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thesis entitled
ISOLATION PURIFICATION AND CHARACTERIZATION
OF CAULIFLOWER LYSOZYME

presented by

KHALIL IBRAHIM EREIFEJ

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of the requirements for

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ISOLATION PURIFICATION AND CHARACTERIZATION
OF CAULIFLONER LYSOZYME

By

“03““

Khalil 10 Ereifej

A THESIS

Submitted to:
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Food Science

1979

ABSTRACT

ISOLATION, PURIFICATION AND
CHARACTERIZATION OF CAULIFLONER LYSOZYME

By
Khalil 1; Ereifej

Fresh cauliflower IBrassica oleracea) heads were bought from

 

a wholesale market in Lansing. The heads were destemmed and cut
into clusters, divided into lots of 400 g each, and kept frozen
in polyethylene bags. Lots were thawed, blended, filtered and
centrifuged. The.lysozyme was isolated from the extract by ion
exchanging on IRC-SO, salted out at 40% ammonium sulfate
saturation, centrifuged, and the precipitate was chromatographed
on a Sephadex G-50 column..

The active fractions were pooled, dialyzed against distilled»
water and freeze-dried.

It was found that the freeze—dried enzyme contained l5.7%N,
with specific activity of 0.337 A 540/min/mg protein. Micrococcus

lysodeikticus cell walls were the substrate. Cauliflower yielded

 

0.9 mg enzyme per loo 9. Purity was checked on SDS polyacrylamide
gel electrophoresis, lysozyme appeared to be a basic polypeptide.

The lysozyme could hydrolyze the cell walls of N, lysodeikticus

bacteria within a wide range of pH, but the maximal rate of
hydrolysis was found to be at pH 6.0. The molecular weight obtained
from $05 gel electrophoresis at pH 7.2 was 2l,700.

Cauliflower lysozyme was free of proteolytic activity. The‘
sedimentation constant (520,”) of the lysozyme was found to be
2.555. The diffusion constant (DZO,w) was found to be 5.l4 x
l0-7 sq. cm. per sec. The partial specific volume (v) was 0.714
cm3/g. The molecular weight of lysozyme calculated by the Svedberg‘
equation was found to be 4l,500. This value is almost twice as

large as the value obtained by SDS-polyacrylamide gel electrophoresis;

this is an indication that lySozyme at pH 6.5 might form a dimer.

TO

My Mother and in
Memory of my Father

ACKNOWLEDGEMENTS

The author wishes to express his sincere appreciation to
Dr. Pericles Markakis for his guidance, aid and encouragement
during the research for this project and in the preparation of
the manuscript. He is also indebted to Dr. Ramesh Chandan,
Dr. Mark Uebersax, and Dr. Everetts Beneke for their advice
and help in the preparation of this manuscript. The author
would like to extend his thanks to Dr. J. Brunner and U. Koch
for their help in determining the sedimentation velocity and

the diffusion coefficients for the cauliflower lysozyme.

ii

TABLE OF CONTENTS

Page
ACKNOWLEDGEMENTS ......................................... ii
LIST OF TABLES ........................................... iv
LIST OF FIGURES .......................................... v
INTRODUCTION ............................................. 1
REVIEW OF LITERATURE ..................................... 2
MATERIALS AND METHODS .................................... 9
RESULTS AND DISCUSSION ................................... 20
LIST OF REFERENCES ....................................... 39

iii

LIST OF TABLES
Table Page
1 Proximate analysis of fresh cauliflower .............. 23

2 Comparison of yields of papaya and cauliflower
lysozyme isolated by different methods ............... 23

iv

Figure

10

ll

12

13

LIST OF FIGURES

 

Page
Amino acid sequence of egg-white lysozyme ......... 3
The cell wall tetrasaccharide with the 8(l-4)
glycosidic linkage hydrolysed by lysozyme ..........
Elution pattern of cauliflower lysozyme
from sephadex G-SO column ......................... 2l
SDS gel electrophoresis at pH 7.2 and 7.5%T ....... 22
Effect of pH on the activity of cauliflower
lysozyme expressed as AA540/min ................... 26
Lineweaver-Burk plot for cauliflower lysozyme ..... 27
Plot of absorbance at 440 nm vs. time of
incubation cauliflower lysozyme with M,
lysodeikticus cell walls for 24 hrs ............... 29
SDS gel electrophoresis pattern at 7.5, 9.0,
l0.5% polyacrylamide. Log relative mobility
vs. % polyacrylamide for different standard
proteins (from Pharmacia) and cauliflower
lysozyme .......................................... 30
SDS polyacrylamide gel electrophoresis pattern
at pH 7.2 and 7.5%T ............................... 31
Log MN vs. Rf for different standard proteins
and cauliflower lysozyme at 7.5, 9.0, l0.5%T
of SDS gel electrophoresis ........................ 32

Time vs. log (x + x0) to determine the sedi-
mentation velocity coefficient of the cauliflower
lysozyme by Beckman model E analytical centrifuge.. 34

Area of the peaks (AZ/4wH2) vs. time (sec) to
determine the diffusion coefficient of the cauli-
flower lysozyme in Perkin-Elmer electrophoretic
apparatus ......................................... 36

Pictures of the cauliflower lysozyme free boundary
taken at different times (sec) .................... 38

INTRODUCTION

Lysozyme was discovered by Alexander Fleming in l922. He re-
ported that a certain protein present in many plants and animals
and biological fluids can clear a buffered suspension of bacteria

such as Micrococcus lysodeikticus. During the past fifty years

 

considerable progress has been made in the study of lysozyme.
The structure of hen egg-white lysozyme has been elucidated by amino
acid sequencing and x-ray crystallography, and its enzymatic action
is fairly well understood. Lysozyme from different sources has
the capability of breaking down the 8(l-4) glycosidic bond of the
N- acetylglucosamine compounds which are part of the bacterial cell
wall. Also lysozyme performs a chitinase activity by breaking down
the glycosidic bond in the oligosaccharides isolated from chitin.
Recently, lysozymes obtained from different sources were
compared with egg-white lysozyme, revealing more information with
respect to protein evolution. The objectives of this work were to

isolate the cauliflower lysozyme and to study some of its properties.

