AN ABSTRACT OF THE DISSERTATION OF
Yawen Wu for the degree of Doctor of Philosophy in Food Science and
Technology presented on March 19. 2004.
Title: Immobilization of Hen Egg White Lvsozyme by the Sole Histidine Residue
to Polystyrene Beads through Peptide Spacers
Abstract approved:
Mark A. Daeschel
Lysozyme is a natural antimicrobial agent that is effective against many
food spoilage and pathogenic microorganisms by disintegrating their cell walls.
Immobilization of lysozyme has attractive applications for use in the food
industry: (1) The enzyme could be readily separated from treated foods and
beverages and re-used while the foods could still be claimed additive-free; (2) It
could impart stable antimicrobial capability to the surface of food packaging
polymers.
In this study, a novel method is described for the preparation of a highly
active immobilized lysozyme system. The method addressed three key issues
in the covalent attachment of a biological active protein to an insoluble support:
1.) The protein should be attached to the matrix by the fewest possible bonds to
minimize conformational change; 2.) The binding site(s) on the enzyme to the
supports should be located as far as practical from its active center and be
nonessential for its tertiary structure; 3.) The binding method should minimize
the steric interference between the support and the immobilized enzyme.
Using polystyrene resin beads as support matrix, peptide spacers of
various lengths composed of 6-aminocaproic acid were synthesized with the
solid phase peptide synthesis method. Then the amino terminals of the spacers
were derivatized with bromoacetyl bromide and coupled to the protein's only
histidine residue (His-15) that is nonessential for its lytic activity.
Immobilized lysozyme with a spacer composed of three 6-aminocaproic
acid units displayed the best lytic result against lyophilized M. lysodeikticus
cells: 2736 U/g resin with a protein load of 2.21 mg/ resin. Retained activity was
14.2% of that of the free enzyme. Preparations with longer spacers yielded
higher protein load yet the retained activity remained at about 14 % level. A
control consisted of random coupling of lysozyme to polystyrene beads without
spacer gave an activity of 158U/G with a protein load of 1.24 mg/g resin and
1.4% of retained activity,
Properties of the immobilized lysozyme system were studied, including
stability, effect of pH, surface characteristics of the support. A kinetics study of
the system using Eadie-Hofstee plot demonstrated strong external diffusion
effects, which resulted in deviation from classic Michaelis-Menton kinetic
behavior.
Immobilization of Hen Egg White Lysozyme by the Sole Histidine Residue to
Polystyrene Beads through Peptide Spacers
by
Yawen Wu
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirement for the
degree of
Doctor of Philosophy
Completed March 19, 2004
Commencement June, 2004
Doctor of Philosophy dissertation of Yawen Wu
Presented on March 19. 2004.
APPROVED:
t
Major Professor, representing Food Science and Technology
c?
Head of the Department of Food Science and Technology
T3T-
U.
Dean of the Graduate School
I understand that my dissertation will become part of the permanent collection
of Oregon State University libraries. My signature below authorizes release of
my dissertation to any reader upon request.
en Wu, Author
ACKNOWLEDGEMENTS
I would like to express my sincere appreciation to my major adviser,
Dr. Mark Daeschel, for his constructive guidance and continuous support during
this research and in the preparation of this dissertation.
Special thanks and appreciation are also extended to: Professor Floyd
Bodyfelt, Dr. Joseph McGuire, Dr. Ronald Wrolstad, Dr. Mina McDaniel, and Dr.
Charles Boyer for kindly serving as committee members when this project
started eight years ago, and especially, for their continual support and
encouragement during the writing of this dissertation five years after I left the
program! (Please feel free to use me as an example for any of your graduate
students who dares to think about taking a job before finishing his writing. Don't
even think about it!)
I would like to thank the Eckelman Foundation for granting me the
fellowship to pursue this project. And I am also grateful to Kerry Group, for
supporting me financially to complete this project through its employee
continual education program.
I wish to thank Mr. Dustan Doud, my dear colleague at Kerry, for proof
reading this dissertation and his insightful comments.
I wish to thank my wife, Tianying Jiang, for her love, help, and
encouragement during these long years; my six year-old son, Tommy; four
year-old daughter, Tina, for the enormous joy, love and meaning they have
added to my life (giving them a piggy-back ride was my favorite break after
hours of writing, or, hair-pulling, on a weekend day; the problem part, though,
was who got to ride first).
Finally, I want to thank my parents for kindling my passion for science
and their never-ending support of my dreams and quests. Without their
unconditional love and help, I would never have been able to come this far.
TABLE OF CONTENTS
Page
1. INTRODUCTION
1
I. Lysozyme
1
Structure of Lysozyme
Lytic Mechanism of Lysozyme
Properties of Lysozyme
Assays of Lysozyme
Application of Lysozyme in Foods
II. Enzyme Immobilization - An Overview
Physical Adsorption
Entrapment
Covalent Bonding
Properties of Covalently Immobilized Enzymes
1
7
10
17
19
23
24
27
28
34
III. Immobilization of Lysozyme
40
IV. Approach of This Study-The His-15 and Spacer Strategy
43
2. MATERIALS AND METHODS
I. Solid Phase Spacer Synthesis
Preparation of t-(Boc)-6-Aminocaproic acid
Purity of t-(Boc)-6-Aminocaproic acid TLC
Attachment Boc-6-Aminocaproic Acid to the Resin
Stepwise Synthesis of 6-Aminocaproic Acid peptide on the Resin
Quantification of 6-Aminocaproic acid by HPLC
II. Attachment of Lysozyme to the Spacer thorough His-15
47
47
49
49
50
51
53
55
Bromoacetylation of 6-Aminocaproic Acid
55
Coupling of Lysozyme onto the Support via Bromoacetylated Spacers- 57
Amino Acid Compositions of the Immobilized Lysozyme
58
III. Controls
Covalent Binding via EDC Coupling As Controls
58
58
TABLE OF CONTENTS (Continued)
IV. Determination of Lysozyme Activity
63
Activity against M. lysodeikticus cells
Free Enzyme
Immobilized Enzyme
Activity against Glycol Chitin
Free Enzyme
Immobilized Enzyme
63
63
64
64
65
65
V. Others
66
Effects of pH on the Activity of Lysozyme - Free and Immobilized
66
Effect of Inhibitor Pentane on the Activity of Immobilized Lysozyme —66
Kinetics Study of Immobilized Lysozyme
66
3. RESULTS AND DISCUSSION
67
I. Solid Phase Spacer Synthesis
67
II. Attachment of Lysozyme
69
Covalent Coupling through Carboxyl Groups of Lysozyme
Covalent Coupling through His-15
Efficacy of the His-15 with Spacer Strategy
Activity against Soluble and Insoluble Substrates
Kinetics Study of the Immobilized System
Effect of pH on Activity of the Immobilized Lysozyme
Hydrophobicity of the Polystyrene Support
Stability of the Immobilized Lysozyme
69
72
74
78
78
86
88
89
4. CONCLUSION
93
BIBLIOGRAPHY
95
APPENDICES
107
Appendix A. On the Initial OD Rise in the First Few Seconds
in the Turbidity Assay of Lysozyme - the Puff Theory
107
Appendix B. HPLC Assay of Lysozyme in Wine and Beer
111
LIST OF FIGURES
Figure
Page
1.
Lysis of the NAG-NAM polysaccharide composing cell walls
2
2.
Amino acid sequence of hen egg-white lysozyme
4
3.
Two representative cartoon maps of the three dimensional
structure of hen-white lysozyme
5
4.
Lytic mechanism of lysozyme (part one)
8
5.
Lytic mechanism of lysozyme (part two)
8
6.
Possible modes of enzyme immobilization
25
7.
Schematic illustration of conformational changes and
steric hindrances
37
8.
Proposed model for the lysozyme immobilization scheme
46
9.
The basic plan and chemical reactions of solid phase peptide
spacer synthesis
10. The modified seperatory funnel used as the reaction vessel
for solid phase spacer synthesis
11.
Chemical reactions involved in attaching 6-aminocaproic acid
to the £2-nitrogen of His-15 of lysozyme
12. Coupling of lysozyme to the reactive amine group on the support
via 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC)
activation of carboxyl groups of the enzyme
13.
Eadie-Hofstee plot of immobilized lysozyme (100 mg SAA.His-IS)
against M. lysodeikticus (0.05-0.175mg/ml)
14. Lineweaver-Burk plot from the same data set of Figure 13.
15.
Effect of pH on the lytic activity of free and bound
lysozyme (3AA,His-15)
48
52
56
60
81
83
87
LIST OF FIGURES (Continued)
16. Stability of bound and free lysozyme over a period of 14 days
when stored at 40C
91
A 1. The OD rise observed during the first few seconds of turbidity assay
lysozyme activity of samples with three different concentrations:
top line - 2.5 ppm, middle line - 1 ppm , bottom line - 0.1 ppm
108
A 2. The initial OD rise of a lysozyme sample at extremely low
concentration - 0.001 ppm
110
B 1. Detected lysozyme concentration of lysozyme samples without
heat treatment from the HPLC and Turbidity methods
114
B 2. Detected lysozyme concentration of lysozyme samples
treated at 750C for 1,2,3, and 4 hours
115
B 3. Detected lysozyme concentration of lysozyme samples
treated at 95C for 1,2,3, and 4 hours
116
LIST OF TABLES
Table
Page
1.
Properties of hen egg-white lysozyme
11
2.
Reactive residues of proteins
30
3.
Average amino acid composition of proteins reactive residues
4.
Number of reactions in which amino acid participates
32
5.
Reactivity of low-molecular-weight reagents used to chemical
Modification of amino acid residues of enzymes
33
6.
Desired characteristics for a protein support
35
7.
Summery of reported studies on lysozyme immobilization
42
8.
Deprotection and coupling cycles for solid-phase peptide synthesis
using Na - Boc-6-aminocaproic acid and ester linkage
to polystyrene-resins
54
Amino acid analysis of the peptide spacers synthesized on
the polystyrene beads
68
10. Activity of lysozyme covalently attached polystyrene beads
via 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC),
with and without NAG protection
71
11. Amino acid compositions of lysozyme immobilized through His-15
73
12. Characterization of polystyrene-lysozyme catalysts
75
13. Specific activity of lysozyme against two different substrates
79
14. The inhibition effect of 4%(v/v) pentane on the activity of free and
immobilized lysozyme against M. lysodeikticus
90
9.
32
B 1. Comparison of Lysozyme activity in red wine and beer samples
detected with HPLC and turbidity methods, respectively
117
B 2. Releasing lysozyme from lysozyme-tannin complex in red wine
with addition of NaCI at 2% and 8% levels
118
Immobilization of Hen Egg White Lysozyme by the Sole Histidine Residue
to Polystyrene Beads through Peptide Spacers
CHAPTER 1
INTRODUCTION
I, Lysozyme
Structure of Lysozyme
Lysozyme [EC 3.2.1.17] is an enzyme that catalyzes the hydrolysis of a
polysaccharide that is the major constituent of the cell wall of certain bacteria.
