Lysozyme

Fleming found the lytic action of human nasal mucus and tear on Micrococcus
cells in 1922 and he reported the similar lytic action of egg white constituent on
Gram-positive bacteria. He proposed the name lysozyme for the enzyme and
Micrococcus lysodeikticus for the organism serving as the substrate (1).
Lysozyme cleaves the -(1,4)-glycoside linkages between N-acetylglucosamine
and muramic acid in bacterial cell wall and chitin or other oligomers. In addition
to glycosidase activity, this enzyme has transglycosylation activity (2) and
esterase activity (3). Thus, numerous names have been proposed for this enzyme.
The Commission on Enzyme Nomenclature in 1964 recommended ‘‘E.C.
3.2.1.17. Systematic name: mucopeptide N-acetylmuramyl hydrolase; trivial
name: mucopeptide glucohydrolase, lysozyme.’’ Lysozyme is easily crystallized
from chicken egg white, and a large amount of the enzyme can be obtained.
Therefore, lysozyme has been one of the most intensively investigated and
characterized enzymes.

II. LYSOZYME IN FOODS

Lysozyme exists abundantly in chicken egg white and constitutes 3.5% of the
total egg white proteins when calculated on the basis of lytic activity. Since
lysozyme interacts with ovomucin gel in egg white, the content may be > 3:5%.
The lysozyme activity in hen egg white decreases during storage of egg dependent
on the temperature, as shown in Figure 1. The activity is retained > 80% during
storage at low temperature for 1 week, while it is decreased < 60% during storage
at room temperature for 1 week (4). The loss of lysozyme activity results from the
increases in pH during storage of egg. The pH increases up to 9.3 within several
days’ storage at room temperature. On the other hand, lysozyme is stable in acidic
pH in the presence of CO2 gas, as shown in Figure 1. Lysozyme interacts with
ovomucin in thick egg white. It is well known that the interaction is responsible
for the gel structure of thick egg white (5). The ovomucin-lysozyme interaction
decreases with egg white thinning.

III. UTILIZATION OF LYSOZYME IN FOODS

Hen egg white lysozyme is utilized as a food preservative with antimicrobial

effects without any food toxicities. The bactericidal action of lysozyme is
intensive on Gram-positive bacteria but much weaker on Gramnegative bacteria,
because the former expose the cell wall consisting of peptidoglycan, while the
latter has a unique cell envelope, which consists of the outer membrane, inner
membrane, and peptidoglycan layer. Therefore, the perturbation of the outer
membrane structure is needed to enhance the susceptibility to lysozyme of the
peptidoglycan layer in Gram-negative

bacteria. Despite this disadvantage, lysozyme is generally used as a food

antiseptic. The antimicrobial action may be efficient on Gram-positive bacteria
but ineffiCopyright . All Rights Reserved.cient on Gram-negative bacteria. Some
attempts to enhance the antimicrobial action for Gram-negative bacteria have been
done by our colleagues. in 1991 found that the covalent attachment of palmitic
acid residues to the lysyl residues of lysozyme greatly enhanced the bactericidal
action on E. coli (6). Nakamura et al. in 1991 reported that the Maillardtype
lysozyme-polysaccharide conjugate also enhanced the bactericidal action on
various Gram-negative bacteria (7). The antimicrobial action of lysozyme-dextran
conjugate is shown in Figure 2. The bactericidal action is enhanced by heating at
50 C. The polysaccharide attachment seems to stabilize lysozyme and to have
stronger affinity to the outer membrane of Gram-negative bacteria. These
modified lysozymes can be used as food antiseptics effective on various bacteria.