REVIEW OF LITERATURE

Lysozyme

Alexander Fleming (l922) first found that a certain protein
present in several biological systems is capable of lysing a
suspension of bacteria in a buffer solution. Fleming (1932)
called that protein lysozyme (EC. 3.2.l.l7). Abraham and Robinson
(l937) isolated and crystallized the hen egg-white lysozyme.
Lysozyme occurs in many tissues -- goose egg, dog and rabbit spleen,
human secretions, bacteriophage and milk.

Since l958, Jolles (1963) and Canfield (I963) had been engaged
in elucidating the primary structure of the hen egg-white lysozyme
which is shown in Figure l.

Osserman, gt_al,, (l974) compared the amino acid composition
of lysozyme obtained from different animal sources.

Chandan, gt al,, (I968) reported the lysozyme activity in
milk of different species. The concentration of lysozyme in human
milk was found to be 3,000 times that of cow's milk. Because of
this high concentration in human milk they assumed that lysozyme
might have physiological or nutritional implications in infant
feeding (Chandan, gt_gl,, l968, Cavalieri, 1957, and Seleste, 1953).

Parry, et_al,, (l965) suggested a rapid and sensitive assay of

muramidase (lysozyme) obtained from bovine milk.

 

Figure l.

Amino acid sequence of egg-white
lysozyme. (Based on R.E. Canfield,
and A.K. Liu, (l965) and D.C.
Phillips, (1966).)

 

 

Chandan, gt_al,, (1964, 1965) showed that the purified bovine
and human milk lysozyme can catalyse the lysis of several gram-
positive and gram-negative bacteria by hydrolysing 8(1-4) links
between muramic acid and glucosamine in the glycopolysaccharides
which are present in bacterial cell walls Figure 2. Also they
observed lysing activity in several biological system such as
tears, nasal secretions, blood, saliva, spleen, and bacteria in
addition to milk.

Teotia and Miller (1975) studied the destruction of Salmonellae
on poultry meat by lysozyme, ethylenediaminetetra-acetic acid
(EDTA), x-rays, microwaves, and chlorine. Lysozyme and microwaves
were found more efficient in the destruction of cells than the
other agents.

‘The plant origin lysozyme became of interest after being
reported in some plant latices in appreciable quantities.

Meyer, gt_al,, (1936) extended the studies initiated by
Fleming in 1922 and found lysozyme activity in crude preparations
of fig and papaya. They studied the properties of the isolated
lysozyme and found that the lysozyme is a basic polypeptide that
can be inactivated by alkali, peroxide, iodine and cuprous oxide
and could be reactivated by hydrogen sulfide, sulfite, and hydrogen
cyanide.

Meyer, gt_al,, (1946) showed that crude extractions of ficin

from Ficus glabatra and Ficus doliana have a lytic enzyme which

 

 

depolymerizes a mucopolysaccharide fraction prepared from organisms

and liberates acetylhexosamine. They also showed that the plant

LVSOZVITIE
NH ocwc OH NHC H,c OH
H
c 0H NHCOCH3 CHon NHCOCH,
H’ CH3CHCOOH CH3CHOOH
NAG NAM NAG NAM

Figure 2. The cell-wall tetrasaccharide with the 8(1-4)

glycosidic linkage hydrolysed by lysozyme shown
by an arrow.

NAG

N-acetylglucosamine.

NAM N-acetylmuramic acid.

lysozymes have a more limited range of antibacterial activity than
the egg-white lysozyme.

Smith, gt_al,, (1955) isolated a mercury derivative of lysozyme
from dried papaya latex. They described two methods for crystal-
lizing the mercury derivative of lysozyme. The Hg++ was inhibitory
and had to be removed for full activity. The enzyme lysed Sarcina
lugga_and was optimally active at pH 4.65. Its molecular weight
was found to be 24,000, and its amino acid composition was established.
The papaya lysozyme has N- terminal glycine, unlike the animal
lysozyme that has N- terminal lysine.

Shukla and Krishna (1961) compared the bacteriolytic activity
of different plant latices such as papaya, fig, jack fruit, and
poinsettia. They also partially purified a bacteriolytic enzyme
from the latex of Calotropis procera.

Howard and Glazer (1967) undertook to extend the investigations
on the chemical and physical properties of the papaya lysozyme and
to compare it with egg-white lysozyme and other glycosidic enzymes.
They prepared the lysozyme according to Smith, gt_al, (1955) and
called it 3X-lysozyme because it was crystallized three times.

Ion exchange chromatography of the 3X-lysozyme on IRC-50 showed
five different peaks designated A, B, C, D and E. Fraction E was
the major component. Its molecular weight was 27,500 and it had
the same amino acid composition as the 3X-lysozyme. The sedimen-
tation equilibrium studies of the 3X-lysozyme suggested that it
consists of a single polypeptide chain. The activity of papaya

and egg-white lysozyme toward Micrococcus lysodeikticus cell walls

 

was compared. The papaya lysozyme showed only one-third of the egg-
white lysozyme activity; but the papaya lysozyme displayed a 400 times
higher chitinase activity toward tetra-N-acetyl-D-glucosamine than

the egg-white lysozyme.

Further, Howard and Glazer (1969) studied the enzymatic proper-
ties and the terminal sequences. They found that gly-ile-ser-lys-ile
was the N- terminal sequence. Ser-phe-gly was found to be the
carboxylic sequence. Papaya lysozyme like egg-white lysozyme
releases the reducing group of N- acetylmuramic acid from the cell
walls of Micrococcus lysodeikticus. They also found that the papaya
lysozyme hydrolyses more glycosidic bonds initially than the egg-
white lysozyme. Papaya lysozyme was stable in the pH range of 1.8 -
10.0 at room temperature. Maximum activity was found to be at

pH 4.6 when Micrococcus lysodeikticus cells were the substrate, also,

 

Howard and Glazer (1969) found that the papaya lysozyme was inhibited
by histamine and histidine as in the case of egg-white lysozyme.
Dahlquist, gt_al,, (1969) investigated the mechanism of papaya
lysozyme and compared it with the mechanism of egg-white and human
lysozymes. Although human, egg-white, and papaya lysozymes hydro-
lyse glycosidic bonds, only the human and egg-white lysozymes
catalyze extensive transglycosylation as well as hydrolysis.
Glazer, gt_al,, (1969) isolated a plant lysozyme from dried
fig latex and compared its physical, chemical, and enzymatic proper-
ties with those of the egg-white and papaya lysozymes. Fig lysozyme
appeared to consist of a single polypeptide chain with a molecular

weight of 29,000. The fig lysozyme showed enzymatic properties and

amino acid composition similar to those of the papaya lysozyme. Both
had similar pH optima for chitin and Micrococcus lysodeikticus cell
walls hydrolysis. Glysine is the N- terminal amino acid residue in
both fig and papaya lysozymes,. but they differ in the C- terminal
residue. In the fig enzyme the C- terminal residue is isoleucine;

in the papaya enzyme it is glycine.

Heneine and Kimmel (1972) investigated the sulfhydryl groups of
the papaya lysozyme. They found four free -SH groups present in
the papaya lysozyme and suggested that one of these -SH groups is
necessary for the biological activity of the enzyme. They also
reported a new method of isolation of lysozyme, a method of isolation
of N-acetyl-D-glucosamine oligosaccharides, and colorimetric method
for monitoring the chitinase activity.

Bradley (1976) investigated the papaya lysozyme isolated and
purified by the modification of the Smith and Kimmel (1955) procedure
and by a new grinding method. This investigation revealed a lot about
the role of the free sulfhydryl and the two disulfide bridges in

the papaya lysozyme molecule.

METHODS AND MATERIALS

Cauliflower (Brassica oleracea) heads were bought from a whole-

 

sale market in Lansing. The heads were destemmed; the curds were
broken into clusters; the clusters were weighed out into lots of
400 g each which were placed in nylon bags and kept frozen until the
time of isolating the enzyme. Proximate analysis was run on the

fresh cauliflower according to AOAC (1975) procedures.
Isolation, and Purification of Cauliflower Lysozyme

1. Isolation of cauliflower lysozyme.

Cauliflower lysozyme was isolated according to Parry, gt_al,,
(1969). Four hundred grams of frozen cauliflower were thawed and
blended with 500 ml of deionized water for two minutes in a waring
blender. The extract was filtered through three layers of cheese-
cloth. The solids were discarded and the filtrate was centrifuged
at 14,500 9 (11000 rpm) for one hour. The supernatant was saved
(fraction l-a). The precipitate was dispersed in 100 ml of water
and centrifuged again. The supernatant (fraction 2-a) was saved.
Fractions l-a and 2-a were pooled (fraction 3-a). Twenty grams of
Amberlite IRC-50 (previously equilibrated with 0.2 M phosphate buffer
pH 6.5 for 4 hours at 4°C) were added to fraction 3-a and stirred

for 4 hours at 4°C. The supernatant was discarded. The resin was

10

poured into a column and washed with deionized water until the eluate
was clear. The adsorbed enzyme was eluted with 0.8 M phosphate buffer
pH 6.5. The active fractions were collected and pooled. Salting out
of the enzyme was achieved by adding 25 g of ammonium sulfate per

100 ml (40% saturation). The solution was kept at 4°C overnight to
assure complete precipitation, then centrifuged at 14,500 g for one
hour. The supernatant was discarded. The precipitate was dissolved
in 20 ml of 0.1 M NaCl — 0.1 M NaAc buffer pH 6.0. That was the

crude cauliflower lysozyme solution.

11. Purification of the enzyme.

The crude cauliflower lysozyme was fractionated on a 1.5 x 50 cm
Sephadex G-50 column. The sample volume applied on the column was
20 m1, flow rate was 18 ml per hour. The volume of each fraction was
2.5 ml. Absorbance of fractions was monitored at 280 nm in a Beckman
spectrophotometer-24. Lysozyme activity in each fraction was checked
according to Parry, et_al,, (1965). The active fractions were pooled
and dialyzed against distilled water at 4°C. The dialyzed enzyme was
freeze-dried and kept in the refrigerator. Nitrogen content was
determined in all the active fractions throughout the isolation and

purification according to Kjeldahl.

III. Nitrogen determination - micro-Kjeldahl.

An aliquot of one ml of each active fraction was digested for
one hour in duplicate according to AOAC (1975). Sulfuric acid of
1.84 specific gravity was used for digestion. Potassium sulfate and

mercuric oxide were added as catalysts. After cooling the flasks,

11

the sides were rinsed with deionized water and digestion continued
for another hour. The digests were transferred into the distillation
apparatus by using approximately 10 ml deionized water. The digested
mixture was neutralized with 15 ml of 50% NaOH solution, containing
5% sodium thiosulfate. The liberated ammonia was steam-distilled
into 5 m1 of 5% boric acid solution, containing 4 drops of methyl
red-methylene blue indicator (2 parts of 0.2% methyl red in alcohol
with one part of 0.2% methylene blue in alcohol). The distillation
was continued until the volume in the receiving flask reached 25 ml.
The ammonium borate complex was titrated with 0.02 N HCl which had
been accurately standardized against tris-hydroxy amino methane
(THAM) as a primary standard. Nitrogen was calculated from the
following formula:

(ml HCl-ml blank) (Normality of HC1) (14.007)

%N= X 100
mg of sample

 

Protein content was calculated as follows:

% Protein = % N X 6.25

IV. Electrophoretic studies.
1. Purity Check.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SOS-page) was performed according to Weber and Osborn (1969) under

both denaturing and non-denaturing conditions.

12

Solutions for SOS-page were prepared as follows:

Gel Buffer:
To obtain 0.2 M phosphate buffer of pH 7.2 and
0.2% SDS, dissolve 7.8 g NaH2P04. H20 and 20.2 g
of anhydrous NazHPO4 and 2.0 9 SDS in enough de-
ionized water to make one liter of buffer.

Sample buffer:
To obtain 0.01 M phosphate buffer pH 7.0, 1%
mercaptoethanol, dissolve 0.138 g NaH2P04. H20
in about 80 ml deionized water, add 1.0 9 SDS
and one ml 2-mercaptoethanol and make the volume
to 100 ml.

Stock acrylamide solution (18%T):
To prepare 18% polyacrylamide solution, dissolve
43.88 g of polyacrylamide and 1.13 g N, N]-
methylenbisacrylamide in 100 ml deionized water
and make to 250 m1. Store in a cark bottle at
4°C.

Gel formation:
To prepare 7.5%T gel concentration mix the
following: 20.8 ml of 18%T stock acrylamide
solution, 1.5 ml of 1.5% ammonium persulfate

solution, and 0.045 ml Temed (N, N, N1 1 -

, N
tetra-methylethylene diamine), then make up

the volume to 50 ml with gel buffer.

13

Glass tubes which had been fitted with rubber
stoppers to about 6.5 cm level were filled with the
gel mix using a syringe and needle or a disposable
pipet. Each gel was carefully overlayed with water.
The gel polymerized in about 30 minutes.

Sample preparation:
Nith SOS-page 0.1-0.2% protein solution was pre-
pared in sample buffer, then boiled for 5-10
minutes in a water bath. After cooling, 8
sucrose crystals were added to increase the
density of the protein. Also, 2-3 drops of
1% bromophenol blue solution were added as a
reference marked dye.

The tubes were placed in the electro-
phoretic cell and both chambers, the upper
(Cathodic) and the lower (anodic), were filled
with reservoir buffer which was diluted 1:1
gel buffer: water. The current was 8mA/tube.
Electrophoresis proceeded for 5-6 hours.

Gels were removed from the tubes by rimming
the edges with a needle and the gels were
flushed with deionized water while the tubes
were submerged in water. Gels were forced out
with a pipet bulb. Gels were then stained with
coomassie brilliant blue G-250 (0.04 g/100 ml
5.5% perchloric acid) for 12 hours. Gels were

destained in 7% acetic acid solution.

l4

Cauliflower lysozyme was electrophoresed in different gel
concentration (7.5, 9.0, 10.5%T). The standard proteins (from
Pharmacia) were electrophoresed in similar gels. A standard curve
was obtained by plotting Rf vs. log. molecular weight. Rf for each
standard protein was the corrected relative mobility and was calcu-
lated as follows:

distance of protein migration length of gel before staining

 

R - Fex
f length of gel after staining distance of dye migration

The Rf value of the cauliflower lysozyme was calculated in the same
manner.
2. Activity of the enzyme bands on the gel.

In this experiment the cauliflower lysozyme sample was
applied on four gels without denaturing agents 503 and mercapoethanol.
The gel and sample buffer was the same 0.2 M phosphate buffer pH 6.0.
One of the gels was stained and the rest were not. Gels were removed
carefully by "rimming" and the nonstained gels were kept refrigerated
until the destaining procedure was completed on the stained gel. Bands
from the nonstained gels were removed by cutting at distances similar
to those of the visualized bands and crushed in one ml water. The
activity of the extracted enzyme was checked according to Parry, gt_al,
(1965).

3. Molecular weight determination of cauliflower lysozyme by

SOS-page.

Determination of the molecular weight of freeze-dried lyso-
zyme was performed by SOS-page according to Weber and Osborn (1969).

Three concentrations of polyacrylamide gels were prepared

(7.5, 9.0, 10.5%1). The lysozyme sample and standard proteins of

15
known molecular weights were electrophoresed as described previously.
Concentration of gels (%T) was plotted vs. log Rf to obtain the
Ferguson plot. Also, log of molecular weight was plotted against

Rf, to estimate the molecular weight of the cauliflower lysozyme.

V. Proteolytic activity.

Detection of the presence of proteolytic fragments of cauliflower
lysozyme or the presence of proteolytic enzymes was done according
to Kakade, gt_al, (1969).

Two substrates were employed:

a. N-a-Benzoyl-D-arginine-p-nitroalanine

hydrochloride (BAPA).

b. N-Benzoyl-L-tyrosine-p-nitroani1ide (BTPA).

Forty milligrams of each substrate were dissolved in 1 ml of
dimethyl sulfoxide in a 25 m1 volumetric flask, and the volume was
completed to the mark with 0.05 M tris buffer, pH 8.0. Tris buffer
contained 0.1 M KCl and 0.05 M CaClz. The pH was adjusted with IN
HCl solution. The experiment was conducted as follows:

One ml of the cauliflower lysozyme solution (10 mg/25 ml) was
added to 1 ml tris buffer in a test tube, and the pH was adjusted
to 8.0. The test tubes were incubated at 37°C in a water bath.

Five ml of the substrate BAPA were added to the tubes and the reaction
was allowed to proceed for 15 minutes at 37°C. One ml of 30% acetic
acid was added to each tube in order to stop the reaction.

The blank was prepared exactly the same way as the sample, but
1 ml of 30% acetic acid was added to the reaction mixture before
incubating at 37°C. The same experiment was performed using the

BTPA substrate.

16

VI. Determination of the pH optimum of cauliflower lysozyme.

The effect of pH on the bacteriolytic activity of cauliflower
lysozyme was demonstrated according to Smith, gt_al, (1955). Two
buffers were employed. First, sodium acetate buffer solutions of
pH's 3.5, 4.0, 4.5, and 5.0 were prepared. The second buffer was
a sodium phosphate buffer solution of pH's 5.5, 6.0, 6.5, 7.0, 7.5,
and 8.0.

The enzyme solution was prepared immediately before the assay by
dissolving 10 mg of cauliflower lysozyme in 25 m1 deionized water.
The substrate used in this assay was Micrococcus lysodeikticus cell
walls. Two and a half mg substrate were suspended in 5 m1 of each
buffer solution. The activity of the enzyme at each different pH was
determined by adding 1 ml of the substrate to 1.9 ml of the buffer
and 0.1 m1 enzyme solution. The reaction mixture was placed in a
1-cm-path Beckman cuvette, and the enzyme solution was added with
a square teflon plunger provided with a groove and three orifices
and connected to a stainless steel handle.

The change in the absorbance per minute was monitored at 540 nm

with a Beckman Spectrophotometer-DB-24 connected to a recorder.

VII. Determination of Michaelis-Menten constant of cauliflower
lysozyme.
Twenty mg of the cauliflower lysozyme were dissolved in 25 ml
of sodium phosphate buffer pH 6.0, and 25 mg of Micrococcus

lysodeikticus cell walls were suspended in 50 ml of the same buffer.

 

Then 0.1, 0.2, 0.3, 0.4, 0.5, 0.8, 1.0 and 1.2 ml of substrate were

17

added to l cm-path Beckman cuvettes. Enough buffer was added to

make the final volume 3 m1 when 0.1 m1 of the enzyme solution was
added. The enzyme solution was added with a square teflon plunger

and the reaction mixture was mixed well. The change in the absorbance
per minute at 540 nm was monitored with a Beckman Spectrophotometer-

DB-24.

VIII. Measurement of the enzymatic activity.
The activity of the cauliflower lysozyme was measured in
terms of microbial destruction by a turbidimetric method according

to Bradley (1976), and Smith, §t_al, (1955). Ten mg of Micrococcus

 

lysodeikticus cell walls were suspended in 50 ml of 0.1 M sodium

 

phosphate buffer pH 6.0. Ten m1 of the substrate were incubated
with the enzyme (about 10 mg/ml) at 37°C. At a specific time
interval, 2 m1 aliquot of the reaction mixture were removed and
placed in a test tube, and 0.2 ml of 4M NaOH was added to stop the
enzymatic reaction. After standing at room temperature for 30
minutes, the absorbance of the reaction mixture was monitored at
440 nm. The absorbance corresponding to 100% lysis was determined
by incubating 10 ml substrate with one mg of the enzyme at 37°C

for 24 hours.

IX. Quantitation:

The amount of lysozyme in cauliflower was estimated according
to Parry, gt_al, (l965). Hen egg-white lysozyme (from Difco) was
employed as a standard lysozyme. The cell walls of Micrococcus

lysodeikticus killed by ultra violet radiation (Difco) were the

 

l8

substrate. A standard curve was obtained by plotting the change

of absorbance per minute at 540 nm against different concentrations
of the standard enzyme. The assay was done by pipeting 0.5 ml
substrate (50 mg of bacteria cell walls were suspended in 100 ml

of 0.15 M phosphate buffer) into a l cm-light 1 cm-path Beckman
cuvette, 0.5 m1 of 0.3 M NaCl, different amount of hen egg-white
lysozyme (10 pg/ml) ranging from 0.1 to 1 ml, and sufficient
phosphate buffer to make the volume 3 ml. The enzyme solution was
added to the mixture with a teflon plunger, mixed well and the change
in the absorbance per minute at 540 nm was recorded.

The change in the absorbance was plotted against the concen-
tration of the standard lysozyme. A straight line was obtained and
used for reference. The cauliflower lysozyme was assayed in the
same manner, using 1 ml of fresh cauliflower extract instead of the
standard lysozyme. By means of the reference curve the activity of
the cauliflower lysozyme was converted to the equivalent standard
lysozyme per unit volume. The yield and recovery were calculated

after freeze-drying the pure cauliflower lysozyme.

X. Determination of sedimentation velocity coefficient and
diffusion coefficient.

Sedimentation coefficient was determined by a Beckman model E
analytical ultracentrifuge according to thantis (1964). The
cauliflower lysozyme was dissolved in phosphate buffer to yield
0.4% protein solution. After exhaustive dialysis against the
solvent, the lysozyme solution was run in a Beckman model E

analytical centrifuge at 59,780 rpm at 20°C. Lysozyme boundary

19

observation was made with the Schlieren optical system. Time (t)

in minutes was plotted against log (x + xo). (x is the distance

between the lysozyme solution boundary and the survace of the meniscus,
X0 is the distance between the center of the rotor and the surface

of the meniscus). To obtain a straight line, the slope was

calculated. Then the sedimentation velocity was calculated as follows:

 

 

2.303
s= 2 x the slope, where w= angular
w .60 velocity in rpm
log(x + x0)
The slope was found to be =
t

XI. Determination of the diffusion coefficient of the cauliflower
lysozyme.

The diffusion coefficient was determined by the use of Perkin-
Elmer electrophoretic apparatus. Enough dry lysozyme was dissolved
in phosphate buffer of pH 6.5 and ionic strength 0.1 to give 0.4%
protein solution. The protein solution was dialyzed against the
same buffer until equilibration. The protein solution was injected
to fill both the ascending and descending arms of the Tiselius cell.
The free boundary of the protein in the solvent was photographed at
different time intervals using a Polar-Pan-42 film. The tempera-
ture was controlled at 4°C. The areas of the peaks on the film
plates were measured, averaged and plotted against time in seconds.
A linear relationship was obtained. The slope represented the dif-

fusion coefficient of the cauliflower lysozyme.

RESULTS AND DISCUSSION

Isolation and Purification of Cauliflower Lysozyme

The cauliflower lysozyme was isolated from the frozen cauli-
flower as described earlier and further fractionated by gel filtra-
tion chromatography which resolved into one distinct compound, Figure
3. This result was reproducible with more than 35 preparations and
more than 60 batches of the frozen cauliflower. The protein content
determination was run on the salt free freeze-dried lysozyme.

The protein content was 98.3% and the nitrogen content was 15.7%.

Furthermore, the purity of the lysozyme was determined by 505
gel electrophoresis. The results are shown in Figure 4. One band
was visible on the gel; the lysozyme appeared to be pure and con-
sisting of one polypeptide.

The yield of the pure lysozyme was 0.9 mg/100 g of fresh
cauliflower. The yields of other lysozymes obtained in different
methods were compared and are shown in Table II. Recovery of the
enzyme was 96%. Bradley (1976) reported five fractions of papaya
lysozyme designated A, B, C, D and E. Fraction E was the major
compound. Furthermore, Fraction E fractionation on Sephadex G-25
resulted in 3 peaks, and papaya lysozyme E represented only 25%
of the total protein applied on the column. The presence of

contaminating proteases in lysozyme E was determined, and in some

20

21

 

 

 

1 5 _ 11.5
. O'----°C‘ Activity
..___. Absorbance
ICC.
’ ":3.
/;%0\ :
W:
‘00 P /.'O .\ it .1100
/P ‘ ..
¢C> 3 g
I C a.
[,1 'x‘ o, E
. C '5
1‘ ¢
2 4,. . ., ..
N / i _ 4
< Z \ \‘ Q
/,r'\l 2‘ "0'5
0.5 T /. ‘1 ‘\ l
I \‘
z i 9‘
C i :
I ,
,/ I at...
f f ‘3 '1ro~o
o . ‘.
4121/ :75 fl; 5 °
05 i0 13 20 25 30
TuboNumbor

Figure 3.

Elution pattern of cauliflower lysozyme from
The column dimensions were

sephadex G-50.
Fractions of 2.5 ml each were

1.5 x 50 cm.
collected at a flow rate of 18.5 mN/hr.

Void volume was 32.5 ml.

22

    

Cauliflower
lysozyme

Figure 4. SDS gel electrophoresis at pH 7.2 and 7.5%T
gel concentration.

23

TABLE I.

Proximate analysis of fresh cauliflower.

 

 

Moisture (%) 92.3
Lipids 1.3
Proteins 2.5
Fiber 0.5
Ash 1.0
Carbohydrate (by difference) 2.4
TABLE II.

Comparison of yields of papaya and cauliflower
lysozymes isolated by different methods.

 

Method Sample (9) Total Enzyme (mg)

 

Howard and Glazer, (1967)
for papaya 180 500

Modified Kimmel and Smith,
(1955) for papaya 180 350

Bradley, (1976)
for papaya 180 1380

Parry, 591. (1969)
for cauliflower 400 3.6
(employed in this work)

 

24

preparations it was found to be 10% by weight (Bradley 1976). Also,
Glazer, gt_al, (1969) reported that five compounds (A, B, C, D and
E) were obtained from chromatographing a crude preparation of fig
lysozyme, but only Fraction 0 presented lysozyme activity which
represent 80% of the material applied on the column. Furthermore,
the IRC-50 ion exchange pattern resulted in 3 fractions. Only
fraction 3, which accounts for 75% of the material on the column,
exhibited lysozyme activity. The other two minor peaks were free
of lysozyme activity. In this work only one peak with lysozyme
activity was obtained from the Sephadex G-50 column pattern. This
peak resulted in one band on $05 gel electrophoresis as it is shown
in Figures 3 and 4 respectively. When this active protein was

tested for proteolytic activity it was free of proteolytic activity.
Characterization of Cauliflower Lysozyme

Measurement of enzymatic activity.

 

The activity of the enzyme was measured by a turbidimetric
method was described before. The isolated freeze-dried cauliflower
lysozyme had a specific activity 0.337 A 540/min/mg protein with
Micrococcus lysodeikticus as a substrate. This result is in agreement
with that reported by Bradley (1976) for papaya lysozyme E.
Detection of proteolytic activity.

The freeze-dried cauliflower lysozyme was tested for proteo-
lytic activity against two substrates, N-a-Benzoyl-D-arginine-p-
nitroalanine hydrochloride (BAPA) and N-Benzoyl-L-tyrosine-p-

nitroanilide (BTPA). No such activity was detected. Since proteolytic

25

enzymes are the most common contaminants of lysozyme, the purity of
the isolated cauliflower lysozyme must be high.

pH Optimum.

 

The cauliflower lysozyme exhibited a sharp pH activity profile.
This enzyme was active in a wide pH range: from 3.0 to 8.0, but
the maximum activity was obtained at pH 6.0 (Figure 5).

Howard and Glazer (1968), found that 4.6 was the optimum pH for

papaya lysozyme when M, lysodeikticus cells were the substrate, and

 

the enzyme was inactive above pH 6.5. When chitin was used as a
substrate, the maximal activity was observed to be at pH from 4.5
to 6.0. The enzyme was partially inactive at pH 7.5.

Generally, according to Glazer, et_al, (1968) and Meyer, gt_al,
(1936), the optimum pH for lysis of M, lysodeikticus was 4.5 - 4.7
and the optimum pH for hydrolysis of chitin was 4.25 - 6.4. The
difference in the pH dependence of hydrolysis of chitin and lysis
of the cell walls of the bacteria appears to result from the presence
of carboxyl groups in cell walls (Glazer, gt_al, 1968). In this
work, the optimum pH was found to be higher than the above mentioned
values. Also, the enzyme was partially inactive above pH 7.5.

The result of determination of the Michaelis-Menten constant
is shown in Figure 6. The maximum velocity of bacterial cell
walls lysis was found to be 0.5 absorbance unit per minute at
540 nm wave length, and the Km was found to be 0.33 mg of cell
walls per m1 under the given conditions. M, lysodeikticus cell

walls were used as a substrate. The time required to lyse half of

lLAfiAO/MUL

Figure 5.

26

010’

008

.06

004

002

 

 

 

Effect of pH on the activity of
cauliflower lysozyme expressed as
AA540 per minute. The buffers
employed were 0.1M acetate pH

3.0 to 5.0 and 0.1M phosphate

pH 5.5 to 8.0, ionic strength

was 0.2 in both buffers.

Figure 6.

27

1.0?
Vans 8 0-5. Almifl.

Km=0.33mg/ml

Char! unit/““0-

 

 

?" flaw...

Lineweaver-Burk plot for cauliflower
lysozyme, substrate employed was
Micrococcus lysodeikticus cell walls
Bacteria. Absorbance was monitored
at 540 nm, chart speed 2 inch per
minute, span equivalent to 2A.

28

the cell walls when the cauliflower lysozyme was incubated with the
substrate at 37°C for 24 hours at pH 6.0 was found to be 40 minutes.
Results are shown in Figure 7.

Molecular weight determination.

 

The molecular weight (MW) of cauliflower lysozyme was estimated
by two different methods. One estimate is based on the comparison
of the electrophoretic mobility of known MN proteins with the
cauliflower lysozyme. The other estimate is based on the Svedberg
formula which relates MN to diffusion and sedimentation coefficients.

For the electrophoretic comparison, six standard proteins
obtained from Pharmacia Co. and the cauliflower lysozyme were run
on polyacrylamide gels of three different concentrations; 7.5, 9.0,
10.5% polyacrylamide. Sample preparation and gel electrophoresis
procedures were described previously. The relative mobility (Rf)
of each standard protein and cauliflower lysozyme was measured and
the log of Rf was plotted against the polyacrylamide concentration
(%T). Results are shown in Figures 8 and 9. Then the logs of MNs
of the standard proteins were plotted against the relative mobility,
linear relationships were obtained and regression equations were
computed; the results are shown in Figure 10.

On the basis of these relationships, the MN estimates for the
cauliflower lysozyme were obtained, each based on three replicate
gels. The estimates were 21,700, 21,800, 24,200. The last value
was discarded because of the large deviation of the standards from
the regression line. An average value for the MW of cauliflower

lysozyme would be 21,700.

Figure 7.

 

 

29

L

1 2 24
TIM. ( kn)

Plot of absorbance at 440 nm vs. time of
incubation cauliflower lysozyme with M,
lysodeikticus cells for 24 hours. The
half-time (t 1/2) is the time required
for one-half of the initial reactants

to be consumed.

30

0.4
0.0 - \o
u‘: .
-0.4 r
(La. 7

Log: Rf

'1.2 II \

'106H

 

 

IM—
0511-
m1-
d.-
O
.5
”I.

°/. T

o——4 a-Lactalbumin

o——o Trypsin Inhibitor
¢e—4} Cauliflower Lysozyme
o——o Carbonic Anhydrase
,___, Ovalbumin

~__c Albumin

C-—<3 Phosphorylase b

Figure 8. SDS gel electrophoresis pattern at 7.5, 9.0, 10.5%
polyacrylamide. Log relative mobility versus %
polyacrylamide for different standard proteins
(from Pharmacia) and cauliflower lysozyme.

Cauliflower
Lysozyme

Figure 9.

 

31

Phosphorylase b

Albumin
Ovalbumin

Carbonic Anhydrase

Trypsin Inhibitor

a-Lactalbumin

SDS polyacrylamide gel electrophoresis pattern
at pH 7.2 and 7.5%T to determine the MW of the
cauliflower lysozyme. Gels A show the location
of cauliflower lysozyme bands. Gels B show the
location of the standard protein bands (Pharmacia).

Log.M.wt.

Figure 10.

32

     
 

 

 

5.0 9‘“ Phosphorylase b
.‘..\
45- Carbonic Anhydrase
‘ Cauliflower
\ :1 lysozyme
'x 5‘ Trypsin Inhibitor
\
a \
\\. \\
b a-Lactalbumin
An) 0'4 0:8 T2
Rf(¢"‘-)

._. 7.5%T, Y = 0.958 x + 5.130 r = 0.9995
2.... 9.0%T. Y = 0.9630 x + 5.040 r = 09976
o""‘310-5%T. Y = 1.011 x + 4.997 I" = 0_9943
C3 cauliflower lysozyme

SDS gel electrophoresis pattern at 7.5, 9S0, 10.5%
polyacrylamide. Log MN vs. relative mobility for
different standard proteins (from Pharmacia) and
cauliflower lysozymes.

33

It is interesting to note that the lines connecting the log of
Rf to %T for the standard proteins and the cauliflower lysozyme
converge to the same point (Figure 8). This is evidence that the
cauliflower lysozyme is a simple protein similar in nature to that
of the standards. Smith, gt al_(l955) found that the papaya lysozyme
had a MN of approximately 25,000. Howard and Glazer (1969) had
indicated that the papaya lysozyme E had a MW of about 25,000 based
upon the amino acid composition. The MW of the cauliflower lysozyme
obtained in this experiment is close to the MW of the papaya lysozyme.

The second method for cauliflower lysozyme MN determination is
based on the Svedberg equation, which relates the MN to the sedi-
mentation velocity and diffusion coefficients.

A Beckman model E ultracentrifuge was used for determining the
sedimentation velocity coefficient of the cauliflower lysozyme.
The enzyme concentration was 0.4% at phosphate buffer pH 6.5.
The runs were made at 59,780 rpm and 20°C. Results are shown in
Figure 11. The sedimentation data are given in Svedberg units
corresponding to water at 20°C ($20,w)' In this study, cauliflower
lysozyme SZO’w was found to be 2.55s. This is in reasonable agree-
ment with the value 2.57s reported for the papaya lysozyme by Smith,
_t_al, (1955) and the value of 2.705 for papaya lysozyme reported
by Howard and Glazer (1967), and the value of 2.735 for fig lysozyme
reported by Glazer, gt_al_(l969). The partial specific volume (v)

of the cauliflower lysozyme was calculated according to Greenberg

(1951). Using a 5 ml pycnometer, 0 was found 0.714 cm3/g.

34

    
 

 

 

 

0-8140F'
as 0-3110F. olepo:0.80819
25
.9
030301 . , , . g
0 4 8 12 16 20

Timelmln-I

Figure 11. Time vs. log (x + x,) to determine the
sedimentation velocity coefficient of
the cauliflower lysozyme by the Beckman
model E analytical centrifuge at
59,780 rpm. The lysozyme concentration
was 0.4% in phosphate buffer pH 6.5 at
20°C.

35

The diffusion coefficient was determined in a Perkin—Elmer
electrophoresis apparatus. The concentration of the enzyme was 0.4%
in a phosphate buffer pH 6.5 and ionic strength of 0.1 and temperature
of 4°C. The free boundary of the enzyme was scanned by a schlieren
optical system. Eight photographs were taken at 8 different inter—
vals over 22 hours after establishing the protein boundary. The
results were calculated by the height-area method from the formula

D=A2/4nH2 2

t, where A is the area in cm under the refractive index
curve, H is the maximal height, t is the time in seconds and D the
.diffusion constant in sq. cm. per sec. Results are shown in Figure 12,
and photographs are shown in Figure 13. The average value for D
was corrected to 020,w' 020,w was found to be 5.14 x 10"7 sq. cm.
per sec.

The molecular weight of the cauliflower lysozyme was calculated

by the Svedberg formula:

RTs
MN=—-—————-
D(1-vp)

where R is the gas constant, T is the absolute temperature K°, s is
the sedimentation coefficient, 0 is the diffusion coefficient, 0 is
the density of solvent at 20°C and v is the partial specific volume
of the cauliflower lysozyme. The molecular weight from this formula
gave a value of 41,500 which is almost twice as high as that obtained
from the electrophoresis experiment. This variation in results leads
us to believe that the cauliflower lysozyme at the indicated con-

ditions might form a dimer.

36

163X24r

20.

     

16 _ 5109.24.7“67

2
A2/4r1H

12.

 

 

0 1‘0 2"—"""""_o 30 ' "'36X103
Tim9( sec.)

Figure 12. Area of the peaks (AZ/4nH2) vs. time (sec.)
to determine the diffusion coefficient of the
cauliflower lysozyme in Perkin-Elmer electro-
phoretic apparatus. Lysozyme concentration
was 0.4% in phosphate buffer pH 6.5 at 4°C.

37

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Canfield, R.C., 1963. The amino acid sequence of egg-white
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39

40

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41

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