The polymer is formed from p (1 —> 4) linked alternating units of Nacetylmuramic acid (NAM) and /V-acetylglucosamine (NAG). The enzyme
cleaves the glycosidic bond between the C-1 atom of NAM and 0-4 of NAG of
in the bacterial cell wall polysaccharide (Figure 1.) This action weakens the
covalent structure of the wall and reduces its resistance to osmotic swelling, as
a result of which the cells burst.
First discovered in 1921 by Fleming in the mucous of humans (Fleming,
1922), lysozyme has since been found widely distributed in animals, plants and
microorganisms - hen egg albumin and other avian egg-whites (Fukamizo et
al. 1983), animal tissues and serum (Jolles and Jolles, 1961; Wardlaw, 1962;
Pavlovskii et al., 1976), fruits and vegetables (Chandan and Ereifej, 1981;
GlcNAc
MurNAc
GlcNAc
MurNAc
Glc NAc
MurNAc
CHj
0,=— CH,CH
R, = —NH— C — CH,
I
^= — 01— CO^H
Figure 1. Lysis of the NAG-NAM polysaccharide composing cell walls. The
hexasaccharide composed of alternating A/-acetylmuramic acid (NAM) and Nacetylglucosamine (NAG) residues as found in the natural cell wall
peptidoglycan of gram(+)bacteria. The saccharine units are thought to bind to
successive sites on lysozyme labeled A through F.
Law and Goodenough, 1995), and bacteria (Richmond, 1959). It is considered
a natural defense mechanism of its host system against bacterial invasion
(Boiler etal, 1983)
Hen egg white lysozyme is the best-characterized member of the
lysozyme family. Some unique properties of the enzyme such as abundant,
easily crystallized, and highly stable have made it an excellent model for
extensive protein and enzyme studies.
The solution of the structure of lysozyme is one of the triumphs of X-ray
crystallography. In 1963, an investigation using X-ray crystallography at 6 A
(1 A = 10nm) resolution revealed that hen egg white lysozyme consists of
relatively small single polypeptide chain of 129 amino acids cross-linked by four
disulfide bridges, which contribute to its high stability (Canfield, 1963). The
enzyme has a mass of 14.6 kdal and its amino acid sequence is shown
In Figure 2. Two years later, three-dimensional structure of hen egg white
lysozyme was determined through 2 A resolution X-ray crystallography (Blake et
al., 1965). Results showed that lysozyme is a compact molecule, roughly
ellipsoidal in shape, with dimensions 450 X 300 X 300 nm.
The high resolution X-ray crystallography also revealed that the oval fold
of the protein can be divided into two lobes or domains with the active site of
enzyme being situated in a well-defined cleft between them for binding the
substrate (Figure 3). One of the domains is almost entirely (3-sheet structure
(encompassing residues 40 to 85), whereas the other is comprised of the N and
C-terminal segments (residues from 1 to 39 and 101 to 129) and
Figure 2. Amino acid sequence of hen egg-white lysozyme. (Adapted from
Canfieldand Liu, 1965)
ACTIVE SITE
Figure 3. Two representative cartoon maps of the three dimensional structure
of hen-white lysozyme (Adapted from Acharya et al., 1989)
is more helical in nature. The two domains are linked by an a-Helix (residues
89 to 99). The cleft in between is partly lined with nonpolar side chains of amino
acids for binding the nonpolar region of the substrate, and it also has hydrogenbonding sites for the acylamino and hydroxyl groups (Fersht.igSS).
Subsequently, the structure of hen egg-white lysozyme has also been
determined from tetragonal crystals under a variety of conditions of temperature
and pressure (Kundrot and Richards, 1987) and from triclinic, monochlinic and
orthorhomic cystal forms (Joynson et al., 1970; Moult et al., 1976; Artymiuk et
al., 1982; Hodsdon et al., 1990). All of these studies indicate that the
conformation of lysozyme is essentially identical under the different conditions
and crystalline environments.
In the meantime, studies have also been done to compare the structures
of the enzyme in crystalline state and in solution. NMR studies of hen eggwhite lysozyme in solution gave data consistent with its crystal structure
(Dobson, 1977; Blake et al., 1981). A more recent NMR study (Smith et al.,
1992) indicated that the main-chain fold of hen egg-white lysozyme is very
similar between the two states. Further studies using Raman spectroscopy,
NMR and inelastic neutron scattering have shown that the incorporation of
water produces only small changes in protein structure and flexibility necessary
for its enzymatic activity (Poole, 1994). These research results suggest that the
structural data obtained from crystallized lysozyme can be applied to lysozyme
molecules in solution with good confidence.
Lytic Mechanism of Lysozyme
The currently accepted mechanism of lysozyme action was elucidated
mainly from examining the 2 A resolution X-ray crystallography data of the
native enzyme and the complexes with inhibitors through model building (Blake
et al., 1967). The catalysis begins when the enzyme's active site or the cleft
selectively meshes with a six sugar segment (termed A through F in Figure 1.)
of the alternating NAG-NAM polysaccharide substrate. When the segment
binds, the D residue is distorted into a half-chair form from its normal chair form
to fit into the active site, thereby weakening the glycosidic bond. This is an
important aspect of the mechanism because the half-chair form is assumed to
be the conformation of the transition state. In other words, in the process of
binding, the enzyme forces the substrate to assume the geometry of the
transition state.
The remaining catalytic mechanism is comprised of the following five
steps:
1 The -COOH group of Glu-35 donates an H+to the bond between C-1
of the D residue and the glycosidic oxygen atom, which thereby cleaves the
bond (Figure. 4);
2.This creates a positive charge on C-1 of the D residue or a carbonium
ion;
Glu
35
V
V
V
;c- Asp
52
Asp
52
Figure 4. Lytic mechanism of lysozyme (part one). A key step in the catalytic
mechanism for lysozyme is the transfer of an hTfrom Glu 35 to the oxygen
atom of the glycosidic bond. The glycosidic bond is thereby cleaved, and a
carbonium ion intermediate is formed.
Glu
35
1/H
oA
D
V
NAG,
Glu
35
<\
Asp
>- 52
-o
>
A~-H
k
r-\
H0N
H
/s
D
V
0
>-\3\
|N;*GI|
Figure 5. Lytic mechanism of lysozyme (part two). The hydrolysis reaction is
completed by the addition of OH" to the carbonium ion and of H+ to the side
chain of Glu 35.
3. Negatively charged Asp 52 stabilizes the carbonium ion by interacting
electronically with the positive charge on C-1 of the D residue;
4. The carbonium ion intermediate then reacts with OH" from the solvent.
The hydrolyzed product diffuses away from the enzyme (Figure 5)
5. Glu 35 becomes protonated, and the enzyme is ready for another
round of catalysis.
The crucial roles of Glu 35 and Asp 52 in the catalytic mechanism have
been proved by selective modification experiments. Lysozyme derivatives
modified at Glu 35 and Asp 52 were totally inactive, whereas it remained active
when all of its carboxyl groups except those of Glu 35 and Asp 52 were
esterfied (Parsons et al., 1969; Eshdat et al., 1973,1974; Yamada et al., 1982).
When lysozyme was pretreated with NAG oligosaccharide - a substrate analog,
however, Glu 35 and Asp 52 could not be modified by the esterfying reagents
and the enzyme remained active (Lin and Koshland,1969).
In the meantime, genetic modification of the two amino acids has been
reported to examine their roles in the proposed mechanism. Site-directed
mutagensis in which Glu-35 and Asp-52 were converted to the corresponding
Asn and Gin demonstrated the critical requirement for Glu-35 - 0% activity from
the Gin mutant, and slightly lesser essentially of Asp 52 - 0.5% activity from the
mutant (Malcolm, et al., 1989).
Studies also showed that the electrostatic force of attraction between
lysozyme molecule and cell wall of M. lysodeikticus plays an important role in
the initial binding process. The cell wall of the bacteria is negatively charged
10
when the cells are suspended in buffer solutions with normal pH range
(Yamasaki et al. 1968; Maurel and Douzo, 1976; Price and Pethig, 1986).
Electrophoresis measurements on the bacterial cells have shown that the net
surface charge density on the cell wall is constant at around -1 .SpC/cm for the
pH range 4-8. Since lysozyme has an isoelectric point around pH 11.2 (Imoto et
al., 1972), it carries a net positive charge in the same pH range.
Therefore from a macroperspective, the lysis of bacterial cells by
lysozyme consists of the following three steps: The first step is an interaction
between the positive charges of lysozyme and the negative charges on the
surface of the bacterial cells. The second step of lysis is hydrolysis of p-1,4glucosaminide linkage in the polysaccharide of the cell wall. The last step is
dissolution of the damaged cell wall.
Properties of Lysozyme
Just like other enzymes the stability and activity of lysozyme are affected
by temperature, pH, ionic strength, and specific inhibitors and enhancers.
Some of the general properties of lysozyme are summarized in Table 1.
Factors Affecting Lytic Activities of Lysozyme
Lysoyzme is highly heat stable. In phosphate buffer at pH 6.2 highly
purified lysozyme showed only 5% loss of lytic activity after being exposed to
11
Table 1. Properties of hen egg-white lysozyme. Notice the two pH optima
depending on the ionic strength (/).
MW
(kDa)
14.6
Shape/Size
(nm)
Oval/450x300x300
pi
11.2
Opt.pH
Opt./*
Opt.Temp
(0C)
6.2
0.06-0.07
25-28
But heat stable
up to 100 at
Opt.pH & Opt./
9.2
0.04-0.05
' / = Vz Z C/Z,2, where c = ion concentration in moles; z = valence.
12
80 0C for 80 min. When exposed to100 0C for 20 min it lost only 25%
(Smolelis and Hartsellf1952). Increasing the proton concentration has shown
to increase the heat stability of the lysozyme. Beychock and Warner (1959)
reported that stability in the temperature range of 85 to 950C was maximum at
pH 5.5, 5% more active than that at pH 6.5.
The extraordinary heat stability of lysozyme is mainly attributed to the
four disulfide bridges formed between Cys64 - Cys80, Cys76 - Cys94, Cys30 Cys115, and Cys6 -Cys127. It has been shown that the Cys6-Cys127 bridge,
which basically ties the head and tail of the polypeptide together (remember
lysozyme has 129 amino acid residues), is not required for the complete folding
of the protein since the protein already folds efficiently to its native state with
other three disulfide bridges (Eyles et al., 1994). The role of the disulfide bridge
appears to be solely strengthening the structure of the already fully functional
enzyme rather than inducing structure into the polypeptide chain. In addition,
the hydrophobic core of the enzyme is also thought to contribute to its stability,
which is formed with hydrophilic amino acid side chains protruding toward the
surface.
The lytic activity of lysozyme varies markedly with the ionic strength of
the solution in which is dissolved. When ionic strength is at 0.06-0.07, it has an
optimum pH of 6.2; when at 0.04 - 0.05, it has a second optimum pH of 9.2
(Davies et al., 1968). In the complete absence of salts, or at an ionic strength
of 0.005 or less, lysozyme has little or no activity on cell walls. This observation
13
was confirmed by Chang and Carr (1971) who found lysozyme to be inactive in
distilled water.
