lysozyme discovery and structure

1. Submitted by;
B.Devadatha
M.Sc2nd sem
Dept of Biochemistry
and Molecular Biology.
Submitted to;
Dr. R. Rukkumani,
Assistant Professor,
Dept of Biochemistry
and Molecular Biology.
LYSOZYME

2.  Lysozyme: is 129 aminoacid residues enzyme
 (EC 3.2.1.17), hydrolase which catalyzes hydrolysis of 1,4-
beta-linkages between N-acetylmuramic acid and N-acetyl-D-
glucosamine residues in peptidoglycan and between N-acetyl-
D-glucosamine residues in chitodextrins.
 Molecular weight of Lysozyme is an approximately 14.7 kDa.
LYSOZYME

3. An enzyme found naturally in
• egg white
• human tears
• saliva (as well as other body fluids)
Capable of destroying the cell walls of certain
bacteria.
Acts as a mild antiseptic
OCCURENCE

4. Historical background
 Lysozyme was the first enzyme structure solved with X-ray
crystallography (Structure 2LYZ in the PDB, deposited 1975 by
Diamond and Phillips). It is now the most common enzyme in the
PDB (966 structures), most of which are engineered mutants from
the work of Matthews .
Lashtchenko (1909) observed the antibacterial
properties of hen egg white. Hen egg white lysozyme
(HEWL) has become the “classic” lysozyme.
Fleming (1922) noted the antibacterial property of
nasal mucus.

5. FLEMINGS DISCOVERY OF LYSOZYME
 The lysozyme was first noticed during some investigations made on
a patient suffering from acute coryza
 The nasal secretion of this patient was cultivated daily on blood
agar plates and for the first three days of the infection there was no
growth, with the exception of an occasional staphylococcus colony
 The culture made from the nasal mucus on the fourth day showed in
24 hours a large number of small colonies which, on examination
proved to be large gram-positive cocci .
 The microbe has not been exactly identified alluded as
 Micrococcus lysodeikticus.

6. PRELIMINARY EXPERIMENTS SHOWING THE ACTION
OF THE LYSOZYME
 Nasal mucus from the patient, with coryza, was
shaken up with five times its volume of normal salt solution, and the
mixture was centrifuged.
 A drop of the clear supernatant fluid was placed
on an agar plate, which had previously been thickly planted with M.
lysodeikticus, and the plate was incubated at 37° C. for 24 hours.
 It showed a copious growth of the coccus, except in the region where
the nasal mucus has been placed.
 There was complete inhibition of growth, and this inhibition extended
for a distance of about 1 cm. Beyond the limits of the mucus.

7. The effect of the lysozyme on bacteria
Inhibitory Action:
 A small portion of the agar is removed from
an ordinary agar plate making a cup into
which some material rich in lysozyme
 A drop of liquid agar, at a temperature
of about 50° C., is placed on the material in
the cup and is allowed to solidify after
which the cup is filled with the liquid agar
 Liquid agar is then poured all over the plate
to make a thin layer over the original
surface

8. • The whole surface of the medium is now
thickly planted with the M. lysodeikticus and
the plate is incubated for 24 hours.
• when it will be seen that there is copious
growth of the coccus except in the region of
the implanted material.
• It is evident that the lytic substance has
dissolved the cocci for a distance of 3 or 4
mm.

9. Properties of lysozyme
 Lysozyme has the characters of a ferment. The rapidity of its
action increases up to 60° C, but at temperatures over 65° C. it
is destroyed more or less rapidly.
 It acts best in a neutral medium.
 Peptic or tryptic digestion does not destroy lysozyme.
 Stability—When kept dry, lysozyme can be preserved for a
long time. It was noted that commercial dried egg albumen
was very rich in lysozyme .

10. Distribution of lysozyme
 In the human body:- Many tissues and secretions have been
examined for lysozyme .
 Of the secretions and body-fluids, all contained lysozyme except
normal urine, sweat, and cerebrospinal fluid.
 In the tissues of animals :- Florey, who titrated the lysozyme
content of extracts of many tissues of several animals and
compared them with human tissues.
 Cat’s tissues, with the striking exception of the salivary gland,
are relatively deficient in lysozyme.

11. CONCLUSION
 lysozyme is a widely distributed antibacterial ferment which is
probably inherent in all animal cells and constitutes a primary
method of destroying bacteria
 while acting most strikingly on non-pathogenic bacteria yet can,
when allowed to act in the full strength in which it occurs in
some parts of the body, attack pathogenic organisms.

12.  That it is very easy to make bacteria relatively resistant to
lysozyme, so that any pathogenic microbe isolated from the
body where it has been growing in the presence of a non-lethal
concentration of lysozyme must have acquired increased
resistance to the ferment.
 There are some differences in the lysozyme of different tissues
and in different animals whereby bacteria are susceptible to
different lysozymes in varying degrees.
CONCLUSION

13. The Hen Egg-white Lysozyme Molecule
 The three-dimensional structure of the hen egg-white lysozyme molecule
has been determined by X-ray analysis of the tetragonal crystals grown at
pH 4.7 from 1 M sodium chloride solution.
 The phases of the X-ray reflections were determined by the method of
multiple isomorphous replacement developed to take systematic account
of anomalous scattering effects .
 It has been interpreted with reference to the known amino acid sequence
of lysozyme as determined independently by Jolls and Canfield and their
colleagues.
 crystals of Lysozyme diffract X-Ray beam to a very high resolution,
currently the highest resolution structure, presented in Protein Data
Bank, was solved at resolution 0.94 Angstrom

15. Interpretation of the Electron Density
Map
• Hen egg-white lysozyme has a molecular weight of
about 14,600 and each molecule comprises 129
amino acid residues.
• The region of highest electron density, at lower right
of center, was found to correspond to the half-
cysteine residue 30.
• since it clearly forms part of a helical conformation
of the polypeptide chain, the axis of which is marked
HH‘
• Four residues removed from an identifiable
phenylalanine residue (34) nearer the carboxyl end
of the chain

17. Role in disease
• Lysozyme is part of the innate immune system.
• Reduced lysozyme levels have been associated
with broncho pulmonary dysplasia in newborns.
• Since lysozyme is a natural form of protection from
gram-positive pathogens
like Bacillus and Streptococcus
• In certain cancers (especially myelomonocytic
leukemia) excessive production of lysozyme by
cancer cells can lead to toxic levels of lysozyme in
the blood.
• High lysozyme blood levels can lead to kidney
failure and low blood potassium.

18. Reference
• On a Remarkable Bacteriolytic Element
found in Tissues and Secretions.
• By ALEXANDER FLEMING, M.B.,F.R.C.S.
• THE HEN EGG-WHITE LYSOZYME MOLECULE
• BY DAVID C. PHILLIPS

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