PROPERTIES AS PROTEIN

Lysozyme consists of 129 amino acids and the molecular weight is 14,307
daltons. The protein contains many more basic amino acids (11 arginines and six
lysines) than acidic amino acids (six aspartic acids and two glutamic acids), and
the isoelectric point is 11.2. The amino acid and nucleotide sequences of
prelysozyme are shown in Figure 3. The sequence 1 to 18 is the signal peptide,
and the N-terminus of mature lysozyme is the position 1 (lysine). Lysozyme has
four disulfide bonds: Cys6–127, Cys30–115, Cys64–80, and Cys76– 94. Human
lysozyme, which consists of 130 amino acids, is very similar to hen egg white
lysozyme in its primary and tertiary structures and the positions of disulfide
bonds. -Lactalbumin, which consists of 123 amino acids, is also homologous to
lysozyme in its primary and tertiary structure and in the position of disulfide
bonds. The x-ray analysis of hen egg white lysozyme was carried out by Blake et
al. in 1965 as the first enzyme to have its tertiary structure determined by x-ray
crystallography (8). The ribbon drawing model is shown in Figure 4. As shown in
Figure 4, it divides the molecule into two parts—a predominantly helical core, and
an irregular -strand core. The helical core consists of four -helices (5–15, 25–35,
88–99, and 108–115), and the -strand core consists of three strand regions (38–
46, 50–54, and 57–60). The two structural domains ( and ) of lysozyme appears to
be formed prior to docking of the domains to generate the native closed-packed
structure (9). The domain forms persistent structure during folding more rapidly
than the domain. In addition to a -helical domain, lysozyme contains two 310
helices (79–84 and 119–124).
V. PROPERTIES AS ENZYME

The most striking feature of the molecule is the crevice running across the waist
of the egg-shaped molecule. The crevice is the active site of lysozyme which
has the binding area for the substrate of hexasaccharide (N-acetylglucosamine/N-
acetylmuramic acid)3. The space-filling CPK model of lysozyme (left) and
its interaction with substrate are shown in Figure 5 (10). The substrate is
compactly bound to the crevice of the lysozyme molecule. A schematic view of
the substrate hexamer, NAG-NAM-NAG-NAM-NAGNAM, and its interactions
with the enzyme are shown in Figure 6 (10). The view is nearly straight
into the crevice, with the heavy edges of the sugar rings on the exterior. The site
of catalysis was identified to be between D and E rings. When a search was
made in the vicinity of the D/E ring for possible alytic groups, two candidates
came to light: Asp52, in a polar environment where it will be ionized, and Glu35,
in largely hydrophobic surroundings where it might easily remain protonated. The
mechanism of cleavage of the polysaccharide bond in lysozyme is suggested as
shown in Figure 7 (10). The proton of Glu35 first attacks the linkage oxygen and
weakens the C1-O bond (a). If the bond is cleaved, ring D forms a carbonium ion.
The nearby charged Asp52 helps to stabilize the carbonium ion (b). The Glu35
proton is replaced by another from an ionizing water molecule, and the resultant
hydroxyl ion then attacks the carbonium ion and completes the reaction (c).
This proposal of Phillips and coworkers (11–13) has been supported by a number
of chemical and kinetic experiments. The enzyme activity of lysozyme can be
easily measured by the lytic action on M. lysodeikticus (M. luteus). However, the
change in the turbidity of M. luteus does not necessarily reflect the real enzyme
activity, because the lysis is a rupture of the bacterial cell wall and is not equal to
the cleavage of glycosidic bonds. Nevertheless, the lytic action is generally used
for the measurement of lysozyme activity because of the ease of the assay. The
decrease in the turbidity of the suspension of M. luteus cell wall is followed by
the absorbance at OD450 and the enzyme activity can be represented as the initial
rate. In order to get a linear curve for the decrease, a small amount of lysozyme (4
g) should be added into the cell suspension (OD450 ¼ 0:7). When the enzyme
reaction is done at room temperature for 1 min, a linear decrease curve is
obtained and used to determine the initial rate. The optimal pH is 7:0 using lytic
activity. On the other hand, when the activity is measured using synthetic
substrates, p-nitrophenyl N-acetylglucosamine oligomer (14) and glycol chitin
(15), the optimal pH is 5 and 5.3, respectively. Therefore, the optimal pH of
lytic activity is different from that of the glycosidic activity. The glycosidic
activity is determined by measuring the reducing power produced by the
glycolysis of ethylene glycol chitin (15). One milliliter of 0.5% ethylene glycol
chitin is added to 0.5 mL lysozyme solution in sodium acetate buffer (pH 4.5).
The mixture is incubated at 40 C for 30 min. After the reaction, 2 mL of the color
reagent (prepared by dissolving 0.5 g potassium ferricyanide in 1 L of 0.5
M sodium carbonate) is added and the mixture is immediately boiled for 15 min to
estimate the reducing power resulting from the hydrolysis of ethylene glycol
chitin.
VI. PURIFICATION OF LYSOZYME
Lysozyme is easily crystallized from fresh chicken egg white. A 5% sodium
chloride (w/w) is added to the homogenized egg white adjusted to pH 9.5–9.8.
The egg white is stirred for 48 h at 4 C. The resulting crystals are collected and
solubilized in a half-volume of acetate buffer (pH 4.5). The solution is
recrystallized at least five times. Thus, the purified lysozyme is obtained
and the purity is 100%. On the other hand, lysozyme can be purified by cation
exchange chromatography, because the enzyme is a basic protein. For example,
when the recombinant lysozyme is expressed in S. cerevisiae, the yeast medium is
applied to CM-toyopearl equilibrated with a buffer of low ionic strength, and
the adsorbed basic proteins are eluted in a gradient manner.
VII. RECENT BREAKTHROUGH STUDIES ON LYSOZYME

