Lysozyme is an enzyme that breaks down bacterial cell walls. It is commonly found in egg whites and tears. The document discusses lysozyme's structure, properties, commercial applications, and potential modifications to increase its antimicrobial efficacy. Lysozyme has value for its use as a preservative in foods and pharmaceuticals due to its ability to inhibit bacterial growth. It can also be chemically modified through processes like lipophilization and glycosylation to broaden its antimicrobial range.
1. LYSOZYMe- a lucrative
enzyme
PRESENTED BY:
Susan Ann Jayan
1st M.Sc., Biotechnology
Bharathiar University
1
2. Also known as muramidase or N-
acetylmuramide glycanhydrolase (EC 3.1.2.17)
The first enzyme to be sequenced and have its
structure elucidated by X-ray crystallography
Coded by the LYZ gene in humans
The dimeric form of lysozyme has different and
extremely valuable properties
Can be chemically modified to increase its
efficacy in antimicrobial action by
lipophilization, glycosylation, heat denaturation, etc.
2
3. Lysozyme from Hen’s Egg
White is 129 amino acids long with
14.3 kDa molecular weight
It comprises 2 domains joined by a
long α helix between which lies the
active site for antimicrobial
activity:
*N-terminal domain(residues
40-88) has some helices and β-
parallel sheets
*The second domain(1-39 and
89-129) has mostly α-helical
structure
4 sulphide bonds in locations
between Cys 6 - Cys 127, Cys 30 -
Cys 115, Cys 64 - Cys 80 and Cys 76
- Cys 94 lend stability and unusual
compaction 3
4. WHERE IT IS FOUND..
It is ubiquitous in nature
Most abundant and commercial source – Hen Egg White (3.5% of total
egg protein)
SOURCE OF LYSOZYME AMOUNT OF LYSOZYME
Hen Egg White 2500-3500 μg/ml
Duck Egg White 1000-1300 μg/ml
Tears 3000-5000 μg/ml
Human Milk 55-75 μg/ml
Cow Milk 10-15 μg/ml
Spleen 50-160 μg/ml
Thymus 60-80 mg/kg
Pancreas 20-35 mg/kg
Cauliflower Juice 25-28 μg/ml
Papaya Juice 8-9 μg/ml
4
5. Relatively small secretory protein, with pI 10-11
Types: C-type in Hen, G-type in
Goose, Bacterial, Phage and Plant lysozymes
It is a very stable enzyme
The overall kcat for lysozyme is about 0.5/sec
Stable between pH 5-9, most stable at 5
Damages bacterial cell walls by catalyzing
hydrolysis of 1,4-beta-linkages between N-
acetylmuramic acid and N-acetyl-D-
glucosamine residues in a peptidoglycan
Cleaves the same linkages between NAG
residues in Chitodextrins
5
8. 1. ANTIMICROBIAL ACTIVITY
PHARMACEUTICAL
INDUSTRY
Prophylactically for
dental caries
Lozenges for sore
throat
In contact lens
decontamination
solutions and eye drops
Creams for topical
reparation of certain
dystrophic and
inflammatory lesions
of skin and soft tissues
8
9. FOOD INDUSTRY
As preservative in foods
Food packaging biofilm –
extends shelf-life of non-
sterile or minimally
processed foods eg., poultry
meat, sausage
Added to infant formula
CHEESE INDUSTRY:
Prevent growth of
Clostridium tyrobutyricum
which causes late-blowing
and undesirable flavors
Hastens ripening of cheese
9
10. WINE INDUSTRY :
Due to its lytic action on
Gram-positive Lactic Acid
bacteria
Good alternative to
sulphites in microbiological
stabilization after malo-lactic
fermentation
Preventive control of the
onset of malo-lactic
fermentation
10
11. ENZYMOLOGY:
Stability and activity is
enhanced when bound
inside nanopores; 3D
structure is preserved as well
CLINICAL RELEVANCE:
Prognostic marker of Male
Breast Cancer
Lysozyme dimer
Stimulates synthesis of
interleukins and α and γ
interferons
Modulates generation of
TNFα
Induces phagocytizing cells
Prevents excess generation of
free radicals
11
12. Lipophilization broadens Gram-negative bacterial
action
Glycosylation:
Glycolization with Palmitic acid gives strong activity
against E. coli
Lysozyme-dextran conjugates and Lysozyme-xyloglucan
conjugates exhibit good emulsifying properties and heat
stability
Chemical and thermal modification helps extend
antimicrobial activity over Gram-negative bacteria
without forfeiting action on Gram-positives
12
Good aftnoonrespectd sir n my friends!My topic for today is ‘lysozyme- a lucrative enzyme’ and so I want to present b4 u some very interesting facts abt this relatively small enzyme, lysozyme.Th antibacterial activity of this enzyme was first observd in 1909 by russian scientist, laschtschenko and th name lysozyme was coined by alexanderfleming in 1922. its 3dimensional struc was explained by davidchiltonphillips in 1965. this, is th brief timeline of lysozyme
Another name for lysozyme is,… bcoz It has th property of cleaving th bond btw n-acetyl muramic acid n n-acetyl glucosamine residues in peptidoglycan layers.Lysozyme has th honor of beinth 1st enzyme to have its struc completely determind by x-ray crystallography and also sequenced, in 1965
Structural analysis revealed tat the enz has 129 aa, and a mol wt of 14.3 kDa
Sources of lysoz are wide..Luk at thconc of lysoz in tears.. We can be thankful for it bcoz it is principally responsible for fighting off infections in th eye, else it cud result in diseases like conjunctivitis..Take a good look at th concentrations of lysoz in human milk n cow milk.. Bcoz we shall come to it later
.It has a very high isoelectric point at pH10-11, which is exploited in its isolation from egg albumin.Besides th human lysozyme, other naturally occurring types are……………..Tis enzyme is very stable coz of its 4 sulphide bonds. And atleast 2 of th 4 sulphide bonds must interact to maintain the enzymatic activity n also its thermal stability . Tis makes lysoz stable for 1-2 min at 100 degree celsiusHowever, thprescence of thiolgrps inactivate thenzmakin HEW inactive @60’ celsius due to presence of free Sulfhydrylgrps..And finally th central prop around which most of th apps of this enzyme revolves is tat it cleaves th 1-4 beta bonds btw NAM n NAG. Overall kcat for lysozyme is about 0.5/sec, which is quite slow (Table 14.4) compared with that for other enzymes. On the other hand, the destruction of a bacterial cell wall may only require hydrolysis of a few polysaccharide chains. The high osmotic pressure of the cell ensures that cell rupture will follow rapidly. Thus, lysozyme can accomplish cell lysis without a particularly high kcat
THIS IS to show u where th cleavage occurs. I’m not going into th details of thphillipsmechm of cleavage as we’ve already heard tat in class. I’m focussin rather on th various ways in which this relatively small peptide enzyme has been exploited to improve th quality of human life.