While many other investigators only observed the first pH optimum of
6.2-6.7 using strong buffer solutions, the appearance of the second pH
optimum at lower ionic strength was also reported by Rupley (1967) and
Neuberger and Wilson (1967). It was suggested that at pH 9.2 the zammonium group of Lys-97 acts as a proton donor to form the glycoside
carbonium ion and that the negative charge on the carboxyl group of Asp-101
stabilizes the intermediate carbonium ion in accordance with the Glu-35 and
Asp 52 mechanism at lower pH of 6.2 (Neuberger and Wilson, 1967, Davies et
al. 1968)
Inhibitors
Knowledge on the inhibitors and activators of lysozyme is very important
for studying its properties as well as its practical application. Lysozyme has
been shown to be inhibited by a serious of agents which can be summarized
into three groups: (1) surfactants, (2) nonpolar hydrocarbons, and (3) acidic
polymers. When lysozyme is used as an antimicrobial agent, efforts should be
made to minimize the presence of these agents in the system.
Surface-active reagents inhibit lysozyme by binding to the enzyme at or
near the active site. Lysozyme has been shown to be inhibited by sodium
dodecyl sulfate (Meyer et al., 1947) and in general by alkyl sulfates, fatty acids,
and aliphatic long-chain alcohols of 12 carbons or more (Smith and Stocker,
14
1949). It has been reported that 2.8% sodium dodecyl sulfate abolishes
interaction between the substrate tri-A/-acetylchitotriose and lysozyme (Rupley,
1967). In addition, this concentration has been shown to produce no change in
the helix content of the protein, but to alter the character of its tryptophan
chromophores through specific interaction with them (Glazer and Simmons,
1965). A Tryptophan residue has been shown to be in involved in the formation
of the enzyme-substrate complex between lysozyme and poly-A/acetylglucosamine (Hayashi et al., 1968).
The second group inhibitors are nonpolar hydrocarbons, such as
isobutane and pentane. They interact with the hydrophobic regions of
lysozyme by hydrophobic bonding, thus render the enzyme inactive due to
conformational change (Watanabe and Takesue, 1972, 1974). The higher the
concentration of pentane and the lower the ionic strength, the stronger the
inhibition.
The last group of inhibitors are acidic organic polymers, including
submaxillary mucoid (Simmons, 1952), hyaluronic acid, pneumococcus
polysaccharide, glutamyl polypeptide, DNA, RNA (Skarnes and Watson, 1955;
Wang et al., 1990), E. coli lipopolysaccharide (Ohno and Morrison, 1989), and
tannin (Green, 1995). It is established that this group of agents inhibits
lysozyme by forming lysozyme-polymer complex through electrostatic
interaction between the basic enzyme and the acidic polymers. The inhibition
tends to be very strong and yet usually pH sensitive and can be reversed by
concentrated urea or sodium chloride solutions. It is worth pointing out that
15
majority of the above polymers are produced by bacteria which serve as their
defense against lysozyme.
Lytic activity of lysozyme on Gram-negative bacteria
While lysozyme lyses many, though not all, Gram-positive bacteria in
their vegetative forms, it is generally inactive against gram-negative bacteria
because their outer membranes prevent access to the underlying peptidoglycan
that is the enzyme's substrate. Many efforts, however, have been made to
broaden the antimicrobial spectrum of lysozyme, by either changing the
structure of the enzyme or pretreating the target microbial cells. All these efforts
have potential value in food preservation and safety.
Conjugation of lysozyme with dextran through the Maillard reaction
increased its activity against both gram-positive and gram-negative bacteria
(Nakamura et al., 1990). Further study from the same group indicated that the
lysozyme-dextran conjugate also has greater heat stability and emulsifying
properties than native lysozyme. In addition, its activity against gram-negative
bacteria significantly increased if the temperature was raised during the
conjugation treatment (Nakamura et al., 1992). Lysozyme modified with four
perilaldehyde residues exerted a markedly enhanced antimicrobial activity
against both gram-negative bacteria (Escherichia coli K12) and gram-positive
bacteria (Staphylococcus aureus) (Ibrahim et al., 1994)
More recently, Ibrahim et al. (1996) reported an interesting finding that
partial unfolding of lysozyme with heat treatment at certain pH could switch its
16
antimicrobial activity to include Gram-negative bacteria without a significant
detrimental effect on the inherent bactericidal effect against Gram-positive
ones. It was found that heat denaturation of lysozyme at increasing
temperatures for 20 min at pH 6.0 resulted in progressive loss of enzyme
activity which greatly promoted its antimicrobial action to Gram-negative
bacteria. The most potent antimicrobial lysozyme to either Gram-negative or positive bacteria was that heated at 80°C and pH 6.0, retaining 50% of the
native enzymatic activity. The partially unfolded enzyme exposed two thiol
groups and exhibited a 14-fold increase in surface hydrophobicity. Addition of
the heat-treated enzyme to E. coli phospholipid vesicles resulted in a blue shift
in the intrinsic tryptophan fluorescence accompanied by an increase in the size
of the vesicle, indicating enhanced protein-membrane binding and subsequent
fusion of liposomes. The authors claimed that the unique antimicrobial action of
unfolded lysozyme attributed to membrane binding and subsequent
perturbation of its functions.
According to Samuelson et al.(1985), pretreatment with
ethylenediaminetertraacetate (EDTA) significantly sensitized Salmonella to the
action of lysozyme. Gram-negative bacteria, including salmonellae, became
lysozyme-sensitive following a quick osmotic downshift - achieved by sudden
dilution of a salt solution. This process has been proposed as the basis for a
dip- or spray-decontamination treatment for poultry and other animal carcasses
(Chatzolopou etal., 1993). After being treated with 0.5 to 5mM trisodium
phosphate for 10 min, Campylobacterjejuni, Escherichia coli, Pseudomonas
17
fluorescens, and Salmonella enteritidis showed greatly increased susceptibility
to lysozyme (Carneiro et al., 1998)
Assays of Lysozyme
A survey of literature reveals that most methods for determining
lysozyme activity involve measuring turbidity change of its substrate
spectrophotometrically. The lysis of bacterial cells suspended in a buffer
system results in decreasing solution turbidity. Under proper conditions, there is
linearity between the concentration of cell suspension and the turbidity, or
absorbance at certain wavelength. Therefore the absorbance-changing rate of
a cell suspension can be used as an indirect measure for the activity lysozyme
in the system.
Most of the assays were developed in the 1950s. Some workers have
used Sarcina lutea cells as the substrate and a wavelength of 440 nm to follow
the cell lysis (Dickman and Proctor, 1952; Smith et al. 1955), while others have
used Micrococcus lysodeikticus cells as substrate and wavelengths of 450nm
(Shugar, 1952) and 540nm (Smolelis and Hartsell, 1952; Kerby and Eadie,
1953).
The Shugar (1952) method is a simple assay that measures initial
velocity and has good reproducibility (± 3%). It has become the most common
method used or adapted by most of the lysozyme research groups nowadays.
One unit of enzyme decreases the absorbance at 450 nm by 0.001/min at 250C
18
in 66mM phosphate buffer, pH 7.1, using a suspension of live Micrococcus
lysodeikticus as substrate in 3 ml reaction mixture (0.2mg/ml; 1 cm light path).
Two modifications are often made to improve the original method: (1)
replacing the vegetative Micrococcus lysodeikticus cells with standardized
lyophilized cells that are commercially available; (2) changing the buffer pH
from 7.1 to 6.24, which is closer to the optimum pH for the enzyme under the
assay condition. It has also been reported that preparing the cell suspension at
least 6 hours prior to use can improve reproducibility of the method after the
cells had been allowed to equilibrate with the buffer for this period (Parry et al.,
1965).
A limitation for the spectrophotometric method, however, is that it
requires that the enzyme sample must be in a clear solution, which often makes
it impractical for measuring lysozyme in many food systems, such as milk and
cheese. An extracting and decolorizing process might destroy the lysozyme
being measured. When this is the case, an agar plate method developed by
Gosnell et al. (1975) is especially useful to measure lysozyme in non-clear
background. Agar plates are prepared consisting of 1.0 agarose with 0.1g NaCI
in 100 ml of a pH 7.0 phosphate buffer seeded with 0.020 g lyophilized
Micrococcus lysodeikticus. Several equidistant holes are punched into the agar
and filled with lysozyme at various concentrations. After being incubated at
47°C for 3 hours, clear zones of lysed bacterial cells will appear around those
wells containing lysozyme. The base 10 logarithms of the clear zone diameters
are directly proportional to lysozyme concentration. As low as 5pg egg white
19
lysozyme per 100 ml of samples could be determined by this method. The agar
plate method can be modified by seeding the agar with living bacterial cultures
and using filter disks instead of punching holes (Vakil et al. 1969).
Another more fundamental limitation for the turbidity assay is that the
polymer substrate -cell walls is insoluble in buffer, albeit a homogeneous
suspension. Theoretically, we want to measure the glycosidic cleavage, but
what is actually measured is the reduction in turbidity of a suspension of dried
cells of Micrococcus lysodeikticus in aqueous buffer solutions. The change in
turbidity has not been correlated directly with the number of glycosidic bonds
the enzyme splits and therefore use of an insoluble substrate is especially
unsatisfactory for kinetic studies.
Efforts have been made to address the ill-defined insoluble substrate
issue. Assays have been developed using soluble oligo or poly NAG derivatives
as the substrate, such as carboxymethylchitin (Marzotto and Galzigna, 1969),
and glycol chitin (Imoto and Yagishita, 1971). These two assays were
monitored by viscosimetry and reducing group analysis.
Application of Lysozyme in Foods
Hen egg white lysozyme has been the most commercially used animalderived antimicrobial to improve the safety and shelf stability of foods.
Lysozyme constitutes 3-4% of the total egg white protein, so that it is readily
available and relatively inexpensive (Powrie and Nakai, 1986). In addition, it's
20
natural origin is very appealing to the food industry due to growing public
concerns about the safety of chemical preservatives and additives. Thus
numerous studies and applications have been developed to use lysozyme as
natural food additive to a wide range of foods with promising results.
While most of the pioneer work on using lysozyme as a preservative in
foods was done in Japan in late 1960s and early 1970s, concentrating on sea
food, meat, beverage, and vegetables, European researchers have mainly
focused on its application in dairy products in the past two decades. Lysozyme
is a legal and popular preservative in cheese and dairy products in Germany,
Italy and France (Anon, 1987). Lysozyme is recently granted GRAS status in
USA.
When lysozyme is used as a preservative in meat products, synergies
are often found between the enzyme and other conventional preservatives.
Akashi (1969) found that cured ground beef was preserved more effectively
with the combination of 3% of NaCI, 12.5 ppm of NaN02, and 50 or 200 ppm of
lysozyme than either by lysozyme alone or by the salts alone. In later work with
Vienna sausage Akashi (1971) found that dipping them in 500 ppm lysozyme in
pH 6.5 phosphate buffer gave the best results than using lysozyme alone, or
nitro-furylacrylamide and sorbic acid.
