Penicillin are the best known and probably the most important antibiotics.They have been approved for human use and they account for over most of the antibiotics produced worldwide.Penicillin was discovered by Fleming in 1929. Natural penicillin are effective against numerous gram positive bacteria. They are produced by many fungi, particularly Penicillin and Aspergillus species. They present the most favourable characteristics of being almost non toxic to mammals.

Penicillin, being β-lactum antibiotics, are specific inhibitors of bacterial cell wall synthesis. They target the synthesis of peptidoglycan by specifically binding to penicillin binding protein (PBP) of bacterial cell wall. This binding inhibits the enzyme activity of this protein. The penicillin have a common chemical nucleus and differ principally in the chemical structure of side chain attached to this nucleus. The various penicillin fermentation also are unusual in that various compounds resembling side chain can be added as precursors to the fermentation medium, and these compounds through microbial action, are directly incorporated in the penicillin molecule. Also, the side chain can be enzymatically removed liberating the penicillin nucleus, so that unnatural side chains can be chemically added to the nucleus in order to create new penicillin.

Chemical Structure of Penicillin:

The basic structure of penicillin is 6-aminopenicillanic acid (6-APA). it consists of a thiazolidine ring with a condensed β-lactum ring. The 6-APA ring carries valuable acyl moiety in position 6. If the penicillin fermentation is carried out without addition of side chain precursors, the natural penicillin are produced. From this mixture, only benzylpenicillin is therapeutically useful. About 38% of the penicillin produced commercially are used in human medicine, 12% in veterinary medicine and 48% are used as starting material for the production of semi synthetic penicillin.

The fermentation of penicillin can be better controlled by adding a side chain precursors, so that only one desired penicillin is produced. Addition of corn steep liquor to the medium increases the total yield of penicillin. Corn steep liquor contains phenylalanine and its breakdown products phenylethylamine and phenyl acetic acid, which when added as precursors directs the mold synthesis towards required penicillin. It also minimises the recovery problem of separating the unwanted penicillin.

Bio-synthesis of penicillin:

The β-lactum ring is constructed from L-cysteine and L-valine. Bio-synthesis occurs in a non-ribosomal process by means of L-α aminoadipic acid and L-cysteine.

The penicillin are unstable molecules when present in free acid form and therefore are usually prepared as much more stable salts or esters. At low pH values, the penicillin rearrange to give biologically inactive isomers, and at strongly alkaline pH, particularly in presence of zinc and copper, penicillin is hydrolyzes to penicillianic acid.

Selection of culture:

1) Many strains of Penicillium notatum and Penicillium chrysogenum were tested in an effort to find which one would produce good yields in submerged culture process. One of these Penicillium chrysogenum NRRL 1951 was found to be a superior producer.

2) The strain was adopted by most of the penicillin manufacturers and the monospores isolated from it are the parent cultures of those now in use.

3) Descendants of these cultures have many properties, they produce 10 times as much penicillin.

4) The penicillin molds are characterized by unusual variability, the greater the productivity of strain, the less stable it is.

5)  Stock cultures can be maintained on agar slants, in dry soil, in lyophilized form or as spore or as cell suspension stored in liquid nitrogen.

6) Stocks carried on agar most liable to variation. Frequent transfer tend to propagate selectively those portions of the culture population that sporulate more readily.

Culture medium:

The medium of  a typical feed batch culture may vary depending on strain and usually consists of corn steep liquor, an additional nitrogen source such as soy meal, yeast extract, or whey, a carbon source such as lactose and various buffers. Phenyl acetic acid are used as precursors. Inoculum medium is similar to production medium except lactose and precursors are not included. The medium constituents have profound effect on penicillin yield. The corn-steep liquor provides peptides, amino acids and amines which are deaminated to provide the ammonia required in the early stages of fermentation. The glucose is rapidly utilized to provide mycelial growth but allows very little penicillin production. About 65% of the metabolizes carbon source is utilized for maintaining energy, 25% for growth and only 10% for penicillin production.

The lactose is only slowly degraded to glucose and it is this slow glucose availability for lactose that allows starvation conditions required for penicillin production. Lipid nutrients are also utilized by fungus during penicillin production and fatty acids and fatty oils are also effective. Some oils are added as anti-foam reagent. These nutrients increases both the amounts of mycelium and yields.

Production of penicillin:

1) Commercial production of penicillin is usually via a fed batch process carried out aseptically in stirred tank reactor.

2) The fermentation involves an initial vegetative growth phase followed by antibiotic production phase.

3) For inoculum production, spores from heavily sporulated working stocks are suspended in water.

4) These spores are then added to flasks of wheat bran plus nutrient solution and these are incubated for 5-7 days at 24°c, so as to provide heavy sporulation. The resulting spores are then used directly to inoculate inoculum tanks. These tanks are equipped with air spargers, agitators, cooling coils for temperature control and anti-foam addition devices.

5) The inoculum tanks are incubated for 24-48 hrs with aeration and agitation in order to obtain heavy mycelial growth.

6) After several stages of growth, the production culture is ready. In a typical penicillin fermentation, there is growth phase of about 40 hrs of duration with a doubling time of 6 hrs. During this period, the greatest part of the cell mass is formed. During the first 20-30 hrs, the fungal growth becomes very thick and heavy. The O2 supply in the growing culture is critical, since the increasing viscosity hinders O2 transfer.

7) This resulting inoculum is then inoculated in the production tank. These tanks are equipped with devices for continuous addition of sterile glucose syrups, pH control, foam sensing devices to activate automatic addition of anti-foams and metering pumps for continuous addition of sterile phenyl acetic acid.

8) The production tanks are inoculated by employing air pressure to force inoculum in the tank.

9) During production, periodic samples are removed for determination of penicillin yields and for contamination checks. This contamination checks are important, because penicillin fermentation are quite sensitive to contamination by penicillinase producing organisms.

10) The pH remains constant at the start of fermentation. But as soon as the carbon compounds becomes depleted and some of the lactic acid of corn steep liquor is utilized, ammonia is liberated and pH rises. The mold uses lactose to produce penicillin and a very little further growth occurs.

11) At the end of fermentation, the pH rises to a more higher level because depletion of lactose causes autolysis of the mycelium. the penicillin fermentation is harvested at this time.

Purification and recovery:

Penicillin in the acid form is solvent extractable, and the antibiotic dissolved in an organic solvent can be back extracted as a salt in aqueous solution. these considerations in general are made use of for the recovery and purification of penicillin from harvested broth.

Steps of purification and recovery:

1) The penicillin broth is harvested from the fermenter and chilled at 5-10°c.

2) The Penicillium chrysogenum mycelium is then filtered on a rotary vacuum filter to remove the mycelium and the other solids.

3) Phosphoric acid are added to lower the pH, in order to convert the penicillin to the anionic form.

4) The broth is immediately extracted in counter current extract with an organic solvent such as butyl acetate.

5) The penicillin is then back extracted into water from the organic solvent by adding enough potassium or sodium hydroxide to form a salt of the penicillin.

6) the resulting aqueous solution is again acidified and re extracted with butyl acetate. this shift between water and solvent aid in purification of penicillin.

7) The solvent extract finally is back extracted with aqueous potassium  to crystallize penicillin as potassium penicillin salt.

The penicillin thus crystals thus obtained are mixed with volatile solvents to remove further impurities. the crystals are collected by filtration and air dried. At this stage the penicillin obtained is 99.5% pure.

 

 


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