SECONDARY METABOLITES
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Metabolites are involved in the growth oforganisms through the process of metabolism.The metabolism is referred to as the sum ofthe all the biochemical reactions carried outby an organism. Depending on the origin andfunction, metabolites can be divided into twomajor categories; namely, Primary andSecondary metabolites.
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Primary metabolites
Primary metabolites are essential to the growth ofthe cell. They are produced continuously during thegrowth phase and are involved in primary metabolicprocesses such as respiration and photosynthesis.Primary metabolites, which are identical in mostorganisms, include sugars, amino acids, tricarboxylicacids, the universal building blocks, and energysources. Other than the above compounds, proteins,nucleic acids, and polysaccharides are alsoconsidered as primary metabolites.
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Secondary metabolite
Secondary metabolites are the compounds which arederived by pathways from primary metabolic routes,and are not essential to sustain the life of cells. Thesecompounds do not have a continuous production.Very often secondary metabolites are producedduring non- growth phase of cells. Secondarymetabolites are the end products of primarymetabolites such as alkaloids, phenolics, steroids,essential oils, lignins, resins and tannins etc.
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Differences between primary andsecondary metabolites
• Unlike secondary metabolites, Primary metabolites areessential to cell growth, and they are involved directly inmetabolic reactions such as respiration and photosynthesis.
• Most primary metabolites are identical among mostorganisms, whereas secondary metabolites are numerous andwide spread, unlike the primary metabolites.
• Secondary metabolites are derived by pathways in whichprimary metabolites involve. Therefore, secondarymetabolites are considered as the end products of primarymetabolites.
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Cont…
• Primary metabolites are produced during thegrowth phase of cell while secondary metabolites areproduced during the non- growth phase of the cell.
• Secondary metabolites are accumulated by plantcells in very small quantities than primarymetabolites.
• The growth phase where primary metabolites areproduced is sometimes called ‘trophophase’,whereas the phase during which secondarymetabolites are made is called ‘idiophase’.
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Cont…
• Most of the secondary metabolites areinvolved in defense reactions, unlike theprimary metabolites.
• Proteins, carbohydrates, and lipids are themain primary metabolites, whereas secondarymetabolites are alkaloids, phenolics, sterols,steroids, essential oils and lignins etc.
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•Most of the antibiotics were discoveredfrom soil microorganisms especially inmicroorganism Streptomyces spp.
Secondary Metabolites-Antibiotics
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•Antibiotics are chemicals produced by mainlymicroorganisms (also synthetically and semisynthetically) and which in low concentrations arecapable of inhibiting the growth of, or killing, othermicroorganisms.
•Broadened by some authors to include materialsproduced by living things – plants, animals ormicroorganisms – which inhibit any cell activity.
•Antibiotics may be wholly produced by fermentation.
•Semi-synthetic processes, in which a productobtained by fermentation is modified by the chemicalintroduction of side chains.
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•Some wholly chemically synthesized compoundsare also used for the chemotherapy of infectiousdiseases e.g. sulfonamides and quinolones.
•Some antibiotics e.g. chloramphenicol wereoriginally produced by fermentation, but are nowmore cheaply produced by chemical means.
•Only a small proportion of known antibiotics isused clinically, because the rest are too toxic.
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Classification of Antibiotics
•The classification to be adopted here isbased on the chemical structure of theantibiotics and classifies antibiotics into 13groups.
•This enables the accommodation of newgroups as they are discovered.
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Grouping of antibiotics based ontheir chemical structures
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The nomenclature of antibiotics
•The same antibiotic may have as many as 13 differenttrade names depending on the manufacturers.
•Antibiotics are therefore identified by at least threenames:
1.The chemical name, which prove long and is rarelyused except in scientific or medical literature;
2.The group, generic, or common name, usually ashorter from of the chemical name or the one givenby the discoverer;
3.The trade or brand name given by the manufacturer todistinguish it from the product of other companies.
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Some Antibiotics
Produced By
Microorganisms
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BETA-LACTAM ANTIBIOTICS
•The Beta-lactam antibiotics are so-called because theyhave in their structure the four membered lactam ring.
