Penicillin

Penicillin | The First Antibiotic

Penicillin | The First Antibiotic

 The accidental discovery of penicillin was done by  Alexander

Flemming  in the year 1939  which was later cultured by Florey and
Chain in the year 1945.
 It was obtained from Penicillium notatum (now called Penicillium
chrysogenum )
 It is the first antibiotic  to be used clinically.

Structure

Penicillium consists of two rings.

1. Thiozolidone Ring which is sulphur containing with carboxyl group

(RING A)
2. Beta Lactum Ring (RING B)
The side chains are attached through the amide linkages.

Mechanism of Penicillin

 The bacterial cell wall  is made up of peptidoglycan that protects the cell
integrity, shape and prevents macromolecules from entering into the
cell.
 It has cross-linking chains of NAG(N-acetylglucosamine) and NAM (N-
acetylmuramic acid) components covalently attached through small
peptides
 Peptidoglycan is continuously synthesized and remodelled during growth
and division.
 Therefore bacteria assemble them into a single macromolecule by
synthesizing the peptidoglycan components.
 The peptide cross-linkages are formed by the activity of specific
enzymes called transpeptidases or Penicillin-Binding Proteins (PBPs).
 Penicillin contains a four-membered beta-lactam ring inhibits the
transpeptidase.
 By mimicking the last two D-alanine residues of the peptide, penicillin
is able to bind irreversibly the active site of the transpeptidase,
preventing the enzyme from cross-linking the peptidoglycan strands.
  This leads to cell lysis as there won’t be any formation of peptidoglycan
and the cell is no more susceptible to osmotic stress.
Penicillin G

 Penicillin G is a broad-spectrum beta-lactam naturally occurring

penicillin which is also known as  benzylpenicillin
  Benzylpenicillin is more active against  Gram-positive bacteria  in
particular cocci, such as  staphylococci , pneumococci, and
other streptococci , and bacilli, including  Bacillus anthracis, Clostridium
perfringens, and Corynebacterium diphtheriae , but less effective against
Gram-negative bacteria.
 The penicillin is less effective against Gram-negative bacteria as it has
an additional outer membrane, which acts as a selective barrier against
penicillin.
 Also, some Gram-negative bacteria have acquired specific genes which
encode for penicillinases (beta-lactamases) which inactivate penicillin
by hydrolysis of the beta-lactam ring
Second Generation Penicillin

 The Gram-positive strains later emerged a new strain producing

penicillinases.
 This lead to the discovery of second-generation penicillin called  semi-
synthetic beta-lactamase resistant penicillin.
 Ex- Oxacillins, Methicillin, Dicloxacillin
Third Generation penicillin

 The third generation penicillin is more effective against a wider group of

Gram-negative bacteria including  Haemophilus influenzae , Escherichia
coli, Salmonella spp., and Shigella spp
 This included broad-spectrum penicillins known as  aminopenicillins
 Ex-  Amoxicillin and ampicillin
 These have higher stability towards penicillinases.
 This also includes  carboxypenicillins  and ureidopenicillins  which is
effective  against Pseudomonas aeruginosa.
Administration

 Penicillin G can be taken either intravenously or intramuscularly.

 Penicillin G  administration ensures a continuous low dose of penicillin
over 2 to 4 weeks.
 Penicillin G degrades more easily by stomach acid and has a
bioavailability of less than 30%.
Adverse Effect

 Penicillin G has adverse effects that include nausea, vomiting, diarrhea,

rash, abdominal pain, urticaria, muscle spasms, fever, chills, muscle
pain, headache, tachycardia, flushing, tachypnea, and hypotension
Toxicity

 Penicillin has a small risk of toxicity compared to other biologically-

active substances
 The doses involve 5g/kg body weight intravenously to cause convulsions
in a patient.
References

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5369031/
2. https://www.ncbi.nlm.nih.gov/books/NBK554560/
3. https://microdok.com/penicillin-mechanism-of-action/

Production of Penicillin

Production of penicillin began in the United States (1941) by surface culture

fermentation of Penicillium notatum. During World War II, penicillin-
producing fungi were studied extensively to increase yields of penicillin. Now
a day, penicillin is produced from Penicillium chrysogenum by submerged
culture techniques. The morphology of Penicillium species is shown in Fig.
5.1. Penicillin is effective against Gram-positive bacteria and also some large
viruses and Rickettsia. Penicillin is a generic term applied to an entire group
of antibiotics.

The penicillin nucleus contains two amino acids, L-cysteine and D-valine (Fig.
5.2) which are closely related in structure. Penicillins are N-acyl derivatives of
6-aminopenicillanic acid (6-APA). By adding side chains to the penicillin
nucleus, new penicillins are synthesized (Fig. 5.3). The semisynthetic
penicillins are prepared from 6-aminopenicillanic acid (6-APA). Acylation of
the 6-APA is easily and efficiently brought about by chemical reactions (Fig.
5.4) Different derivatives of semisynthetic penicillin are shown in Fig. 5.5.

 
Inoculum development:

The selected strain of Penicillium chrysogenum is maintained in the form of a

master culture and preserved by lyophilization, or by fixing the spores in
sterilized soils and mixtures. For inoculum preparation, spores from working
solid cultures are suspended in water. These spores are added in flasks
containing nutrient solution and incubated for 4 to 6 days at 25°C. The
resulting spores are used directly to inoculate inoculum tank. The inoculum
tanks are incubated for 48 hours with agitation and aeration to grow more
mycelium. The resulting inoculum is used for a production tank or it is added
to a second or even third-stage inoculum tank to produce more inoculum for
large-scale fementation.