In a Japanese patent by the Eisai Company (1971), fresh seafoods were
claimed to be preserved in aqueous solutions containing a lysozyme salt, amino
acids, and NaCI. Later the same company patented another process to
preserve oysters and shrimps by soaking them in aqueous solutions of
21
lysozyme and NaCI (Eisai, 1972). The Eisai Company (1971) also patented a
process in which wine and sake were stabilized by incorporation of lysozyme
together with p-hydroxybenzoic esters.
Lysozyme was added to soy milk to preserve Tofu bean curd (Taiyo
Food Co., 1972) and dried milk compositions for pediatric use were preserved
by addition of lysozyme derived from egg whites along with ovalbumin and
ovomucin (Morinaga Milk Industry Co., 1970). Kanebo Ltd (1973) patented a
process to extend the shelf life of fresh vegetables, fish, meat and fruits by
coating the surface with lysozyme.
The major successful use of lysozyme commercially has been
eradicating cells of Clostridium tyrobutyricum as they outgrow from germinated
spores (Carminati et al, 1985; Bottazzi et al, 1993), thus control the so-called
"late blowing" in certain semi-hard and hard cheeses caused by fermentation of
lactate by the bacterium (Wasserfall and Prokopek, 1976; Carini and Lodi,
1982). The International Dairy Federation (1987) recommended the use of
lysozyme as good practice in controlling late blowing when the number of C.
tyrobutyricum spores in milk is in the order of hundreds. It has been estimated
that more than 100 tons of lysozyme are used annually for this purpose (Scott
etal, 1987).
Lysozyme has been reported to kill resting vegetative cells of C.
tyrobutyricum at room temperature and severely inhibit actively growing ones at
concentration of 500U/ml (Wasserfall and Teuber, 1979; Bester and Lombard
1990). Its effect on the spores involves a two-step process: It first stimulates its
22
germination; then significantly inhibits the outgrowth of spore cells into
vegetative cells (Wasserfall and Teuber, 1979; Bester and Lombard 1990).
Wasserfall and Teuber (1979) suggested that the inhibition of the outgrowth of
spore cells into vegetative cells was the basis for using lysozyme to control late
blowing.
Lysozyme has potential in the wine industry to be used as a control
measure for malolactic fermentation and preservative against bacterial spoilage
during wine conservation (Amati et al., 1992). In the course of winemaking,
malolactic fermentation (MLF) - the conversion of malic acid to lactic acid and
carbon dioxide by mainly Leuconotoc oenos, reduces acidity, thus has
significant effects on the sensory profile and microbial stability of wines
(Henick-Kling 1993). While MLF can be beneficial for high acid wines, MLF may
reduce acidity too much in high pH wines and cause proliferation of spoilage
lactic acid bacteria, such as Pediococcus sp. and Lactobacillus sp.(Rankine
and Bridson, 1971; Radler, 1975)
According to Green et al. (1994), lysozyme concentrations of 500 and
1000 ppm are effective at preventing MLF in red wine. The ability of lysozyme
to stop MLF midway through fermentation was demonstrated in the 300 ppm
lysozyme treatment. Similar results were reported later by Gerbaux et al.
(1997) that an addition of 500 ppm lysozyme to grape must inhibited MLF, while
addition of 250ppm to red wines, after MLF, promoted microbiological stability.
In the wines to which lysozyme was added, there was no increase in the
content of acetic acid and biogenic amines during a period of six months at 18
23
degree C. Whereas the levels of volatile acidity and biogenic amines in the
controls were 20% and 400% higher, respectively.
II. Enzyme Immobilization - An Overview
Enzyme immobilization is a physical or chemical process that fixes
enzymes onto a solid support, or traps them into an insoluble matrix. By so
doing, a soluble enzyme is converted into bound or insoluble form but remains
biologically active with regards to its substrate. The technique has two major
advantages: the enzymes can be recovered and used again for research or
application purposes; the enzyme can be used in a variety of configurations of
bioreactors that allow continuous operation. Immobilized enzymes also often
have improved storage and operational stability.
Enzyme immobilization methods may be divided into three categories:
The enzyme can be (1) adsorbed onto, (2) physically entrapped or
encapsulated within, and (3) covalently bound to a distinctive phase that allows
exchange with, but is separated from, the bulk phase in which substrate is
dispersed. Figure 6 lists possible modes of enzyme immobilization. The
covalent binding approach has been by far the most widely investigated and it
has been the top choice thus far for most of the lysozyme immobilization
studies. Therefore, this overview will rather be strongly oriented towards
methods based on the covalent immobilization of enzymes.
24
Immobilized enzymes are a qualitatively new type of biocatalyst different
from the free enzymes in solution, not only in their physical properties, but also
in many other biochemical characteristics. After being
fixed to an insoluble support the enzyme is in a new state in which its properties
are influenced by different factors related to the chemical and structural nature
of the support, as well as to the way that the enzyme is attached to the matrix
structure. A discussion of the factors affecting the characteristics of
immobilized enzymes is also included at the end of this overview.
Physical Adsorption
The oldest and simplest method of enzyme immobilization is that of
physically adsorbing the enzyme on to a solid matrix. Eighty eight years ago
Nelson and Griffin (1916), reported that invertase retained its catalytic ability
after adsorbed on to activated charcoal. Most of the later developments in this
area took place between 1965 and 1975. Wide ranges of materials have
25
(1) Covolent bonding
(2) Eleclrostotic bonding
(3) Copolymen'zcjfion
(A) Polymei entropment
':;[$ Enzyme molecule
I
(5) Hydrophobic interoction
/A,
Phospholipid
Polymer matrix
Pf©©
(6) Liposmal entrapment
(7) Encapsulation
Figure 6. Possible modes of enzyme immobilization (from Trevan, 1980).
26
been used as absorbents for various proteins. Synthetic ion exchange resins,
though originally designed for chromatographic purposes, such as Dowex 50
(Barnett and Bull, 1956), DEAE- and CM-cellulose (Mitz, 1956), have been the
most extensively used. Other materials often used, but usually with lower
adsorption capacities, include polystyrene resins, kaolinite, collagen, aluminum,
silica gel, and glass.
Immobilization by physical adsorption is a very easy to perform: the
adsorbent and enzyme are stirred together for sometime. Yields (enzyme
bound per unit of adsorbent) for physical adsorption can be high. However, the
process is non-specific and a real or apparent decrease in activity will ensure if
adsorption involves groups at the active site. Protein-protein interaction resulted
from overloading can also lead to apparent loss in catalytic activity (Bernath
and Veith, 1974). During operational use adsorbed enzymes may leak from the
carrier because of the binding force(s) - ionic, hydrophobic, hydrogen bonds, or
Van der Waals' interactions, between the protein and the carrier is weak.
Another factor which often causes desorption is the addition of substrate to the
enzyme preparation (Trevan, 1980). This is particularly problematic, for
although other factors which might cause desorption (such as pH, temperature,
or ionic strength) can be controlled, no enzyme can work without its substrate.
One interesting development of the technique of adsorption is in the use
of an activator of an enzyme as a coupling agent between a support and that
enzyme. Fukui et al. (1975) demonstrated the immobilization of tyrosinase and
tryptophanse by adsorption on to an insoluble derivative of pyridiosal-5'
27
phosphate, an activator of these enzymes. Thus the enzyme is not only
specifically adsorbed on to the polymer, but is also activated by the same
process.
Entrapment
The entrapment technique is a growing development in enzyme
immobilization technology (Kumaraswamy et al., 1981; Stevenson and Sefton,
1987). The method is based on enclosing enzymes in the lattice of a polymeric
matrix or semi -permeable membranes, such that the substrate and products
are able to diffuse through the enclosure while the enzyme proteins are too
large to escape. A minor loss of biological activity is one of the main positive
features of the entrapping technique, simply because the enzyme is not actually
attached to anything. There are none of the steric problems associated with
covalently binding the enzyme in such a way that its active site is obstructed by
a portion of the polymer matrix.
On the other hand, the diffusion limitations are the most negative feature
of the method. This type of enzyme immobilization is obviously not suitable for
enzymes with high molecular weight substrate, such as lysozyme.
28
Covalent Bonding
Compared to physical adsorption and entrapment methods, the
immobilization of enzymes on solid supports by covalent coupling usually leads
to very stable preparations with extended active life. Large scale
experimentation in this field was first carried out by Grubhofer and Schleith in
1953, who used a diazo derivative of poly-p-aminostyrene to immobilize pepsin,
amylase, and carboxypeptidase (Grubhofer and Schleith,1954). Since then
covalent binding has become the most frequently used and investigated
approaches for enzyme immobilization, mainly due to the growing discovery of
various bonding reactions and of matrices with functional groups capable of
covalent coupling, or susceptible to activation to provide such groups.
The compositional and structural complexity of proteins has not allowed,
however, the application of general rules to predict the best method suited for a
specific immobilization task. Accumulated experience in the field has
emphasized the importance of two factors that have to be evaluated when
choosing a method for the covalent immobilization of an enzyme: (1) the type of
functional group on the protein through which the covalent bonds with the
support matrix are formed and hence the chemical reaction to be used; (2) the
physical and chemical characteristic of the support matrix onto which the
reactive groups are to be coupled.
29
Functional Groups and Coupling Reactions
The rule of thumb for choosing the functional groups on the enzyme
through which the covalent bond with the support is to be formed is that they
should be naturally nonessential for the catalytic activity of the enzyme. In the
meantime the binding reactions that can be carried out under relatively mild
condition should be preferred. Such reactions should exhibit, under ideal
conditions, relatively high specificity toward one type of functional group on the
protein and minimal side reactions with other functional groups.
In practice, however, such a situation is seldom if ever realized. For the
immobilization of a given enzyme, the information is often limited on its amino
acid composition, the amino acids involved in the active site, the effects of
specific chemical modifications on activity, the protection of the active site
region by inhibitors, as well as the three-dimensional structure of the enzyme.
Indeed, so far the reaction and support selecting process is still very
much empirical, heavily relying on the methods used in the chemical
modification of proteins. It is not uncommon that several different reactions and
supports have to be tested for a particular immobilization task before a
satisfactory preparation can be achieved (Cabral and Kennedy, 1991).
The selection of methods applied to the chemical modification of
enzymes has been based on knowledge of the reactivity, frequency of
occurrence (relative concentration) and the accessibility of the amino acid
residues of the enzyme. Table 2 summarizes the residue of amino acids that
have functional groups in the side chain suitable for linking to a support.
30
Table 2. Reactive residues of proteins (from Taylor 1991)
f-Amino of r.-lysinc (i.-Lys) and N-tcrminus amino group
Thiol of i.-cysteine (i.-Cys)
Carboxyi of i.-aspartate (i.-Asp) and i.-glutamate (1..-GI11)
and C-terminus carboxyi group
-NH2
-SH
-COOH
<y
OH
Phenolic of L-tyrosine (r-Tyr)
NH
N-C
\
Guanidino of i.-arginine (i.-Arg)
NH,
Imidazole of i.-hislidinc (i.-His)
Disulfide of i.-cystinc
Indolc of i-tryptopluin (i.-Trp)
CH3-S-CH2OH
Thioether of i..-nietl)ionine (i-Met)
Hydroxyl of i.-serine (i.-Scr) and i.-threoninc (i.-Thr)
31
From this list, notice that the amino acids with amide groups (glutamine
and asparagine), those with hydrocarbon side chains (alanine, leucine,
isoleucine, valine, phnenylalanine, and proline), and glycine are absent due to
their relatively low concentration on the exposed protein surfaces and, more
importantly, due to their non reactive hydrophobic nature.