•A lactam is a cyclic amide.
•It is named as such, because the nitrogen atom isattached to the β-carbon relative to the carbonyl.
•The Beta-lactam antibiotics inhibit the formation of thestructure-conferring peptidoglycan of the bacterial cellwall.
•As this component is absent in mammalian cells, Beta-lactam antibiotics have very low toxicity towardsmammal
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•The Beta-lactam antibiotics include the well-established and clinically important penicillins andcephalosphorins as well as some relatively newermembers: cephamycins, nocardicins, thienamycins,and clavulanic acid.
•Except in the case of nocardicins these antibiotics arederivatives of bicyclic ring systems in which thelactam ring is fused through a nitrogen atom and acarbon atom to ring compound.
•This ring compound is five-membered in penicillins(thiazolidine), thienamycins (pyrroline) and clavulanicacid (oxazolidine);
•It is six-membered (dihydrothiazolidine) incephalosporins and cephamycins.
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The commercial productionof penicillin
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•First discovered by Fleming in 1928
•19% of worldwide antibiotic market
•Superior inhibitory action on bacterial cell wallsynthesis
•Broad spectrum of antibacterial activity
•Low toxicity
•Outstanding efficacy against various bacterialstrains
•Excessive use has led to development ofresistant pathogens
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Secondary metabolites (idiolites) are produced from
Substrates provided by primary metabolism.
Characteristics of secondary metabolites:
• They are not essential for growth andreproduction.
•Their formation is extremely dependent ongrowth conditions.
•It is possible to get dramatic overproduction ofsecondary metabolites.
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COMMERCIAL PRODUCTION OF PENICILLIN
Originally used Penicillium notatum , now usePenicillium chrysogenum.
Initially produced via surface mat culture.
Problems!
Inefficient, slow penicillin synthesis andcontamination.
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DEEP LIQUID CULTURE
•Inoculum prepared until it represents ~ 5-10% of thevolume of the fermenter. About 3-5 tonnes of wetmycelial mass will be used to inoculate a 50,000 litrefermenter.
• The fermenters vary from 38,000-380,000 litres.
•Three distinct phases:
1.Trophophase: Rapid mycelial growth (30-40 hrs)
2.Idiophase: Penicillin production via fed batchfermentation (5-7 days).
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3. C and N depletion phase:Carbon andnitrogen sources are depleted, antibioticproduction ceases, the mycelia lysereleasing ammonia and the pH rises.
•Fermenter cooled by internal coils orexternal jackets (25-27oC).
•The pH is maintained between 6.8-7.4 bythe automatic addition of H2SO4 or NaOHas necessary.
•Oxygen added and mixed with mycelium.
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CULTURE MEDIUM
Composition of early media%
corn steep liquor (cotton seeds, peanut,
Linseed or soybean meals)2-4
lactose, glucose or beet molasses2-4
CaCO3 or phosphates (buffer)0.5-1
precursor0.1-0.5
Catabolite repression of the enzymes responsible forpenicillin biosynthesis
Occurs in high concentrations of glucose.
Use of precursors to increase penicillin yield.
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•Precursors of the appropriate side-chain areadded to the fermentation.
•Thus if benzyl penicillin (penicillin G) isdesired, phenylacetic acid is added.
•Phenyl acetic acid is nowadays addedcontinuously as too high an amount inhibitsthe development of the fungus.
•High yielding strains of P. chrysogenumresistant to the precursors have thereforebeen developed.
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Use of precursors:
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Extraction of penicillin afterfermentation
•The broth is transferred to a settling tank.
•Penicillin is highly reactive and is easily destroyedby alkaline conditions (pH 7.5-8.0) or by enzymes.
•It is therefore cooled rapidly to 5-10°C.
•The separation of penicillin is based on thesolubility, adsorption and ionic properties ofpenicillin.
•Since penicillins are monobasic carboxylic acidsthey are easily separated by solvent extraction.
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•The fermentation broth is filtered with arotary vacuum filter to remove mycelia andother solids and the resulting broth isadjusted to about pH 2 using a mineral acid.