Since lysozyme is easily obtained in a purified form, it has been studied

comprehensively as a model protein for structure, dynamics, and folding. The
recent development of recombinant techniques has enabled elucidation of the
molecular mechanism of structural and functional properties of lysozyme. In early
studies, recombinant lysozyme was investigated in the E. coli expression system.
However, since the prokaryotic cells have a different secretion system from
eukaryotic for correct foldings cells, the correctly folded lysozyme could not be
expressed. It seems likely that the folding of disulfide-rich protein such
as lysozyme is difficult in E. coli because of the absence of endoplasmic reticulum
(ER) in which proteins are posttranslationally folded. Although the enzyme is
obtained as an inclusion body, the yield of refolding is at a very low level. The N-
terminus of recombinant lysozyme is methionine, while that of native lysozyme is
lysine. Since the N-terminus lysine is essential for correct folding, the substitution
of Nterminus lysine with another amino acid results in a significant decrease in
the stability of lysozyme. Kumagai et al. in 1987 found that hen egg white
lysozyme was correctly processed and folded in S. cerevisiae (16). The N-
terminus of recombinant lysozyme is lysine and the stability is the same as the
native lysozyme. Since S. cerevisiae is a typical eukaryotic cell with an ER
system, lysozyme is correctly processed and folded in the yeast. Taniyama et
al. in 1992 reported the folding mechanism of disulfide bond–deficient human
lysozyme C77/95A mutant secreted in S. cerevisiae (17). Although the
deficient mutants of other disulfide bonds could not be secreted, only C77/95A
mutant secreted by eightfold greater than wild-type in yeast. The stability of
C77/95A mutant greatly decreased despite the correct folding. These observations
show that the disulfide bond Cys77–95 contributes to the stabilization of
the folded form of human lysozyme. Recently, the identification of human
lysozyme as an amyloidogenic protein which causes serious diseases is of
particular interest. The two unknown natural mutations (Ile56Thr and Asp67His)
in the human lysozyme gene both cause autosomal-dominant hereditary
amyloidosis (18). It has been suggested that the lysozyme amyloid fibril may be
formed by the intermolecular -sheet association due to -strand exposed to the
molecular surface by a single amino acid substitution. We have developed new
approaches for industrial application using genetic modifications of lysozyme.
Nakamura et al. (19) reported that the large molecular size of N-glycosylated
lysozyme with a polymannose chain was predominantly expressed in yeast
carrying the lysozyme gene modified to have N-linked signal sequence Asn-X-
Thr/Ser at the position of the molecular surface. The polymannosyl lysozyme
showed remarkable heat stability in that no coagulation was observed by heating
at 100 C. In addition to heat stability, the lysozyme revealed excellent emulsifying
properties, superior to the commercial emulsifiers (20). On the basis of the idea
that the covalent attachment of palmitic acid residues to the lysysl residues of
lysozyme stabilizes it and makes it more active (6). (21) attempted the genetic
‫‪fusion of a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro), which forms a -‬‬
‫‪strand conformation with the same length as a palmitic acid residue, to its C-‬‬
‫‪terminus. As expected, the hydrophobic peptide fusion greatly enhanced the‬‬
‫‪bactericidal action to E. coli. These modified lysozymes having antimicrobial‬‬
‫‪action against both Gram-positive and Gram-negative bacteria and can be used for‬‬
‫‪industrial applications in future.‬‬

