a peptide bond. The multitude of chemical modifications based on this four-membered ring permits the astonishing array of antibacterial and pharmacological properties within this valuable family of antibiotics.

Clinically useful families of b-lactam compounds include the penicillins, cephalosporins, monobactams and carbapenems. Many new variants on the b-lactam theme are currently being explored. Certain b-lactams have limited use directly as therapeutic agents, but may be used in combination with other b-lactams to act as b-lactamase inhibitors.

Co-amoxyclav, for example is a combination of amoxycillin and the b-lactamase inhibitor clavulanic acid. During cross-linking of the peptidoglycan polymer, one D-alanine residue is cleaved from the peptidoglycan precursor and this reaction is prevented by b-lactam drugs.

More recent studies have shown that the activity of this class of drugs is more complicated and involves other processes as well as preventing cross-linking of peptidoglycan.

slide54

B-lactamase

An increasing number of bacteria are penicillin resistant. Penicillinase-resistant penicillins such as methicillin, nafcillin, and oxacillin are frequently employed against these bacterial pathogens.

Although penicillins are the least toxic of the antibiotics, about 1 to 5% of the adults in the United States are allergic to them. Occasionally a person will die of a violent allergic re- sponse; therefore patients should be questioned about penicillin allergies before treatment is begun.

slide56

MRSA

VRSA

slide57

CEPHALOSPORINS

Cephalosporium: Cephalosporin C

Dihydrothiazine ring (6 member)

B-lactam ring

cefatrioxone

Same mode of action with broader spectrum than penicillins

Resistant to b-lactamases

Hence used to treat infections which are penicillin resistant

Used to treat Nesseria gonorrhea (STD)

slide58

Most cephalosporins (including cephalothin, cefoxitin, ceftri- axone, and cefoperazone) are administered parenterally.

Cefoperazoneis resistant to destruction by b-lactamases and effective against many gram-negative bacteria, including Pseudomonas aeruginosa.

Cephalexineand cefixime are given orally rather than by injection.

7-ACA: 7- aminocephalosporanic acid nucleus structure in all cephalosporins

slide59

G+ = G-

G+ > G-

R1

R2

G+ < G-

slide60

TETRACYCLINES

  • Broad spectrum
  • Effective for G+ and G- (mycoplasmas, rickettesia, chlamydia)
  • Used for combatting stomach ulcer (Helicobacter pylori)
  • Inhibit protein synthesis by blocking binding of amino acyl tRNA to ribosome (A site)

BASIC STRUCTURE

  • Napthacene ring
  • Chlortetracycline and oxytetracycline are most commonly used in human and veterinary diseases and for preservation of meat, fish and poultry

Three members of the tetracycline family. Tetracycline lacks both of the groups that are shaded. Chlortetracycline (aureomycin) differs from tetracycline in having a chlorine atom (blue); doxycycline consists of tetracycline with an extra hydroxyl (purple).

slide61

Streptomyces aureofaciens

20 diff species producing mix of tet

Genetic modification

Polyketide synthesis

TETRACYCLINES

Str. aureus. S.flavus

S. rimosus, S. antibioticus

Antibiotics synthesized by successive condensation of small carboxylic acids

Like acetate, butyrate, propionate, malonate

High doses of tetracycline may result in nausea, diarrhea, yellowing of teeth in children, and damage to the liver and kidneys.

slide62

Oligosaccharide antibiotics

AMINOGLYCOSIDES

  • Structurally all contain a cyclohexane ring and amino sugars bound by glycosidic linkages
  • Bind to the 30S small ribosomal subunit and interfere with protein synthesis in at least two ways. They directly inhibit protein synthesis and also cause misreading of the genetic message carried by mRNA…prolonged use can cause kidney damage and hearing loss

Streptomycin, kanamycin, neomycin, and tobramycin are synthesized by Streptomyces, whereas gentamicin comes from a related bacterium, Micromonosporapurpurea.

