1. Secondary metabolites

2. Metabolites are involved in the growth of organisms through the process of metabolism. The metabolism is referred to as the sum of the all the biochemical reactions carried out by an organism. Depending on the origin and function, metabolites can be divided into two major categories; namely, Primary and Secondary metabolites.

3. Primary metabolites
Primary metabolites are essential to the growth of the cell. They are produced continuously during the growth phase and are involved in primary metabolic processes such as respiration and photosynthesis. Primary metabolites, which are identical in most organisms, include sugars, amino acids, tricarboxylic acids, the universal building blocks, and energy sources. Other than the above compounds, proteins, nucleic acids, and polysaccharides are also considered as primary metabolites.

4. Secondary metabolite
Secondary metabolites are the compounds which are derived by pathways from primary metabolic routes, and are not essential to sustain the life of cells. These compounds do not have a continuous production. Very often secondary metabolites are produced during non- growth phase of cells. Secondary metabolites are the end products of primary metabolites such as alkaloids, phenolics, steroids, essential oils, lignins, resins and tannins etc.

5. Differences between primary and secondary metabolites
• Unlike secondary metabolites, Primary metabolites are essential to cell growth, and they are involved directly in metabolic reactions such as respiration and photosynthesis. • Most primary metabolites are identical among most organisms, whereas secondary metabolites are numerous and wide spread, unlike the primary metabolites. • Secondary metabolites are derived by pathways in which primary metabolites involve. Therefore, secondary metabolites are considered as the end products of primary metabolites.

6. Cont…
• Primary metabolites are produced during the growth phase of cell while secondary metabolites are produced during the non- growth phase of the cell.
• Secondary metabolites are accumulated by plant cells in very small quantities than primary metabolites.
• The growth phase where primary metabolites are produced is sometimes called ‘trophophase’, whereas the phase during which secondary metabolites are made is called ‘idiophase’.

7. Cont…
• Most of the secondary metabolites are involved in defense reactions, unlike the primary metabolites. • Proteins, carbohydrates, and lipids are the main primary metabolites, whereas secondary metabolites are alkaloids, phenolics, sterols, steroids, essential oils and lignins etc.

8. Secondary Metabolites-Antibiotics
Most of the antibiotics were discovered from soil microorganisms especially in microorganism Streptomyces spp.

9. Antibiotics are chemicals produced by mainly microorganisms (also synthetically and semi synthetically) and which in low concentrations are capable of inhibiting the growth of, or killing, other microorganisms.
Broadened by some authors to include materials produced by living things – plants, animals or microorganisms – which inhibit any cell activity.
Antibiotics may be wholly produced by fermentation.
Semi-synthetic processes, in which a product obtained by fermentation is modified by the chemical introduction of side chains.

10. Some wholly chemically synthesized compounds are also used for the chemotherapy of infectious diseases e.g. sulfonamides and quinolones.
Some antibiotics e.g. chloramphenicol were originally produced by fermentation, but are now more cheaply produced by chemical means.
Only a small proportion of known antibiotics is used clinically, because the rest are too toxic.

11. Classification of Antibiotics
The classification to be adopted here is based on the chemical structure of the antibiotics and classifies antibiotics into 13 groups.
This enables the accommodation of new groups as they are discovered.

12. Grouping of antibiotics based on their chemical structures

13. The nomenclature of antibiotics
The same antibiotic may have as many as 13 different trade names depending on the manufacturers.
Antibiotics are therefore identified by at least three names:
The chemical name, which prove long and is rarely used except in scientific or medical literature;
The group, generic, or common name, usually a shorter from of the chemical name or the one given by the discoverer;
The trade or brand name given by the manufacturer to distinguish it from the product of other companies.

14. Some Antibiotics
Produced By
Microorganisms

15. BETA-LACTAM ANTIBIOTICS
The Beta-lactam antibiotics are so-called because they have in their structure the four membered lactam ring.
A lactam is a cyclic amide.
It is named as such, because the nitrogen atom is attached to the β-carbon relative to the carbonyl.
The Beta-lactam antibiotics inhibit the formation of the structure-conferring peptidoglycan of the bacterial cell wall.
As this component is absent in mammalian cells, Beta-lactam antibiotics have very low toxicity towards mammal

17. The Beta-lactam antibiotics include the well-established and clinically important penicillins and cephalosphorins as well as some relatively newer members: cephamycins, nocardicins, thienamycins, and clavulanic acid.
Except in the case of nocardicins these antibiotics are derivatives of bicyclic ring systems in which the lactam ring is fused through a nitrogen atom and a carbon atom to ring compound.
This ring compound is five-membered in penicillins (thiazolidine), thienamycins (pyrroline) and clavulanic acid (oxazolidine);
It is six-membered (dihydrothiazolidine) in cephalosporins and cephamycins.