•It is then extracted with a smaller volume ofan organic solvent such as amyl acetate orbutyl acetate, keeping it at this very low pHfor as short a time as possible.
•The aqueous phase is separated from theorganic solvent usually by centrifugation.
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•The organic solvent containing the penicillin isthen typically passed through charcoal toremove impurities, after which it is backextracted with a 2% phosphate buffer at pH 7.5.
•The penicillin is then acidified once again withmineral acid (phosphoric acid) and thepenicillin is again extracted into an organicsolvent (e.g. amyl acetate).
•The product is transferred into smaller andsmaller volumes, the penicillin becomesconcentrated several times over, up to 80-100times.
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•The penicillin may be converted to a stable saltform in one of several ways which employ the factthat penicillin is an acid:
(a) it can react with a calcium carbonate slurry togive the calcium salt which may be filtered,lyophilized or spray dried.
(b) it may react with sodium or potassium buffers togive the salts of these metals which can also befreeze or spray dried;
(c) it may precipitate with an organic base such astriethylamine.
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NATURAL PENICILLINS
(i)They are destroyed by acid in the stomach.
(ii)Sensitive to the enzyme penicillinase
(iii)Effective against Gram +ve bacteria only.
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SEMISYNTHETIC PENICILLINS
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Chemical and Enzymatic Deacylation
of Penicillins to 6-APA
CH3
R
C
N
H
O
N
O
S
S
O
N
CH3
COOH
Penicillin V or G
NH2
CH3
CH3
COOH
(6-APA)
Penicillin acylase
Alkaline
[Enzymatic]
CH3
R
C
N
H
O
N
O
S
CH3
COOSiMe3
[Chemical]
[R=Ph or PhO]
Pyridine
Me3SiCl
PCl5
ROH
H2O
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6-Aminopenicillanic Acid (6-APA)
Penicillin:
6-APA: Raw material for production of newsemisynthetic penicillins (amoxycillin and ampicillin)
Fewer side effects
Diminished toxicity
Greater selectivity against pathogens
Broader antimicrobial range including G- -ve
Improved pharmacological properties
Gastric acid stability & oral absorbability
Resistance to beta-lactamases
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6-Aminopenicillanic Acid (6-APA)
Chemical method:
Use of hazardous chemicals - pyridine, phosphorous
pentachloride, nitrosyl chloride
Enzymatic method:
Regio- and stereo-specific
Mild reaction conditions (pH 7.5, 37 oC)
Enzymatic process is cheaper by 10%
Enzymes:
Penicillin G acylase (PGA)- Escherichia coli, Bacillus megaterium,
Streptomyces lavendulae
Penicillin V acylases (PVA)- Beijerinckia indica var. Penicillium,
Fusarium sp., Pseudomonas acidovorans
Immobilized Enzyme:
Life, 500-2880 hours
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Penicillin G
6 - APA
Side Chain Modification
Amoxycillin
AUGMENTIN
Clavulanic acid
Penicillinase (E.coli)
-lactamase
resistant
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Antibiotic Production
•Isolation or collection of cultures
•Screening of cultures to detectthose with antimicrobial activity
•Development of methods forsubmerged-culture production
•Development of methods forisolation and purification ofantibiotic
•Determination of antibioticproperties (physical: adsorptionand absorption, chemical: reactions,solubility in solvents, stability toacids, alkalis, heat etc.)
•Evaluation of antibiotic
–Pharmacological tests
–Antimicrobial activity
–Comparison with existing antibiotic
•Development of pilot plantproduction methods
•Submission of licence for clinicaltrials
•Testing of purified antibiotic
•Development of plant scaleproduction methods
•Obtaining a product licence forclinical use
•Other considerations:
–Development of methods to controlproduction of antibiotic
–Development of new applications
–Development of marketing anddistribution system
–Financing of business
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The classical method for searchingfor antibiotics
•By random search in the soil.
•Although the first important commercially producedantibiotic was discovered by chance, most present dayantibiotics were discovered by systematic search.