‫الليزوزيم‬
‫ليزوزيم هو إنزيم يفكك جدار البكتيريا اكتشف انزيم الليزوزيم في عام ‪ 1921‬على يد العالم‬
‫االسكتلندي الكسندر فلمينج ليزوزيم هو المادة االولى التي تم اكتشافها كمضاد حيوي ضد‬
‫البكتيريامن مصدر بيولوجي يتواجد الليزوزيم في إفرازات مختلفة للثديات‬
‫مثل الدموع ‪ ،‬العرق ‪ ،‬اللعاب‪ ،‬وايضا في خاليا الدم البيضاء التي تشكل جهاز المناعة لجسم‬
‫الكائن الحي‪.‬كذلك يتواجد هذا االنزيم في انسجة اخرى مثل بروتين البيض وفي الحليب‪.‬هذا‬
‫اإلنزيم يتسبب لتحلل خاليا البكتيريا وبذلك يساعد في حماية الحيوانات والكائنات من هذه‬
‫البكتيريا‪ .‬يتواجد هذا االنزيم ايضا في انواع معينة من النبات مثل (بابايا و فيكوس) ‪،‬وايضا‬
‫في البكتيريوفاج (فيروسات التي تهاجم بكتيريا) ‪ .‬انزيم الليزوزيم يساعد القيروس‬
‫على اختراق ‪.‬جدار خلية البكتيريا سمي هذا االنزيم بليزوزيم النه يتسبب لتحلل البكتيريا‪،‬حيث‬
‫تنفجر خلية البكتيريا بسبب دخول الماء اليها بعد تفكيك جدارها بفعل اإلنزيم ‪،‬نتيجة لذلك تنتفخ‬
‫خلية البكتيريا ثم نتفجر‪،‬وهذا ما يسمى" بليزيس" وتعني تحلل او تفكك ‪ ،‬ومن هنا جاء االسم‬
‫ليزوزيم‪.‬‬
‫جدار البكتيريا‬
‫لمعظم الخاليا البكتيرية يوجد جدار الذي يحيط بالغشاء‪،‬الجدار يحافظ ويحدد شكل خلية‬
‫البكتيريا ‪،‬وبذلك يساعدها في التواجد والعيش في بيئة ذات تراكيز مخففة مثل المياه‬
‫العذبة‪.‬جدار الخلية يتركب من مادة خاصة تسمى بيبتيدوجليكان‪ .‬البيبتيدوجليكان عبارة عن‬
‫بوليمير صلب الذي يكون مبنى شبكي يتميز بقوة ميكانيكية عالية‪ .‬كال الوحدتين من المبنى‬
‫األساسي للبيبتيدوجليكان هما وحدتين من السكر الخاص بالبكتيريا ‪NAM‬و ‪ NAG‬وحدات‬
‫السكر مربوطة ببعضها البعض بشكل متتالي وتكون معا شبكة من السالسل الطويلة المربوطة‬
‫فيما بينها بواسطة سالسل عرضية قصيرة من الحوامض االمينية‪ .‬هذه الشبكة هي المسؤولة‬
‫عن إعطاء المبنى الصلب للبيبتيدوجليكان‪.‬‬