18. The commercial production of penicillin

19. First discovered by Fleming in 1928
19% of worldwide antibiotic market
Superior inhibitory action on bacterial cell wall synthesis
Broad spectrum of antibacterial activity
Low toxicity
Outstanding efficacy against various bacterial strains
Excessive use has led to development of resistant pathogens

21. Characteristics of secondary metabolites:
Secondary metabolites (idiolites) are produced from
Substrates provided by primary metabolism.
Characteristics of secondary metabolites:
They are not essential for growth and reproduction.
Their formation is extremely dependent on growth conditions.
It is possible to get dramatic overproduction of secondary metabolites.

22. COMMERCIAL PRODUCTION OF PENICILLIN
Originally used Penicillium notatum , now use Penicillium chrysogenum.
Initially produced via surface mat culture.
Problems!
Inefficient, slow penicillin synthesis and contamination.

23. DEEP LIQUID CULTURE
Inoculum prepared until it represents ~ 5-10% of the volume of the fermenter. About 3-5 tonnes of wet mycelial mass will be used to inoculate a 50,000 litre fermenter.
 The fermenters vary from 38, ,000 litres.
Three distinct phases:
 1. Trophophase: Rapid mycelial growth (30-40 hrs)
Idiophase: Penicillin production via fed batch fermentation (5-7 days).

24. 3. C and N depletion phase:Carbon and nitrogen sources are depleted, antibiotic production ceases, the mycelia lyse releasing ammonia and the pH rises.
Fermenter cooled by internal coils or external jackets (25-27oC).
The pH is maintained between by the automatic addition of H2SO4 or NaOH as necessary. 
Oxygen added and mixed with mycelium.

25. Composition of early media % corn steep liquor (cotton seeds, peanut,
CULTURE MEDIUM
Composition of early media %
corn steep liquor (cotton seeds, peanut,
Linseed or soybean meals) 2-4
lactose, glucose or beet molasses 2-4
CaCO3 or phosphates (buffer)
precursor
Catabolite repression of the enzymes responsible for penicillin biosynthesis
Occurs in high concentrations of glucose.
Use of precursors to increase penicillin yield. 

27. Precursors of the appropriate side-chain are added to the fermentation.
Thus if benzyl penicillin (penicillin G) is desired, phenylacetic acid is added.
Phenyl acetic acid is nowadays added continuously as too high an amount inhibits the development of the fungus.
High yielding strains of P. chrysogenum resistant to the precursors have therefore been developed.

28. Use of precursors:

29. Extraction of penicillin after fermentation
The broth is transferred to a settling tank.
Penicillin is highly reactive and is easily destroyed by alkaline conditions (pH ) or by enzymes.
It is therefore cooled rapidly to 5-10°C.
The separation of penicillin is based on the solubility, adsorption and ionic properties of penicillin.
Since penicillins are monobasic carboxylic acids they are easily separated by solvent extraction.

30. The fermentation broth is filtered with a rotary vacuum filter to remove mycelia and other solids and the resulting broth is adjusted to about pH 2 using a mineral acid.
It is then extracted with a smaller volume of an organic solvent such as amyl acetate or butyl acetate, keeping it at this very low pH for as short a time as possible.
The aqueous phase is separated from the organic solvent usually by centrifugation.

31. The organic solvent containing the penicillin is then typically passed through charcoal to remove impurities, after which it is back extracted with a 2% phosphate buffer at pH 7.5.
The penicillin is then acidified once again with mineral acid (phosphoric acid) and the penicillin is again extracted into an organic solvent (e.g. amyl acetate).
The product is transferred into smaller and smaller volumes, the penicillin becomes concentrated several times over, up to times.

32. The penicillin may be converted to a stable salt form in one of several ways which employ the fact that penicillin is an acid:
(a) it can react with a calcium carbonate slurry to give the calcium salt which may be filtered, lyophilized or spray dried.
(b) it may react with sodium or potassium buffers to give the salts of these metals which can also be freeze or spray dried;
(c) it may precipitate with an organic base such as triethylamine.

33. NATURAL PENICILLINS They are destroyed by acid in the stomach.
Sensitive to the enzyme penicillinase
Effective against Gram +ve bacteria only.