production of lactic acid within the biofilm (plaque) produced by bacteria on tooth decalcifies the enamel and leads to dental caries or bacterial infection of the tooth. Thus lysoz which can kill thg+ve lactobacilli is effective here in preventing plaques..Streptococcus mutans produces a filmy plaque on teethThe dextran slime can be depolymerized to glucose for use as a carbon source, resulting in
Cow milk has lesser lysozyme"butyric late blowing", which occurs during the ripening of certain European-type cheeses. This problem is due to the contamination of milk by a naturally occurring, spore-forming bacterium, called Clostridium tyrobutyricum. The origin of the contamination of milk by this bacterium lies in the widespread use of silage as a feed.Lysis of starter bac will release cytoplasmic enzymes tat plays key role in proteolysis for ripenIng
Sulphites are not an ideal technological tool to control the growth of lactic acid bacteria: Sulphites have a non-specific antimicrobial activity: besides inhibiting lactic acid bacteria, they will also inhibit the yeasts responsible for the alcoholic fermentation.Sulphites are mostly active at low pH, which makes them relatively inefficient on wines having a low acidity.When used to stabilise the wine after malo-lactic fermentation, sulphites have a detrimental impact on the organoleptic qualities of the wine ("bleaching effect" on the colour and off-flavours like sulfur tastereduction of the sulphite levels in wine due to:The widespread use of sulphites in other foods (fruits, vegetables, fruit juices), and the related risk of toxicity coming from a too high cumulative intake.The well-known allergenicity of sulphiteswith lysozyme has shown that this enzyme can be a good alternative/ complement to the use of sulphites:Lysozyme has a very specific activity: it is active only on lactic acid bacteria and does not interfere with the alcoholic fermentation.Contrary to the sulphites, the lysozyme activity increases with the pH.Lysozyme is a safe product which has been positively evaluated by several regulatory agencies (WHO, FDA, Scientific Food Committee of the EU), and which has a safe track record during many years of use in the pharmaceutical and food industries.Lactic acid bacteria can indeed play both a positive and a negative role in wine:They are responsible for the often-desirable conversion of the malic acid into the lactic acid: a reaction known as "malo-lactic fermentation".They can become a problem if their growth is uncontrolled, either during the early stages of the alcoholic fermentation, or after the completion of the malo-lactic fermentation, mainly because of the production of excessive volatile acidity and biogenic amines (histamine in particular).Certain spoilage lactic acid bacteria (Lactobacillus sp, Pediococcus sp) can produce particularly negative results, leading to flawed or unmarketable wines.When sulphites are used at this stage, they present the most detrimental effect on the organoleptic properties of the wine (bleaching effect, sulphur taste). An addition of 250 to 300 ppm (25 to 30 g/hl) of lysozyme will allow for a dramatic reduction in the quantity of sulphites necessary to stabilise the wine, down to the minimum level required to ensure the protection against oxidation and the control of the acetic acid bacteria (not inhibited by lysozyme).
One key challenge is the stability of enzymes, a particular type of protein that speeds up, or catalyzes, chemical reactions. Taken out of their natural environment in the cell or body, enzymes can quickly lose their shape and denature. Everyday examples of enzymes denaturing include milk going sour, or eggs turning solid when boiled.confined lysozyme and other enzymes inside carefully engineered nanoscale holes, or nanopores. Instead of denaturing, these embedded enzymes mostly retained their 3-D structure and exhibited a significant increase in activity.Lysoz is naturally synthzd in th mammary ducts . Studies showd tat it was also expressd in male breast cancer cases. Thustisprotein is a potential tumor marker in males with breast cancerWhen I say dimer, u may ask me so how d’udimerizeth monomer.. Well, in nature it exists as a reversible dimer btw pH5 & 9. An irreversible dimer forms in HEW upon extended storage and retains activity of th monomer. Commercially, Lysozyme is dimerized by using a coupling rgt in a buffer of pH10..Regulation of th synthesis of th tumor necrosis factors are essential bcoz their over-prodn is harmful too
Gram-positive bacteria are more sensitive to lysozyme than Gram-negative bacteria because the peptidoglycan is not protected by an outer membrane and it is a more abundant molecule.Gram-negative bacteria are sensitive to lysozyme if pretreated by some procedure that removes the outer membrane and exposes the peptidoglycan directly to the enzyme and this is why we lipophilizethenz with stearic acid/palmitic acid/myristic acid/capric or caproic acidHeat denaturation of lysoz causes it to partially unfold, and tis makes it include g-ves also in its antimicrobial spectrum witout losing its action agnstg+ves