‫مبنى االنزيم‬

‫‪URLloadcancel‬‬ ‫استعمال تقنيات حيود األشعة السينية ساعد في التعرف على مبنى انزيم‬
‫الليزوزيم ‪ ،‬تم التعرف على المبنى الفراغي لإلنزيم عام ‪ 1965‬على يد‬
 ‫‪Export Animated‬‬ ‫العالم "دافيد فيلفيس" وشركائه‪ .‬إنزيم الليزوزيم من أصل بيض الطيور‬
‫‪Image‬‬ ‫يسمى ليزوزيم سي وهو إنزيم صغير نسبيا ‪ ،‬يتركب من سلسلة ببتيدية‬
‫واحدة والتي تتركب من ‪ 129‬حامضا امينياوزنه الجزيئي ‪14500‬‬
‫دالتون تسلسل الحوامض االمينية في االنزيم يشكل المبنى االولي اما‬
‫المبنى الثانوي النزيم الليزوزيم يتألف من التفاف السالسل الببتيدية على‬
‫بعضها البعض بشكل ‪ .‬حلزونات الفا باللون الخمري وصفائح بيتا ‪.‬‬
‫باللون االصفر المبنى الفراغي لإلنزيم هو المبنى الثالثي للسلسلة‬
‫الببتيدية ويكون على شكل دائري والذي يحتوي على ‪ 4‬اربطة‬
‫كبريتية (دي سولفيدية) بين الحوامض األمينية سيستئين باللون االصفر‬
‫‪،‬تعتبر األربطة الكبريتية عامل مهم لتشكيل وتثبيت المبنى الفراغي‬
‫لإلنزيم وعند تفكيكها يحدث تغير في المبنى الفراغي وبذلك تتغير فعالية‬
‫اإلنزيم وقد تتوقف نهائيا ً ‪ .‬وعند مقارنة مبنى البروتين بين أنواع مختلفة‬
‫من الكائنات الحية تبين وجود تشابه ‪ ،‬حيث تتواجد مناطق في‬
‫البروتينات التي حفظت بينها‪،‬نجد مثل هذا التشابه في منطقة الموقع‬
‫الفعال ‪.‬‬
‫فعالية االنزيم‬
‫يقوم إنزيم الليزوزيم بفك الرباط بين وحدتي ‪ NAG‬و‪ NAM‬حيث‬
‫ترتبط وحدتي السكر للبببتيدوجليكان في الموقع فعال لإلنزيم ‪،‬وفيه‬
‫تحدث عملية هيدروليزا ‪.‬عملية كهذه تسبب لكسور في جدار البكتيريا‬
‫وبذلك يفقد الجدار القوة الميكانيكية لتماسكه‪.‬وبما أن البكتيريا تعيش عامة‬
‫في بيئة مخففة التراكيز(هيبوتيني) عندها ونتيجة لفعالية الليزوزيم‬
‫ستحدث اسموزا ويدخل الماء لداخل البكتيريا فتنتفخ وتنفجر ‪.‬‬

‫اعاقة عمل االنزيم‬

‫‪ NAG-3‬مبني من ثالث وحدات من السكر ‪ NAG‬المرتبطة مع بعضها البعض‪ .‬هذا السكر‬
‫يستطيع ان يرتبط لالنزيم ليزوزيم في الموقع الفعال ‪ ،‬لكنه ال يتفكك بفعل االنزيم ‪.‬‬
‫ارتباط المعيق مع االنزيم يمنع ارتباط مادة االساس معه وبذلك يعيق االنزيم‪ .‬ارتباط المعيق‬
‫يكون الحدى الحامضين االمينيين في الموقع الفعال‪.‬‬