•The soil is a vast repository of microorganisms and it isto the soil that search is turned when antibiotics arebeing sought.
•The stages to be discussed below are not necessarilyrigidly followed; they are merely meant to indicate in ageneral manner some of the activities involved in thedevelopment of antibiotics.
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(i) The primary screening
Several methods have been employed inprimary screening.
(a)The crowded plate
(b)The direct-soil-inoculation method
(c)The cross-streak method
(d)The agar plug method
(e)The replica plating method
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(a) The crowded plate
•A heavy aqueous suspension of soil is plated onagar.
•Organisms showing clear zones around themselvesare isolated for further study.
•This method has the disadvantage that slow-growingantibiotic-producing organisms such asactinomycetes are usually over grown and aretherefore hardly isolated.
•The susceptibility of soil organisms to the antibioticsproduced in the test, may be unrelated to thesusceptibility of clinically important organisms.
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(b) The direct-soil-inoculationmethod
•This method is used when the aim is to isolateantibiotics against a known organism or organisms.
•Pour plates containing the test organisms are prepared.
•Soil crumbs or soil dilutions are then placed on theplates.
•Antibiotic producing organisms develop which theninhibit the growth of the organisms in the plate.
•They are recognized by the cleared zone which theyproduce around themselves and they may then be pickedout.
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(c) The cross-streak method
•This method is used for testing individual isolates, especiallyactinomycetes which may be obtained from soil without anyprevious knowledge of their antibiotic-producing potential.
•The organism may come from one of the two methods alreadyindicated above.
•The purified isolate is streaked across the plate containing a mediumwhich supports its growth as well as that of the test organisms.
•A variety of media may be used for streaking the antibioticproducer.
•It is allowed to grow for up to seven days, in which time anyantibiotic produced would have diffused a considerable distancefrom the streak.
•Test organisms are streaked at right angles to the original isolatesand the extent of the inhibition of the various test organismsobserved.
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(d) The agar plug method
•This method is particularly useful when the testorganism grows poorly in the medium of the growth ofthe isolate such as fungi.
•Plugs about 0.5 cm in diameter are made with a sterilecork borer at progressive distances from the fungus.
•These plugs are then placed on plates with pure culturesof different organisms.
•The diameters of zones of clearing are used as ameasure of antibiotic production of the isolate.
•The method may be used with actinomycetes.
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Secondary screening
•Organisms showing suitably wide zones of clearingagainst selected target organisms are cultivated in brothculture in shake flasks using components of the solidmedium in which the isolate grew best.
•Crude methods of isolating the active antibiotic aredeveloped by extracting the broth using a wide range ofextractive methods.
•With each extraction the resultant material is assessed foractivity against the target organisms at various dilutions.
•The extract is either spotted on filter paper discs placedon agar seeded with the test organism or introduced intowells dug out from the seeded agar with sterile corkborers.
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•The most efficient extractive methods andthe spectrum of activity of the organismsare determined.
•Secondary screening is aimed at eliminatingat an early stage any antibiotic which doesnot appear promising either by virtue of lowactivity, other undesirable properties orbecause it has been discovered previously.
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(vi) Plant production:
•The production plant utilizes all theinformation obtained in the pilotexperimentation.
(vii) Certification:
•A government agency must approve theantibiotic before it becomes available forgeneral use.
(viii) Marketing and financing
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Towards a new definition of‘antibiotic’
•The current definition of the term ‘antibiotic’ which restricts themto chemicals produced by microorganisms
•However, the higher organisms have been shown to produce anti-microbial substances.
•Such substances are low molecular weight secondary metabolitesin the same way as regular antibiotics are.
•Due to this, there is now a tendency to extend the term antibioticto all secondary metabolites, irrespective of their origin, which areable to inhibit various growth processes at low concentration.
•Even wholly synthetic antimicrobials such as ciprofloxacin arenow legitimately termed antibiotics.
•The word antibiotic derives from two origins, anti (against) andbios (life). Nothing in the word itself restricts antibiotics both inorigin or in use to microbial life.
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