34. SEMISYNTHETIC PENICILLINS

35. Chemical and Enzymatic Deacylation
of Penicillins to 6-APA H R C N S S
CH3
CH3
NH2
Penicillin acylase
CH3
CH3 O
Alkaline N N
COOH
[Enzymatic]
COOH O O
Penicillin V or G
(6-APA)
[R=Ph or PhO]
[Chemical]
PCl5
ROH
H2O
Pyridine
Me3SiCl H R C N S
CH3
CH3 O N
COOSiMe3 O

36. 6-Aminopenicillanic Acid (6-APA)
Penicillin:
6-APA: Raw material for production of new semisynthetic 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

37. 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, hours

38. Penicillin G
Penicillinase (E.coli)
6 - APA
Side Chain Modification
Amoxycillin
AUGMENTIN
b-lactamase
resistant
Clavulanic acid

41. Antibiotic Production
Development of pilot plant production methods
Submission of licence for clinical trials
Testing of purified antibiotic
Development of plant scale production methods
Obtaining a product licence for clinical use
Other considerations:
Development of methods to control production of antibiotic
Development of new applications
Development of marketing and distribution system
Financing of business
Isolation or collection of cultures
Screening of cultures to detect those with antimicrobial activity
Development of methods for submerged-culture production
Development of methods for isolation and purification of antibiotic
Determination of antibiotic properties (physical: adsorption and absorption, chemical: reactions, solubility in solvents, stability to acids, alkalis, heat etc.)
Evaluation of antibiotic
Pharmacological tests
Antimicrobial activity
Comparison with existing antibiotic

42. The classical method for searching for antibiotics
By random search in the soil.
Although the first important commercially produced antibiotic was discovered by chance, most present day antibiotics were discovered by systematic search.
The soil is a vast repository of microorganisms and it is to the soil that search is turned when antibiotics are being sought.
The stages to be discussed below are not necessarily rigidly followed; they are merely meant to indicate in a general manner some of the activities involved in the development of antibiotics.

43. (i) The primary screening
Several methods have been employed in primary screening.
The crowded plate
The direct-soil-inoculation method
The cross-streak method
The agar plug method
The replica plating method

44. (a) The crowded plate
A heavy aqueous suspension of soil is plated on agar.
Organisms showing clear zones around themselves are isolated for further study.
This method has the disadvantage that slow-growing antibiotic-producing organisms such as actinomycetes are usually over grown and are therefore hardly isolated.
The susceptibility of soil organisms to the antibiotics produced in the test, may be unrelated to the susceptibility of clinically important organisms.

45. (b) The direct-soil-inoculation method
This method is used when the aim is to isolate antibiotics against a known organism or organisms.
Pour plates containing the test organisms are prepared.
Soil crumbs or soil dilutions are then placed on the plates.
Antibiotic producing organisms develop which then inhibit the growth of the organisms in the plate.
They are recognized by the cleared zone which they produce around themselves and they may then be picked out.

46. (c) The cross-streak method
This method is used for testing individual isolates, especially actinomycetes which may be obtained from soil without any previous knowledge of their antibiotic-producing potential.
The organism may come from one of the two methods already indicated above.
The purified isolate is streaked across the plate containing a medium which supports its growth as well as that of the test organisms.
A variety of media may be used for streaking the antibiotic producer.
It is allowed to grow for up to seven days, in which time any antibiotic produced would have diffused a considerable distance from the streak.
Test organisms are streaked at right angles to the original isolates and the extent of the inhibition of the various test organisms observed.

47. (d) The agar plug method
This method is particularly useful when the test organism grows poorly in the medium of the growth of the isolate such as fungi.
Plugs about 0.5 cm in diameter are made with a sterile cork borer at progressive distances from the fungus.
These plugs are then placed on plates with pure cultures of different organisms.
The diameters of zones of clearing are used as a measure of antibiotic production of the isolate.
The method may be used with actinomycetes.

48. Secondary screening
Organisms showing suitably wide zones of clearing against selected target organisms are cultivated in broth culture in shake flasks using components of the solid medium in which the isolate grew best.
Crude methods of isolating the active antibiotic are developed by extracting the broth using a wide range of extractive methods.
With each extraction the resultant material is assessed for activity against the target organisms at various dilutions.
The extract is either spotted on filter paper discs placed on agar seeded with the test organism or introduced into wells dug out from the seeded agar with sterile cork borers.

49. The most efficient extractive methods and the spectrum of activity of the organisms are determined.
Secondary screening is aimed at eliminating at an early stage any antibiotic which does not appear promising either by virtue of low activity, other undesirable properties or because it has been discovered previously.

50. (vi) Plant production:
The production plant utilizes all the information obtained in the pilot experimentation.
(vii) Certification:
A government agency must approve the antibiotic before it becomes available for general use.
(viii) Marketing and financing

51. Towards a new definition of ‘antibiotic’
The current definition of the term ‘antibiotic’ which restricts them to chemicals produced by microorganisms
However, the higher organisms have been shown to produce anti-microbial substances.
Such substances are low molecular weight secondary metabolites in the same way as regular antibiotics are.
Due to this, there is now a tendency to extend the term antibiotic to all secondary metabolites, irrespective of their origin, which are able to inhibit various growth processes at low concentration.
Even wholly synthetic antimicrobials such as ciprofloxacin are now legitimately termed antibiotics.
The word antibiotic derives from two origins, anti (against) and bios (life). Nothing in the word itself restricts antibiotics both in origin or in use to microbial life.