For enzymes with limited information of their structure and amino acid
composition, data in Tables 3, 4 are often used to access the most convenient
residues in the enzyme that is to be covalently immobilized. Comparing the
data in the two tables, it appears that the most convenient residues for
immobilization are the lysyl residues, followed by cystein, tyrosine, histidine,
aspartic acid, glutamic acid, arginine, tryptophan, serine, threonine, and
emthionene.
In practice, most of the common covalent coupling reactions
involve amino groups, carboxyls, or the aromatic rings of tyrosine and histidine.
After the residues are chosen, suitable reactions may be selected
according to the rules governing chemical modification of protein, which is
summarized in Table 5 by Means and Feeney (1971).
Polymeric Supports
After the coupling reaction is selected that would preserve the most
activity, proper support(s) can be chosen to fit the final application
32
Table 3. Average amino acid composition of proteins reactive residues
(from Means and Feeney, 1971).
Residue
Percent
Ser
Lys
Thr
Asp
Glu
Arg
Tyr
Cys
His
Met
Trp
7.8
7.0
6.5
4.8
4.8
3.8
3.4
3.4
2.2
1.6
1.2
Table 4. Number of reactions in which amino acid participates (from
Means and Feeney, 1971).
Residue
Number of Reactions
Cys
Lys
Tyr
His
Met
Trp
Arg
Glu
Asp
Ser
Thr
31
27
16
13
7
7
6
4
4
0
0
Table 5. Reactivity of low-molecular-weight reagents used to chemical modification of amino acid residues of enzymes (from Means and Feeney,
1971).
Reactivc amino acid residue of enrvmc'
Reaecm
-NH,
Acetic anhydride
Aldchyde/NaBHj
N-Carboxyanhydrides
Cyanogen bromide
Diiizonium salts
5,5'-Dithiot!!S(2-ni'roben7.0!c
acid)
Formaldehyde
Clyoxal
Haloacctatcs
Maieic anhydride
p- Mercu riben/oa EC
Sodium borohydride
Succitnc anhydride
Thiols
Wator-solublt carbodiimide
and iuicicophik-
-SH
vL
--COOH
K>
.S--S-
SCH,
z
I
-r -f
+ 4- -•--
+++
+++
' - . • . • -. dnd • - • (ndrc.Hc rciativf rcaciivitirf -^. ± ±. ami •*: ± ~ Ufctfu-tv indicate ttm-.^r f^Aciivittci which toa? nr tn;(;-- n'.« nt :u;;uricd depcndirj; upon the E-i/uthuor^ t?iRpi>tu:J.
' Spunliitieuy.tiy n'vrr*:h:c im-Jirr ihi: reaciion ^vniiuiv-^ or wpon diiuhon. rcgcncrnling origimi! aroup.
Fasiiv rcscrsibir. ri-r.cncrstin* orijiina! jiroiir-.
34
accordingly. A carrier judiciously chosen can enhance the immobilized protein
preparation. The primary base supports for enzyme covalent bonding are
mainly polysaccharides and other polymers, including: agarose, cellulose,
dextrans, chitosan, alginate, collagen polyacrylamide, nylons, and polystyrenes,
silica gel and glass beads, etc. The properties of these supports are thoroughly
reviewed by Cabral and Kennedy (1991).
Although there is no universal carrier, a number of desirable
characteristics should be common to any material considered for immobilizing
enzymes (Table 6). Important support properties, such as morphology (shape,
size, surface, porous vs. nonporous, surface area, etc.), mechanical and
microbial stability, and modification, should be assessed with respect to the
specific application prior to the selection for evaluation.
Properties of Covalently Immobilized Enzymes
While in many cases the stability of enzyme is increased by
immobilization (Zaborskey, 1973) mainly because of the prevention of autolysis
by restricting intermolecular contact, according to most studies the activity of
immobilized of immobilized enzymes has been lower than that of the same
amount of soluble enzyme at a given concentration of substrate and effectors
(Engasser and Horvath, 1976). The changes in the enzymatic behavior
35
Table 6. Desired characteristics for a protein support.
Large surface area
Good permeability
Hydrophilic character
Chemical, mechanical, thermal, microbial stability
High rigidity
Suitable shape and particle size
Regenerability
36
due to immobilization in a heterogeneous medium can be attributed to two main
factors. The first involves changes in the conformation of the enzyme molecule
and steric hindrance dependent on the mode of enzyme binding to the carrier.
The second factor concerns the non-uniform distribution of substrate, products
or protons between the immediate vicinity of bond enzyme, often called the
microenvironment, and the bulk solution, or macroenvironment, where effects
related to mass transfer often arise, such as diffusion restrictions of substrate
and products to the active site of the enzyme and back into the bulk solution.
Conformational Change and Steric Hindrance
The decrease in enzymatic activity upon immobilization is often caused
by conformational changes in the enzyme structure or by steric hindrance s in
the immediate vicinity of the enzyme molecules. These two effects are
illustrated in Figure 7. Multiple covalent bonds between the enzyme and the
matrix can stretch the whole molecule and thus alter the three-dimensional
structure of the active site. Datta et al. (1973) have suggested that such
conformational alterations may account for the significant loss in the lysozyme
activity upon immobilization.
Thus an important consideration in the covalent attachment of a
biologically active protein to an insoluble support is that the protein should be
attached to the matrix by the fewest possible bonds (Cuatrecasas, 1969). This
will increase the probability that the attached macromolecules will retain
37
Enxymo
\
Substrato
a
fl
Enzyme in
free solution
Immobilized enzyme
Conformational
chango
Steric hindrance
Figure 7. Schematic illustration of conformational changes and steric
hindrances that may affect the kinetic behavior of an enzyme upon
immobilization (from Engasserand Horvath, 1976).
38
its native tertiary structure, and its properties may more nearly resemble those
of the native protein in solution.
Steric hindrances are caused by the shielding effect of the matrix, which
makes part of the enzyme molecules less accessible to the substrate. When
an enzyme molecule is randomly bound to a matrix there is no guarantee that it
will bind the correct way round and, often as not, it is likely to be bound with its
active site completely blocked by the support matrix (Figure 7). This steric
effect is particularly troublesome for enzymes with high molecular weight
substrates, such as lysozyme.
In order to reduce the shielding of the active sites, which may
accompany the binding of an enzyme to a carrier, a "spacer" can be used to
keep the enzyme at a certain distance from the matrix. This approach has
found wide application in affinity chromatography, where steric hindrances
would otherwise impair the binding of high molecular weight substances to the
matrix-bound moiety (Steers et al., 1971; Mosbach et al., 1974).
Diffusional Restrictions and Partition Effects
When an enzyme is bound to a support, its substrate diffuses from the
bulk solution to the catalytic sites, and the product of reaction diffuse back to
the bulk solution. Thus concentration gradients are established in the
surroundings of the bound enzyme so that concentrations of substrate and
product both differ between the immediate vicinity of the bound enzyme and the
39
macroenvironment. These processes can cause diffusional resistance -either
external and/or internal diffusional barriers.
The external diffusion barrier is a result of the thin, unstirred layer of
solvent that surrounds the polymer particle (Trevan, 1980). Solutes diffuse in
this layer by a combination of passive molecular diffusion and convection. The
thickness of this layer is affected, to a certain extent, by the speed at which the
solvent around the immobilized enzyme particle is stirred. Increasing the
stirring rate will reduce this external diffusion barrier. Thus an important factor
in any experiment involving an immobilized enzyme preparation is the stirring
speed.
Internal diffusion resistances are limitations to free diffusion within the
polymer matrix imposed by the polymer matrix. Within the polymer matrix
diffusion within usually takes place by passive molecular diffusion only, and is
not affected by stirring speed. Obviously internal diffusion effects will be more
marked if enzyme is immobilized by entrapment within the polymer matrix
rather than attachment to the surface of the polymer matrix. As expected, the
decrease in enzymatic activity due to immobilization has been found to be
greater with high than with low molecular weight substrates (Silman et al.,
1966).
It is well established that electrostatic, hydrophobic, and hydrophilic
interactions among the carrier the substrate and solutes often produce an
unequal distribution of these species between the micro- and
macroenvironment. A good example is partitioning of protons that causes
40
shifting of the pH optimum of the enzyme that is immobilized on polyonic
matrices. Polyanions will tend to concentrate protons (thus lowering pH)
around the enzyme while polycations will tend to expel protons (raising pH).
Goldstein (1972) observed a shift of 1 pH unit towards alkaline values of the pH
optimum of chymotrypsin acting on acetyl-L-tyrosine ethyl ester, when
immobilized on the polyanion ethylene-maleic anhydride copolymer.
Conversely a downward shift of the pH optimum by 1 unit was observed when
chymotrypsin was immobilized on the polycation polyornithine.
III. Immobilization of Lysozyme
A highly active immobilized lysozyme system can be exploited for food
industry application. Some promising advantages of the technology are: (1) the
enzyme can be readily separated from treated foods and beverages and be
repeatedly used while the foods can still be claimed additive-free, which is
something now preferred by more and more consumers;(2) It can provide
stable microbial inhibition capability to surface of polymers to develop
antimicrobial food packaging system to improve shelf life and food safety.
Despite the appealing potential benefits of immobilized lysozyme
systems, however, studies on lysozyme immobilization have been relatively
sporadic since early 1970s. Covalent binding appeared to be the major
coupling method employed by various research groups, and retained specific
41
activities against M. lysodeikticus of the systems were very low - only 0.015.5% of the activity of the free enzyme, hardly of any practical potential (Table
7). It is concluded that diffusion limitation, steric hindrance, excessive coupling,
and coupling through essential amino acid residues are the main reasons
responsible for the significantly low retained activities (Datta et al., 1973; Moser
et al., 1988; Crapisi et al., 1992; Chen and Chen, 1996).
The significant diffusion limitation of immobilized lysozyme likely results
from the insoluble particulate substrate. It is a system of a doubly
heterogeneous nature: a situation where the enzyme attached to one solid
reacting with a substrate located in a separate solid material, the cell wall.
Since the diffusivties of micron sized particles are several orders of magnitude
less than those of proteins (the diameter of the globule protein is about 1/200 of
that of the cell of M. lysodeikticus), the substantial rate loss is at least partially
attributable to collision limitations (Datta et al.1973). Additionally, steric factors
may be more significant than when the substrate is totally soluble.
An reciprocal relation between binding yield and specific activity of the
immobilized protein was, first observed by Datta et al., in 1973, and repeatedly
confirmed by subsequent reports (Moser et al., 1988; Crapisi et al., 1992). In
general, the higher the immobilization yield, the lower the specific activity.
Table 7. Summery of reported studies on lysozyme immobilization. Turbidity reduction rate measurement of M.
lysodeikticus cell suspension is the principle of the assays used by all the studies.