Production media:

The exact composition of penicillin production media used in the industry for
production of penicillin is unknown. These media are considered to be trade
secrets of that particular fermentation industry. A typical medium described by
Jackson (1958) contains fermentable carbohydrates, such as corn steep liquor
solids (3.5%), lactose (3.5%) and glucose (1%) potassium dihydrogen
phosphate (0.4 %), calcium carbonate (1%), edible oil (04%), and penicillin
precursor. The pH of this medium after sterilization is 5.5 to 6.0. Inoculum
media are similar to production media except that lactose and precursors are
not added in the inoculum media. These media compositions may be slightly
changed to increase yields and meet economic changes.

 Fermentation:

The media are placed in a fermentation vessel, sterilized, and inoculated with a
suspension of Penicillium chrysogenum (inoculum). A flowsheet for large-
scale production of penicillin is shown in Fig. 5.6. The fermentation vessel is
equipped with devices that allow continuous addition of nutrients, acids/bases
to maintain the pH (7 to 7.4), and cooling coils to maintain the temperature
(24°C).

The mycelium grows rapidly by utilizing organic nitrogen compounds and

lactic acid as a source of carbon. Ammonia is liberated by deamination of the
amino acids of the corn steep liquor and pH rises. Ammonia is actively
assimilated and maintains the pH. Active penicillin production is associated
with this phase of lactose and ammonia utilization (Fig. 5.7). Maximum
antibiotics are produced within 4 to 5 days.
 
Recovery and purification:

A schematic flow diagram for the recovery of potassium penicillin G (down

stream process) is incorporated in Fig. 5.6. The first step in the recovery
process is the removal of mycelium or cells by filtration (rotary vacuum filter)
or centrifugation. These stages are carried out under aseptic conditions, to
avoid contamination of the filtrate with penicillinase-producing
microorganisms (Bacillus species) which may cause loss of antibiotics.
Penicillin is extracted under controlled conditions of temperature, pH and
sterility to minimize chemical and enzymatic degradation. It is extracted in the
form of acid into amyl acetate, methyl isobutyl ketone, or butyl acetate in a
counter-current solvent extractor at pH 2.5 to 3.0.

A penicillin-containing solvent is treated with active charcoal to remove

pigments and other impurities. The charcoal is separated from the extract on a
precoated rotary vacuum filter and then washed with the solvent. Penicillin
from the solvent is crystallized by the addition of sodium or potassium
hydroxide to form its salt. The end product of penicillin is then crystallized
into sodium or potassium penicillin.
Discovery of Penicillin:
Alexander Flemming discovered penicillin secretion by the mold Penicillium notatum in
1929. He reported that a contaminating colony of the fungus lysed adjacent colonies of
staphylococci; but the lytic agent seemed too unstable to be useful. However, when Chain
purified the active material, called penicillin, in 1939, it proved remarkably effective in
certain infections.

Penicillin is not a single chemical compound, but a group of compounds of related structure
and activity. There are six penicillins – Penicillin G (benzyl penicillin), penicillin V
(phenoxy-methyl penicillin), penicillin F (A2-pentcnyl penicillin), penicillin X (p-
hydroxybenzyl penicillin), penicillin K (n-heptyl penicillin) and penicillin O (allyl-
mercaptomethyl penicillin). Penicillin is selective for gram-positive bacteria, some
spirochaetcs and the gram-negative diplococci (Neisseria).
Commercial Production of Penicillin:
Commercial production of penicillin is depicted in Fig. 40.7. The commercial production of
most of the antibiotics follows a similar plan. The major differences relate to the
microorganism, media composition, and extraction procedure. For penicillin production,
inoculum of a high yielding strain of P. chrysogenum is prepared as follows: inoculum is first
cultured in flasks in wheat bran nutrient solution for 1 week at 24°C.

The culture is then transferred to inoculum tanks, and grown for 1-2 days under aeration; this
supports heavy mycelial growth. The inoculum is now added to very large fermentors
containing the production medium, which consists of 10 per cent total glucose/molasses, 4-
5% corn-steep liquor solids, 0.5-0.8 per cent phenylacetic acid and 0.5% vegetable oil; pH is
adjusted to 6.0 and temperature is regulated at 25- 26°C.

Earlier media contained lactose, but it is no longer used. The fermentation is carried out
under aerobic conditions, and nutrient supply is maintained by regular feeding (fed-batch
culture). The fungus grows in a submerged culture mostly as mycelial balls.
The fermentation is carried out for 7 days. Initially, mycelial growth occurs, and
carbohydrates are used up. This reduces the carbohydrate level in the medium; this favours
penicillin production, which begins from the second day of fermentation. The pH of medium
rises to 8.0 by the 7th day, and penicillin production stops at this stage. Phenylacetic acid is
the precursor for the benzene ring side chain of Penicillin G.

ADVERTISEMENTS:

The presence of precursor promotes the production of Penicillin G. At the end of

fermentation period, the fungal biomass is separated by filtration and used as animal feed
supplement. Penicillin G is present in the broth, and is extracted using an organic solvent.
Penicillin G is finally purified as potassium salt; it is converted to 6-aminopenicillanic acid
(6-APA) cither chemically or biologically.

The side chain of 6-APA is variously modified to obtain a variety of derivatives of penicillin,
which are used in medicine. In addition, several other fungal metabolites are used as
antibiotics (Table 40.3).