Known as reserve antibiotics as they develop resistance quickly

slide64

AMINOGLYCOSIDES producing organisms

Streptomycin Streptomyces griesus

Neomycin B and C S.fradiae

Kanamycin A, B and C S.kanamyceticus

Hygromycin B S.hygroscopicus

Gentamycin Micromonosporapurpurea

SisimicinM.inyoensis

slide65

MACROLIDES

Antibiotics with a large lactone ring (macrocyclic lactone ring)

Which consists of 12-, 14- and 16-membered lactone rings with 1-3 sugars linked by glycosidic bond

Effective agaist penicillin resistant MO, G+ org, inhibitb y binding to 50S ribosome

Clarithromycin (Erythromycin derv)

Used to treat stomach ulcers

Erythromycin : Streptomyces erythreus

14-membred connected to 2 sugars

Genetic modifications by polyketide synthesis

slide66

MACROLIDES

Polyene macrolides: lactone rings in range of 26-28

Eg. Nystatin, amphotericin

Actinomycetes are most common organisms which produce them

Erythromycin is a relatively broad-spectrum antibiotic effective against gram-positive bacteria, mycoplasmas, and a few gram-negative bacteria. It is used with patients allergic to penicillinsand in the treatment of whooping cough, diphtheria, diarrhea caused by Campylobacter, and pneumonia from Legionella or Mycoplasma infections.

Newer macrolides are now in use.

Clindamycinis effective against a variety of bacteria including staphylococci and anaerobes such as Bacteroides.

Azithromycinis particularly effective against Chlamydia trachomatis.

slide67

Aromatic rings in structure

Chloroamphenicol, griesofluvin, novobiocin

AROMATIC ANTIBIOTICS

CHLORAMPHENICOL

Broad spectrum antibiotic against G+ and G- bacteria, rickettesia, chlamydia, actinomycetes

chloramphenicol binds to 23S rRNA on the 50S ribosomal subunit. It inhibits the peptidyltransferase and is bacteriostatic.

Streptomyces venezuelae and S.omiyanesis

This antibiotic has a very broad spectrum of activity but unfortunately is quite toxic. One may see allergic responses or neurotoxic reactions. The most common side effect is a temporary or permanent depression of bone marrow function, leading to aplastic anemia and a decreased number of blood leukocytes. Chloramphenicol is used only in life-threatening situations when no other drug is adequate.

Penicilliumpatulum

GRIESOFULVIN

Maybe attacks chitin biosynthesis hence acts as anti fungal antibiotic

slide68

Following a 40-year hiatus in discovering new classes of antibacterial compounds, three new classes of antibacterial antibiotics have been brought into clinical use:

Cyclic lipopeptides (Daptomycin), Glycylcyclines (tigecycline) and Oxazolidinones (Linezolid)

PEPTIDE ANTIBIOTICS

Daptomycin : Streptomycesroseosporususedtotreat MDR infections

Tigecycline: Tygacil® marketed by Wyeth usedtotreat MDR strainsof

Staphylococcusaureus and Acineotobacterbaumanii. Mechanismsimilartotetracycline.

Also shows suceptibilityto NDML (New Delhi metallo-b-lactamasemultidrugresistantEnterobacteriaceae)

NDML is an enzyme which makes bacteria resistant to broad range of b-lactam antibiotics.

This includes antibiotics of carbapenems for treatment of antibiotics resistant infections.

Termed as “SUPERBUGS” Such bacteria susceptible to polymixins and tigecyclines

slide69

MECHANISM OF DRUG RESISTANCE

Plasmids

R-Plasmids

Superinfection: Clostridium difficile, Candida albicans

Transformation, conjugation, transduction, ABC transporters

Phage therapy

There has been some recent progress in developing new antibiotics that are effective against drug-resistant pathogens.

Two new drugs are fairly effective against vancomycin-resistant enterococci. Synercid is a mixture of the streptogramin antibiotics quinupristin and dalfopristinthat inhibits protein synthesis.

A second drug, linezolid (Zyvox), is the first drug in a new family of antibiotics, the oxazolidinones. It inhibits protein synthesis and is active against both vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus.