PG Department

1. CEPHALOSPORIN
Siddhi M Intwala
Msc Microbiology

2. CONTENT:-
 Introduction
 History
 Chemical Structure
 Mode of Action
 Classification
 Biochemistry
 Cloning Of Cephalosporin
 Strain Improvement
 Fermentation
 Extraction
 Refrences

3. INTRODUCTION
 Penicillin was 1st antibiotic discovered
 Discovery of Cephalosporin
 It is resistant to penicillinase hydrolysis

4. HISTORY
 Penicillin G was considered to be a miracle drug
when discovered in 1940s
 It was quite effective for the treatment of systemic
infection caused by gram positive bacteria but not
effective to the infection of gram negative bacteria.
 So it became important to develop a new drug
which was important for treatment of both gram
positive and gram negative organism.

5.  At about this time a new antibiotic cephalosporin C
was discovered from a species of mold
Cephalosporium acremonium.
 Work of Newton and Abraham at Oxford led to
purification of Penicillin N and Cephalosporin C in
1955.
 In 1951 Cephalosporin was proposed by Abraham
and Newton.
 And this structure was confirmed by Hodgkin and
Maslen by X-ray Crystallography..

6. CHEMICAL STRUCTURE:-
 Cephalosporin is a class of Beta Lactam antibiotics.
 Originally derived from the mold called
Cephalosporium acremonium. (Cephalosporin C)
 It is closely related to Penicillin.
 They have an important role in the modern
treatment of bacterial infections.
 A wide variety of cephalosporins have become
available.

8. MODE OF ACTION OF CEPHALOSPORIN.
 Cephalosporins and other β lactam antibiotics
inhibit the bacterial growth by interfering with
bacterial cell wall synthesis.
 All bacterial cell wall have a protective layer of
peptidoglycan at outer surface of cell membrane.
 The peptidoglycan layer consist of chains of
alternating N acetylglucosamine and N acetyl
muramic acid and are connected by peptides.

9.  The peptide cross-link adjacent peptidoglycan
chains to form a net of peptidoglycan layers.
 In gram positive bacteria the cell wall is made up
of peptidoglycan and techoic acid or techuronic
acid.
 In gram negative bacteria the peptidoglycan layer
has a distinct outermembrane layer consisting of
lipoproteins and lipopolysaccharides.

10.  During course of bacterial cell wall synthesis , the
monomers of N Acetyl glucosamine and N Acetyl
Muramic acid peptide are linked to form
peptidoglycan chain and three enzyme catalyse the
cross linking between these peptidoglycan chain
by forming bridges.
 Binding of β lactam antibiotics to transpeptidases
and carboxypeptidase enzyme interferes the
formation of peptide bridges between peptidoglycan
chain.
 And thus inhibit the final stage of cell wall synthesis.

11. CLASSIFICATION OF CEPHALOSPORINS:-
 Classified into four different generations.
 And the 4 generation is based on the spectrum of
antimicrobial activity.
 It is grouped w.r.t. increase in Gram negative
activity and decrease with respect to gram positive
bacteria.

12. 1. First Generation Cephalosporin
2. Second generation cephalosporin
3. Third generation Cephalosporin
4. Fourth generation Cephalosporin

13. FIRST GENERATION CEPHALOSPORINS
 Best activity against Gram Positive aerobes, with
limited activity against a few Gram-Negative
aerobes.
 Example:-
 Cefazolin (Ancef)
 Cephalexin
 Cephalothin
 Cephradine

14. SECOND GENERATION CEPHALOSPORIN:-
 Mainly effective against Gram Negative bacteria
 Modest activity against Gram Positive bacteria
 Example:-
 Cefoxitin ( mefoxin )
 Cefuroxime ( zinacef )
 Cef. axetil ( zinnat )
 Cefaclor ( ceclor )
 Cefprozil ( cefzil )

15. THIRD GENERATION CEPHALOSPORIN:-
They have enhanced Gram Negative activity.
 Example:-
 Ceftriaxone ( rocephin )
 Cefotaxime ( claforan )
 Cetazidime ( fortum )
 Cefoperazone ( cefobid )
 Cefixime ( suprax )

16. FOURTH GENERATION CEPHALOSPORIN:-
 Works for both Gram Positive and Gram Negative
organisms.
 Example
 Cefipime

17. BIOCHEMISTRY OF CEPHALOSPORIN:-
 Biosynthetic pathway of cephalosporin have been
well characterized.

20.  Biosynthesis of cephamycin C is done by
S.clavuligarus and Nocardia lactamdurans.
 Biosynthesis of cephalosporin C by A.
chyrsogenum.
 All begin with the condensation of 3 amino acids.
i.e. L-α-aminoadipic acid, L-cysteine and L-valine to
form tri-peptide δ ( L-α-aminoadipyl L- cyestinyl D
valine) by using enzyme ACV synthethase or
cyclase.

21.  The racemization of valine from the L- to D-
configuration takes place during synthesis of the
tripeptide .
 The LLD-ACV tripeptide is cyclised to form penam
nucleus Isopenicillin N by isopenicillin N synthetase
or cyclase.
 Both LLD-ACV and isopenicillin N serve as an
intermediate in biosynthesis of penicillins.
 Genes involved in the biosynthesis of Penicillin and
intermediates is termed as pcb.

22.  The genes coding for the biosynthesis of
cephalosporin is designated as cef.
 The isopenicillin N epimerase catalyse the
conversion of the L-α-aminoadipyl side chain of
isopenicillin into the D-α-aminoadipyl side chain of
penicillin N.
 The 5 membered thioazolidine ring of penicillin N is
then expanded to give the 6 membered
dihydrothiazine ring of deacetoxycephalosporin C
by the deacetoxycephalosporin c synthetase.

23.  The next enzyme in this pathway,
deacetoxycephalosporin synthetase catalyse
hydroxylation of deacetoxycephalosporin C at the
C3 methyl group to yield deacetylcephalosporin C.
 In A.chyrosogenum both the ring expansion and
hydroxylation activities reside on same protein
which is coded by one gene.
 Unlike fungi the expandase and the hydroxylase
are too distinct enzymes in S.clavuligerus and
N.lactamdurans encoded by two separate genes.

24.  In A.chrysogenum, the last step in cephalosporin c
biosynthesis catalyse by cephalosporin c
synthetase involves the transfer of acetyl group
from acetyl CoA to C3 hydroxymethyl moeity of
deacetylcephalosporin C.

25. CLONING OF CEPHALOSPORIN IN
BIOSYNTHETIC GENES:-
 DNA transformation of A. chrysogenum is less
efficient then bacterial system, hence
complementation of blocked mutant not feasible.
 Reverse genetics is used for cephalosporin C.
 3 steps occur:-
1.Purification of desired enzyme.
2.determination of partial amino acid
sequence.
3.synthesis of oligonucleotide probes.

26.  Genomic DNA library of A. chrysogenum is constructed in
E.coli.
 Sequencing of cloned DNA will confirm the DNA probe
matches the sequence.
 1985, first β – lactam biosynthetic gene pbc C was cloned
from A. chrysogenum.
 There are no introns & they encodes a polypeptide of 338
amino acids.
 Upon expression in E.coli, the cyclase protein converts LLD
– ACV to isopenicillin N.
 All genes except cefD have been cloned from A.
chrysogenum.

27. STRAIN IMPROVEMENT:-
 Strain Improvement can carried out by three
methods
1. Mutagenesis And Selection.
2. Protoplast Fusion.
3. Genetic Engineering.

28. 1. MUTAGENESIS AND SELECTION
 Improvement in yield of Cephalosporin C is
achieved primarily by increasing productivity of A.
chrysogenum culture by strain mutation and
selection method.
 Chemical mutagens such as N-methyl-N-nitro-N-
nitrosoguanidine and ethyl methane sulphonate or
UV light are used to induce chromosomal
mutations.
 Growth of the mutants on selective agents can be
used to increase probability of isolating improved
clones.

29.  Toxicity of A.chryosogenum of heavy metallic ions,
such as Copper, Manganese and Mercury has also
been exploited for selection purposes.

30. 2.PROTOPLASMIC FUSION:-
 It is a means of combining the advantageous
characters for different culture lineages.
 Mycelia are treated with DTT, followed by
Novozyme in presence of osmotic stabilizer usually
0.7M NaCL.
 PEG is used to induce membrane fusion between
protoplast; the fusants are indentified by
regeneration on selective media.

31.  Auxotrophic markers have been used to select for
fusants.
 Strains that have been transformed with positive
selective markers such as phleomycin and
hygromycin resistance are better candidates for
protoplast fusion.

32. 3.GENETIC ENGINEERING:-
 In past 20 years the cloning of many genes
involved in the biosynthetic pathway of
Cephalosporin s have led to attempt to improve the
strains through genetic enigeneering.
 Transformation of industrial strains with additional
copies of genes encoding for rate limiting enzymes
results in ellivation of metabolic pathway and
increase antibiotic production.
 Skarud et al have transformed industrial strain of A.
chyrosgenum with a plasmid containing the
hygromycin resistant gene.

33.  And because of this gene transformations there
was a 40% reduction of deacetoxycephalosporin C
present in the cephalosporin fermentation broth
compared to parental strain.
 This process was successfully scaled to production
tank.

34. FERMENTATION
 Cephalosporin is produced industrially by Fed
Batch Fermentation.
 pH ranges between 6 to 7.
 Temperature range between 24 to 28.
 Media should be provided with Carbon and
Nitrogen source for growth.

35.  Carbon Source:- Glucose, Sucrose, Starch,
Soluble starch, n-paraffins, Vegetable and Animal
oils, Acetic acid, Methanol, Glycerol, Sorbitol and
Ethanol.
 Nitrogen Source:- Natural nitrogen-containing
meat extracts, peptone, casein, cornsteep liquor,
yeast extracts, soya bean flour, tryptone, cotton
seed meal and wheat bran.

36.  Nitrogen-containing organic or inorganic
compounds may also be used, for example, urea,
nitrates and ammonium salts such as ammonium
acetate, ammonium chloride, ammonium sulphate
and ammonium phosphate.
 Inorganic Salts:- Sulphates, Nitrates, Chlorides,
Carbonates and Phosphates of potassium,
magnesium and calcium.

37.  Growth-promoting substances :- Cysteine,
Cystine, Thiosulphate, Methyl oleate and, in
particular, Methionine and also trace elements such
as iron, zinc, copper and manganese.
 Optimal productivity was achieved when sucrose
and DL Methionine were feed in an exponentially
ramped feed.

38.  With all fungal fermentation , dissolved oxygen is an
important physical parameter.
 And it is important because the enzyme cyclase and
particularly expandase which are helpful in biosynthetic
pathway require oxygen for effective catalysis.
 If dissolved oxygen level drops below 20% the output of
cephalosporin C will be reduced.
 And chemical breakdown of Cephalosporin C results in
the loss of 40% of cephalosporin C production.
 Addition of Acetyl esterase to the fermentation results in
accumulation of deacetylcephalosporin C and increases
the yield by 40%.

39. EXTRACTION OF CEPHALOSPORIN C
 After the fermentation is completed the mycelia and
insoluble media components are removed by filteration
and ultracentrifugation.
 Cephalosporin C must be extracted from fermentation
broth under neutral and slightly acidic condition.
 A temperature dependent chemical conversion of
cephalosporin C to Compound X occurs in fermentation
broth.
 At pH less then 2 Cephalosporin C Lactone is formed.

40.  To minimize this degradation it is important that the
cephalosporin C broth is processed rapidly without the
change in pH and temperature condition.
 2 main strategies are used for the purification of
Cephalosporin C.
 1. strategy:-
Activated carbon column or non ionic resin column is
used to adsorb Cephalosporin C from fermentation broth.
Alternatively a combination of anion and cation
exchange column arranged in series and can be used to
purify Cephalosporin C directly from broth.

41.  2. Strategy:-
It is a subsitution of the amine moiety on the α
aminoadipyl side chain of cephalosporin C.
 Several extraction steps are required to achieve an
acceptable purity of the end product.

42. CEPHALOSPORINS: USES
 Used to treat infections caused by bacteria
 Respiratory
 Ear
 Bone/joint
 Genitourinary tract infections
 Used throughout peri-operative period

43. CEPHALOSPORINS: ADVERSE REACTIONS
 Gastrointestinal reactions
 Nausea; vomiting; diarrhea
 Other body system reactions
 Headache; dizziness; malaise; heartburn;
fever; nephrotoxicity; hypersensitivity;
aplastic anemia; toxic epidermal
necrolysis
 Nursing alert
Allergy: Approximately 10% of people
allergic to penicillin are also allergic to
cephalosporins

44. REFRENCES:-
 Fermentation, Biocatalysis and Bioseperation.-Michael Flickinger.
 A TextBook Of Industrial Microbiology (2 edition)- Wulf Crueger &
Anneliese Crueger.
 United States Patent. Smith et. al
Patent Number: 4,533,632
Date of Patent: Aug. 6,1985

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