Support
Method
Polyacrylamide
Covalent/Diazonium
Nonporous iron particles
Covalent/Glutaraldehyde
Silicate
Covalent/Shift base
Glass beads
Enzyme Load
(mg/g support)
2.85
Retained Activity
(%)
"appreciable"
Reference
Dattaetal. 1973
<0.01
Hailing etal. 1979
2.2
1.5
Moseretal. 1988
Covalent/BrCN
2.0
2.9
Chitosan
Adsorption
2.5
0.24
Silica gel
Adsorption
0.8
0.8
Polystyrene
Ionic Binding
0.2
10.7
Nonporus glass beads
Covalent/Glutaraldehyde
0.4
5.5
AS-La
Covalent/EDC
31.4
14.0b
3
b
Not Reported
Crapisietal. 1992
Chen etal. 1996
An enteric coating polymer which shows reversibly soluble-insoluble characteristics with pH change.
Activity measured when AS-L - lysozyme complex was in the soluble state.
to
43
It is generally observed that with covalent coupling the binding yield is
proportional to the density of protein coupling groups on the support surface
and the number of activated amino acid residues on the surface of the protein.
Therefore, while an highly-derivatized surface and/or an highly-derivatized
protein can yield higher protein load (Datta, 1973; Crapisi et al, 1992), it can
result in excessive bonding between the two as to significantly change or even
denature the enzyme. And lastly, any binding through or close to essential
amino residues would render the protein inactive.
A review of the reported studies suggested that most of the coupling
methods chosen in the studies were directly adopted from the methods used
for other enzymes. The "trial -and -error" approach was often used to choose a
coupling reaction. It is a common observation that several different reactions
and supports have to be tested before a suitable immobilization protocol forms
the basis of experimentation. (Datta et al., 1973; Crapisi et al., 1992).
Moreover, it appears that little advantage was taken of the comprehensive and
detailed information available on lysozyme structure, catalytic mechanism, and
other enzymatic behaviors under different conditions to formulate
immobilization methods .
IV. Approach of This Study - The His-15 and Spacer Strategy
Based on above and earlier discussion, rules for maximizing the activity
of a covalently immobilized enzyme system can be summarized as the
following 1.) The protein should be attached to the matrix by the fewest
44
possible bonds to minimize conformational change; 2.)The binding site(s) on
the enzyme to the supports should be located as far as practical from its active
center and nonessential for its tertiary structure; 3.) The binding method should
minimize the steric interference between the support and the immobilized
enzyme.
The extensive knowledge and understanding of the structure and
properties of lysozyme make it possible to apply these parameters to its
immobilization approaches.. The basic strategy of this study is to establish a
single bond between a single nonessential amino acid residue in lysozyme and
its support to minimize any conformational change of the bound enzyme, and to
introduce a suitable spacer between the enzyme and its support to reduce
steric hindrance.
The IS"1 amino acid residue in lysozyme molecule is the only histidine
(His-15) in the enzyme and has been shown nonessential to its lytic activity
(Kravchenko et al., 1964; Goux and Allerhand 1979). Moreover His-15 is
located on the opposite side of the activity center (Blake et al., 1967) and the
imidazole group is well exposed on the surface and readily reactive to alkylating
agents, such as bromoacetamide derivatives (Yamada et al, 1984). These two
fortuitous facts make His-15 the ideal reaction group on the enzyme for this
study.
The role of the so-called spacer molecules involved in the binding of
biospecifically active groups at the surface of matrices was the subject of
extensive discussion in 1970s (Cuatrecasas, 1970; Cuatrecasas and Anfinsin,
45
1971; O'Carra et al., 1974; Costerton, et al., 1978. Turkova, 1978). The
introduction of flexible spacers lengthens the distance between the protein and
the matrix surface, thus reduce the steric hindrance.
Solid phase peptide synthesis was developed by Merrifield (1963) in
order to simplify and accelerate peptide synthesis. The fundamental premise of
the technique is that amino acids can be assembled into a peptide of any
desired sequence of significant length while one end of the chain is anchored to
an insoluble support. The most commonly used solid support in solid peptide
synthesis is fine polystyrene beads prepared by copolymerization of styrene
with 2 per cent divinylbenzene, which happened to be a common support used
for many enzyme immobilization studies (Grubhofer and Schleith, 1953;
Filippusson and Hornby, 1970; Taylor and Swaisgood, 1972).
Thus solid phase peptide synthesis process is readily adaptable to be
used in this study for spacer synthesis, using polystyrene beads as the support
for lysozyme. With the amino group on the to carbon, 6-amino-caproic acid is a
good amino acid to be used as the building block for the spacer. Each coupling
reaction will increase the length by 6 carbon units, instead of 2 as it will get
from using glycine. The proposed model of the lysozyme immobilization
scheme is given in Figure 8.
46
A
9
?
/^\
^—(O-C-CH,-CH,-CI K-CH^CI-U-NH^C-CM, —Hisf-15 <
'
x
/
Figure 8. Proposed model for the lysozyme immobilization scheme: The His-15
of the enzyme, which is on the opposite side of its active center, is covalently
linked to a spacer that is anchored to a solid support.
47
CHAPTER 2
MATERIALS AND METHODS
I. Solid Phase Spacer Synthesis
The method of spacer synthesis was adopted from the process of Solid
Phase Peptide Synthesis (Stewart and Young, 1969). The procedure and
chemical reactions used in the spacer synthesis are summarized in Figure 9.
The solid support is a synthetic resin, 200-400 mesh, a copolymer of
styrene and divinylbenezene, which bears reactive chloromethyl groups (also
known as Merrifield Polymer, Fluka). Four basic steps involved in the synthesis
of the peptide spacer: (1 )Protect the amino group of 6-Aminocaproic acid with a
f-butyloxycarbonyl(f-Boc) group, (2)The salt of the f-Boc-6-Aminocaproic reacts
with the chloro group on the resin to form an ester bond between the amino
acid and the resin,(3)Activate the amino group of the attached amino acid by
removing the f-Boc protecting group, (4) Attach the carboxyl group of the next tBoc-6-aminocaproic acid to the activated amino group of the first amino acid
along with a coupling agent. Steps (3) to (4) were repeated till the desired
length of the spacer was achieved. Throughout all these steps, reagents and
reaction conditions were chosen so that all reactions went to 100 per cent
completion.
48
0
II
HzN—(CH2)5-C-OH
Step*!.
PROTECT
O
Ph
II
I
(O^C-O-C-O-N^C-CN
O
O
II
II
(CH3)3C-0-C-NH-(CH2)5-C-OH
Step 2.
ATTACH
Cl-CH2-Pofyrrer
O
II
O
II
(CH3)3C-0-C-NH-(CH2)5-C-0-CH2-Polymsr
Step 3.
DEPROTEC
O
II
NH2-(CH2)5-C-0-CH2-Po]yrrer
Step 4
COUPLE
o
II
o
II
(CH3)3C-0-C-NH-(CH2)5-C-OH
OOO
II
II
II
(CH3)3C-0-C-NH-(CH2)5-C-NH-(CH2)5-C-0-CH2-Polyn£r
Figure 9. The basic plan and chemical reactions of solid phase peptide spacer
synthesis.
49
Preparation of t-butyloxycarbonyl (Boc)-6-Aminocaproic acid
The procedure was modified from the Sampson and Barttlett method
(1990). The amount of 5.5 mmol Boc-On (Fisher Chemical) and 5 mmol 6aminocaproic acid (Aldrich Chemical) were suspended in a 300 ml bottomed
round bottle with 200 ml 50% aqueous dioxane and 10 ml triethyl amine. The
mixture was magnetically stirred at room temperature for 22 hours.
Then high vacuum (755 mmHg) was applied to the bottle to evaporate
excessive dioxane and triethyl amine. The remaining content in the bottle was
transferred to a 300 ml seperatory funnel and extracted 3 times with 100 ml
ethyl acetate to remove unreacted reagents. The aqueous phase was acidified
to pH 1 with 12 N HCI and then extracted 4 times with ethyl acetate. The total
of 400 ml ethyl acetate extract was combined into a 1000 ml Erlenmeyer flask
and dried over Na2S04. Finally, the ethyl acetate phase was carefully decanted
into a 1000 ml beaker and let evaporate overnight. White crystals of Boc-6aminocaproic acid precipitated out on the bottom of the beaker.
Purity of the Boc amino acid determination by TLC (Thin Layer
Chromatography)
TLC analysis was performed to check the purity of the Boc-amino acid,
using the Stewart and Young (1969) method. Approximately 1 pi of the amino
acid derivative solution (50 mg per ml in ethyl acetate) was spotted onto a TLC
plate (Brinkman Silica Gel H, 4X5 inch). Same amount of 6-aminocaproic acid
was also spotted as control. Then the plate was run in a solvent composed of
50
chloroform (85), methanol (10), and acetic acid (5) in a sealed glass chamber in
which the atmosphere was saturated with the solvent.
After solvent development the TLC plate was dried in the air and placed
in a closed chamber containing a beaker of fresh, concentrated HCI. After 15
min of exposure to the HCI fumes, the plate was heated in a 105 0C oven for 10
min. Then the plate was sprayed with ninhydrin solution (0.2% in acetone) to
develop color. Finally the purity of Boc-6-aminocaproic acid was assessed
based on the shape and Rf s of the color spots
Attachment Boc-6-Aminocaproic Acid to Chloromethylstyrene Resin
The procedure was adopted from the book "Solid Phase Peptide
Synthesis" by Stewart and Young (1969). Chloromethyl polystyrene resin (5
grams) (Merrifield Polymer, Fluka), 5 grams of Boc-6-Aminocaproic acid and 3
mis of triethyl amine were combined in a 250 ml round-bottom flask with 25 ml
absolute alcohol added to cover the resin. The mixture was refluxed gently for
24 hours at 81 0C in a Clas-Col electric heater with a 500-mm H2O condenser
with a CaCb drying tube attached. The molar ratio for the reactants was 1.0
mole of Boc-6-aminocaproic acid (230.18 g per mole): 1.0 mole of Cl of the
chloromethyl resin (232.6 g per mole ): 0.9 mole triethyl amine ( 0.14 ml per
millimole; sp. gr. 0.723).
The reacted resin was transferred to coarse-fritted Buchner funnel and
washed successively with EtOH, H2O, MeOH, and CH2CI2, three times with
51
each solvent, allowing one minute contact time for the solvent to penetrate the
resin beads and for solutes to diffuse out of the beads.
The washed resin, still suspended in CH2CI2, was then transferred to a
250-ml seperatory funnel, stirred with CH2CI2 (approximately 20 ml per g), and
let stand until the bulk of the resin floated to the top and a fairly sharp
demarcation line appeared at the bottom of the floating resin. The solvent,
carrying the suspended finest particle of resin, was run out of the funnel and
discarded. This separation process was repeated three times to prevent
troublesome clogging of the glass wool filter in the synthesis vessel. After
removing all volatile solvents with a water aspirator, the resin was dried
overnight in a desiccator with silicon gel. The amount of 50 mg resin was
weighed and hydrolyzed to determine the degree of substitution of the amino
acid on the resin.
Stepwise Synthesis of 6-Aminocaproic Acid peptide on the Resin
Figure 10 shows the apparatus used for the synthesis of the peptide.
The reaction vessel was modified from a 250-ml seperatory funnel with the
upper-half of the hole in the stopcock enlarged into a "pit" - 1/4 inch in diameter
and 1/4 inch deep by an electric drill, The lower half of the hole was left intact.
Glass wool was pressed into the pit to form a filter such that liquid could pass
through but not the resin beads when the stopper turned to the drain position.
52
N
^
Drilled pit stuffed with glass wool
Figure 10. The modified seperatory funnel used as the reaction vessel for solid
phase spacer synthesis. A pit was drilled in the stopcock and filled with glass
wool to serve as filter to keep the resin beads inside the seperatory funnel
when the stopcock was turned to drain position to drain the solvents.
53
Boc-6-Aminocaproic acid-resin (1 gram) was loaded into the reaction
vessel. The sequence of steps used to add each amino acid to the resin is
outlined and explained in Table 8. Temporary co-amino protection is provided
by the Boc group, removed at each step by the moderately strong acid
trifluroacetic acid (TFA)
Quantification of 6-Aminocaproic acid by Reverse-Phase High-Performance
Liquid Chromatography (HPLC)
The amino acid was quantitatively determined based on the HPLC
amino acid analysis originally developed by Heinrikson and Meredith (1984),
using precolumn derivatization with phenylisothiocyanate (PITC).
The analysis was performed at room temperature with a Shimadzu LC10AS HPLC system using an Econosil C18 column (250 mm X 4.6 mm, 10 (jm)
and Shimadzu SPD-6A UV spectrophotometric detector at 254 nm. Isocratic
mobile phase was composed of 0.1 M ammonium acetate, pH6.8 in methanol:
water (80:20)v/v. Pump speed was set at 1.0 ml/min. PITC reagent and 6aminocaprioc acid were purchased from Aldrich Chemical Co. All other
chemicals were analytical grade.
Standard Preparation
The amount of 10[jl of 2.5 mM standard 6-aminocaproic acid was
vacuum dried in a small test tube and dissolved in 100 pi of coupling buffer
(acetonitrile: pyridine: triethylamine:H20, 10:5:2:3). To this solution was added
5 pi of PITC. After a 10-min reaction at room temperature, the solution
54
Table 8. Deprotection and coupling cycles for solid-phase peptide synthesis
using Na - Boc-6-aminocaproic acid and ester linkage to polystyrene-resins.
Step No.
1.
Reagent and Solvent
Tinne(nnin) X Treatment No.
Dichloromethane(DCM) wash
3X 1
2
TFA-DCM
1 X3
3
Dichloromethane wash
4X1
4
Diisopropylethylamine-DCM (1:19)
2X2
5
DCM wash
6 X1
6a
Boc-amino acid (3 equiv.) in DCM
5 (no filtering)
6b
DCC (3equiv.) in DCM
60
7
DCM wash
6X2
55
was evaporated to dryness by rotary evaporation under high vacuum. The
PITC-derived amino acid was then dissolved in 250 pi of 5 mM ammonium
acetate water solution. Quantity of 20 pi of the solution was injected as
standard to the HPLC system described above.
Sample Preparation
Dioxane (5 ml) and 5 ml concentrated hydrochloric acid were added to
50 mg resin sample in a 50-ml round glass flask. The mixture was re-fluxed
over night in a Clas-Col electric heater with a 0.5 m air condenser. A converter
such controlled the temperature dialed that mild simmering was maintained.
The resulting hydrolysate was filtered through a 0.45 pm filter and 10 pi of the
filtration was derivatized with PITC by the procedure described above.
II. Attachment of Lysozyme to the Spacer thorough His-15
Bromoacetylation of 6-Ammocaproic Acid.
The Yamada method (Yamada et al. 1984) was used to bromoacetylate
the amine group of 6-aminocaproic acid at the end of spacer. The
bromoacetylated 6-aminocaproic acid was then coupled to the imidazole group
of His-15 of lysozyme. Figure 11 summarizes the chemical reactions involved.
After step 5 in Table 8, the resins were washed with distilled water 4 times, each
for 1 minute. The resins were transferred to a 100 ml beaker containing 20 ml
distilledwater. The beaker was cooled in an ice bath and 500 pi bromoacetyl
56
0
II
H2N—(CH2)5-C—O—CH2-Potymer
O
II
BrCH2C—Br
0
II
O
II
BrCH2C—NH—(CH2)5-C—0—CH2-Po^mer
His-i5
NH
Nc-y
0
II
His-i5
0
N-CH2C-N—(CH2)5-C—0—CH2-Po^mer
NW
Figure 11. Chemical reactions involved in attaching 6-aminocaproic acid to the
£2-nitrogen of His-15 of lysozyme.
57
bromide (5.7 mmol; MW 201.86, d 2.317) was added dropwise over 15 min with
vigorous stirring. During the reaction the pH of the suspension was maintained
at 7 with 4 N NaOH. After addition of all bromoacetyl bromide, the suspension
was stirred for another 15 min. Finally the resins were transferred to Buchner
funnel with coarse fritted disc, washed thoroughly with distilled water, and
stored at 4°C.
Coupling of Lysozyme onto the Support via Bromoacetylated Spacers
Coupling reactions were performed between lysozyme and resins with
spacer lengths of one 6-aminocaproic acid (1AA), 2AA, 3AA and 4AA
respectively, using the following procedure.
A mixture of 20 mg lysozyme (Fordras Sa, Switzerland) and 400 mg
resin with bromoacetylated 6-aminocaproic acid in 10 ml of 0.5 M sodium
acetate-acetate acid buffer (pH 5.5) containing 20% methanol was incubated at
40oC for 12 hr. The resin beads were then submerged in 6 M urea solution for
30 min with gentle stirring to dissociate any adsorbed protein; followed by
washing with 1.0 liter 66mM phosphate buffer, pH 6.24. From monitoring
absorbance at 280nm of the wash and its volume, the amount of enzyme
removed by washing was calculated. Accordingly, the amount of immobilized
lysozyme was determined.
Beads (100 mg) with immobilized lysozyme were reserved for amino acid
composition analysis of the immobilized protein by HPLC to verify the bonding
through His-15 of lysozyme.
58
Amino Acid Compositions of the Immobilized Lysozyme
Amino acid analysis of the immobilized lysozyme was performed by
Protein Structure Core Facility of University of Nebraska Medical Center,
Omaha, Nebraska, using the following procedure: 2000 pmoles of noreleucine
was added to the samples as internal standard. The samples were then
evaporated to dryness in a speedvac. The samples were then hydrolyzed in
HCL vapor under argon atmosphere. (6N HCI with 1% phenol and 0.5% sodium
sulfite were used in the hydrolysis mix). After 20 hours of hydrolysis at 11CC,
the samples were dissolved in 200 (jl of Beckman Na-S sample buffer. The
samples were then brought up in 200 pi Na-S. Sample of 50 pi was injected
automatically onto the Beckman 6300 Amino Acid Analyzer. In data analysis,
correction was made to the amount of the internal standard to minimizing
dilutional errors.
III. Controls
Lysozyme was also immobilized to the polystyrene beads as controls
with three different methods: (1) Covalent binding via 1-ethyl-3dimethylaminopropyl carbodiimide (EDC) coupling without spacer; (2) Covalent
binding via EDC coupling with one 6-amino-caproic acid as spacer, and (3)
Covalent binding through His-15 without spacer.
Covalent Binding via EDC Coupling As Controls
The carboxyl groups in the enzyme were activated by 1-ethyl-3(3-
59
dimethylaminopropyl)carbodiimide (EDC) in 0.1 M phosphate buffer, pH 4.75 at
25 0C to form O-acyl isourea derivatives. Then the pH was raised to 7 and the
temperature lowered to 4 0C to allow the highly reactive intermediates to
condense with the amine groups on the surface of the beads to yield the
corresponding amides, or peptide bonds. The chemical reactions involved in
the coupling process are given in Figure 12.
1. Preparation of the Support - Attachment of Boc-Glycine
Boc-Glycine (Sigma Chemical) was attached to the chloromethyl
polystyrene beads to introduce amine groups. Five grams of the resin, 3.8
grams of Boc-Glycine, and 2.7 ml of triethyl amine were mixed in a 250 ml
round-bottom flask with 20 ml absolute alcohol added to cover the reactants.
The mixture was refluxed gently for 24 hours at 81 "C in a Clas-Col
electric heater with a 500-mm H2O condenser with a CaCb drying tube
attached. The molar ratio for the reactants was 1.0 mole of Boc-Glycine (175.2
g per mole): 1.0 mole of Cl of the chloromethyl resin (232.6 g per mole ): 0.9
mole triethyl amine (0.14 ml per mMole; sp. gr. 0.723). The reacted resin was
transferred to coarse-fritted Buchner funnel and washed successively with
60
o
II
Protein—C—OH
CH3CH2-N=C=N—(CH2)3—N;
EDC
,CH3
*CH3
O
Protein—C—O
I
/CH3
CH3CH2—HN-C=N—(CH2)3—N^
H2N
Polymer
O
Protein—C—NH—Polymer
and
O
CH3CH2-HN-C-NH-(CH2)3—N:
,CH3
CH,
Figure 12. Coupling of lysozyme to the reactive amine group on the support via
1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) activation of carboxyl
groups of the enzyme.
61
EtOH, H2O, MeOH, and CH2CI2, three times with each solvent, allowing one
minute contact time for each solvent to penetrate the resin beads and for
solutes to diffuse out of the beads.
The washed resin, still suspended in CH2CI2, was then transferred to a
250-ml seperatory funnel, stirred with CH2CI2 (approximately 20 ml per g),
and let stand until the bulk of the resin floated to the top and a fairly sharp
demarcation line appeared at the bottom of the floating resin.
The solvent, carrying the suspended finest particle of resin, was run out
of the funnel and discarded. After removing all volatile solvents with a water
aspirator, the resin was dried overnight in a desiccator with silicon gel. The
amount of 50 mg resin was weighed and refluxed in 20 ml HCI - Dioxane
(50:50, v/v) for 24 hr. The amount of glycine in the hydrolysate was determined
HPLC (Heinrikson and Meredith (1984) to determine the degree of substitution
of the amino acid on the resin.
2. Deprotect the Amine Groups on the Resin Beads
Five hundred mg of the treated beads were soaked and stirred in a
trifluroacetic acid - CH2CI2 mixture (1:4) for 30 min to remove the Boc protection
on the amine groups. The beads were then transferred to a coarse-fritted
Buchner funnel and washed with C^C^and later with distilled water. Finally
the washed beads were suspended in 20ml of 0.1 M phosphate buffer, pH 7.0,
for 1 hr at 4 0C.
62
3. Protection of the active site of lysozyme with N-acetylglucosamine NAG
NAG oligosaccharide (50 mg) (trimer, tetramer and pentamer. Sigma)
was added to 50 ml 2 mg/ml solution of lysozyme in 0.1 M phosphate buffer, pH
4.75. The mixture was stirred gently at room temperature for 30 min to protect
the carboxyl groups of Glu-35 and Asp-52 from being reacted to EDC (Lin and
Koshland, 1969).
4. Activation of lysozyme with EDC
To activate the carboxyl groups in lysozyme, 500 mg EDC were mixed
into the 50 ml lysozyme solution treated with NAG. After stirring gently at 25 0C
for 2 hr, the pH of the mixture was raised to 7 and the temperature dropped to
40C.
As control, 10 ml of 2 mg/ml solution of lysozyme not treated with NAG
was also activated with EDC under the same condition.
5. Coupling of Lysozyme to the Resin Beads.
Same coupling reactions were carried out for both the NAG-protected
and non-NAG-protected lysozymes. Four hundred milligrams (400 mg) glycine
derivatized beads were transferred to 10 ml buffer containing 20 mg activated
lysozyme. The mixture was stirred at 4 0C for 2 hr. The resin beads were then
soaked in 6 M urea solution to remove any adsorpted protein and dialyzed
against water for 4 hours (to remove the protecting substrate in the NAG
treated experiment and for control purpose in the non-treated experiment)
63
followed by washing with 1.0 liter 66mM phosphate buffer pH 6.24. From the
absorbance at 280nm of the wash and its volume, the amount of immobilized
lysozyme was determined.
The same coupling procedure was performed for the resin beads with
one 6-amino-caproic acid as spacer.
The third control - covalent binding through His-15 without spacer, was
prepared with the same protocol as with spacers except for using the glycine
attached resin beads as support.
IV. Determination of Lysozyme Activity
Activity against M. lysodeikticus cells
The activity of both free lysozyme and immobilized lysozyme was
determined by measuring the absorbance decrease rate of lyophilized M.
lysodeikticus cells in a 66 mM pH 6.24 phosphate buffer at 25 0C. One activity
unit is defined as the decrease of one milli-absorbance per minute.
Free Enzyme
Lyophylized M. lysodeikticus (Sigma, No. M-3770) cell suspension of
0.02% (w/v) was prepared in the 66 mM phosphate buffer, which gave an
A450nm between 0.7 and 0.8. The lytic reaction was carried out in a 3 ml
quartz cuvette with 1 cm light path at 25 0C using a Shimatzu UV-Vis 2100
spectrophotometer. Assay was started by adding 0.1 ml of sample to the
64
bottom of the cuvette and then 2.5 ml of the substrate suspension was added
and mixed well. The difference of /Wjnm between 20 and 40 seconds (linear
part of the curve) of reaction was recorded with the spectrophotometer and the
activity was calculated using kinetic software (Shimadzu Corp.). A blank assay
using 0.1 ml of the phosphate buffer instead of lysozyme was also performed to
account for any autolytic degradation of the substrate.
Immobilized Enzyme
The activity of immobilized lysozyme was assayed by addition of 100mg
of lysozyme beads into a 5 ml suspension of 0.02% w/v lyophilized M.
lysodeikticus cells in 66 mM phosphate buffer at 25 0C, and agitated at 240 rpm
in a 25-ml centrifuge tube. After one minute, the cell suspension was quickly
aspirated into a 25-ml syringe to separate it from the enzyme beads through a
small coarse-fritted glass Buchner funnel. The A45onm of the enzyme-free cell
suspension was then determined with the same method for free enzyme assay
and subtracted from the initial A45onm before the enzyme was added. The
difference of A45onm was recorded to calculate the activity - units/g resin. A
blank assay with 100mg polystyrene beads without lysozyme was done to
account for any A45onm loss due to non-enzymatic reasons.
Activity against Glycol Chitin
Glycol chitin, a water-soluble polymer of substituted Nacetylglucosamine with P-1,4-glucosminide linkages, was also used as
65
substrate to measure the activity of lysozyme of both free and immobilized,
based on the method of Imoto and Yagishita (1971)
Free Enzyme
For free enzyme, 0.5 ml of lysozyme solution was added to 1 ml of
0.05% glycol chitin (Sigma) solution and incubated for 30 min at 37 0C. Both
solutions were buffered in 0.1 M acetate at pH 4.5 and preincubated at 37 0C.
To this reaction mixture was added 2.0 ml of 0.025% potassium ferricyanide in
0.5 M sodium carbonate, and the mixture was immediately placed in boiling
water for 15 min to develop color in a test tube stopped with aluminum foil.
After cooling to room temperature, the A42onm was measured against water in
the same Shimadzu spectrophotometer used for the cell lysis method. The
difference (A A42onm) between a given sample and the blank was referred to as
the amount of reducing power generated by the tested lysozyme.
Immobilized Enzyme
For immobilized enzyme, in a continuously stirred 15-ml beaker, 100 mg
beads mixed with 5 ml of 0.05% glycol chitin (Sigma) solution buffered in 0.1 M
acetate at pH 4.5 and incubated for 30 min at 37 0C. The mixture was then
filtered and 1.5 ml of the clear solution was mixed with the color reagent and
completed the remaining steps the same as for the free enzyme.
66
V. Others
Effects ofpH on the Activity ofLysozyme - Free and Immobilized
The effect of pH on both free and immobilized lysozymes was conducted
by measuring activities against lyophilized M. lysodeikticus substrate (0.02%
w/v) suspended in 66mM phosphate buffer of pH values from 4.0-9.2. Buffer pH
was adjusted with 0.1 N HCL and 0.1 N NaOH accordingly.
Effect of Inhibitor Pentane on the Activity of Immobilized Lysozyme
Lyophilized M. lysodeikticus substrate solution (0.25 mg/ml in 66mM
phosphate buffer, pH 6.24), containing 4% (v/v) pentane (Sigma Chemical)
(%,v/v) was used to study its inhibition effects on the lytic activity lysozyme free (2 ppm) and immobilized (3 AA, His-15).
Kinetics Study of Immobilized Lysozyme
The turbidity assay described earlier in this chapter was used to
measure the initial activity of immobilized lysozyme (3 AA, His-15) for six
substrate concentrations: 0.05, 0.075, 0.1, 0.125, 0.15, and 0.175, mg/ml. The
data were evaluated with the Eadie-Hofstee plot and the Lineweaver-Burk plot,
respectively.
67
CHAPTER 3
RESULTS AND DISCUSSION
I. Solid Phase Spacer Synthesis
The amount of 6-aminocaproic acid attached to the polystyrene resin
beads and the length of the spacers determined by HPLC are summarized in
Table 9. The data indicate that the adopted solid phase peptide synthesis
protocol worked successfully for synthesizing the spacers with different
predetermined lengths on the polystyrene bead support. The achieved spacer
lengths were very close to the desired integer numbers, indicating all the
reactions throughout the synthesis process went to completion.
Several properties of polystyrene beads make it a good candidate as
solid support to immobilize enzyme. It is both mechanically and microbially
stable. It is relatively inexpensive and its fine particle size offers large surface
area to volume ratio to bind large quantities of enzyme as necessary.
Polystyrene has been reported as a preferred support for immobilizing Pfructofuranosidase (Filippusson and Hornby, 1970) and trypsin (Taylor and
Swaisgood, 1972). Besides, the "coincidence" that polystyrene has been used
as the solid support for solid phase peptide synthesis (Stewart and Young,
1969) made it an ideal support for the purpose of this search.
The solid support is composed of fine beads (70-140 pm in diameter) of
a synthetic resin prepared by copolymerization of styrene with 2 per cent
68
Table 9. Amino acid analysis of the peptide spacers synthesized on
the polystyrene beads.
Spacer length designed
(No. of amino acid)
Amino acid attached
(mM/g resin)
Spacer length synthesized
(No. of amino acid)
1
0.86
1.00
2
1.69
1.97
3
2.67
3.10
4
3.37
3.92
69
divinylbenzene. The long alkyl chains bear a phenyl ring on every second
carbon. These chains are cross-linked at approximately every fiftieth carbon by
p-diethylphenyl residues derived from the divinylbenzene.
The crosslinking causes the polymer to be completely insoluble in all
ordinary solvents, yet swell extensively in the organic solvents used in the
peptide synthesis. Because of the swelling, many of the peptide chains on the
resin are attached within the pores of the beads. Therefore the spacers
synthesized in this study (0.86 mM/g resin) should be viewed as evenly
distributed throughout whole matrix, not just on the surface of the beads.
However, since the polystyrene matrix should not be swollen under the
lysozyme attachment condition, the enzyme is presumed only bound to the
spacers on the surface.
II. Attachment of Lysozyme
Covalent Coupling through Carboxyl Groups ofLysoyzme
EDC coupling is a fairly common covalent bonding method that has been
used for immobilizing a variety of enzymes: rabbit muscle lactate
dehydrogenase (Cho and Swaisgood, 1971), trypsin (Taylor and Swaisgood,
1972), and lysozyme (Chen & Chen, 1996)
In order to get a consistent support environment between the test and
control experiments, the same polystyrene resin beads were used as the
70
support for controls in this study to match that of the spacer and His-15 specific
binding system. Since amino groups are the reactive groups at the tip of the
spacers, carbodiimide or EDC coupling was chosen as the covalent binding
method for the controls in this study, which allowed the carboxyl groups on the
surface of lysozyme molecules attach to the amino groups on the support
surface or at the tip of spacers.
When lysozyme was directly attached to the polystyrene support through
its carboxyl groups without the pretreatment of NAG oligosaccharide, the bound
enzyme suffered great activity loss: only 0.7% of the activity was retained
compared to the activity of the same amount of free enzyme (Table 10). When
pretreated with NAG oligosaccharide, the retained activity increased from 0.7%
to 1.8%, while the protein load remained at about the same level. This result
suggests the very low activity observed for the bound enzyme was at least in
part due to some bonding through the key carboxyl groups - i.e. Glu- 35 and/or
Asp-52 involved in the catalysis. However, even the improved 1.8% retained
activity is still a very disappointing result.
When free lysozyme was derivatized with a carbodiimide-nucleophile
procedure very similar to the EDC reaction employed in this study, the
presence of NAG oligosaccharide protected Asp-52 and Glu-35 and over 50%
activity was retained (Lin and Koshland, 1969). Therefore there should be
other adverse changes happened during the immobilization process that
caused the major activity loss.
71
Table 10. Activity of lysozyme covalently attached polystyrene beads via 1ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), with and without NAG
protection .
Method
of
Attachment
Lysozyme
bound
(mg/g resin)
Activity
(U/g resin)
Retained activity
compared Jto free
enzyme*
(%)
EDC w/o NAG protection
1.24
158
0.7
EDC with NAG protection
1.30
213
1.8
Based on the specific activity of free lysozyme at 9.09 U/pg protein.
72
Since the 11 free carboxyl groups (2 glutamic acid residues, 8 aspartic
acid residues, and 1 COOH-terminal leucine residue) are all on the surface of
the molecule, it is very likely that lysozyme will have multiple attachment sites to
its support through EDC coupling reaction. Multiple attachment could strain or
even disrupt the tertiary structure of the enzyme and make steric hindrance
more evident. In addition, it has been reported that chemical modification of the
carboxyl group of Asp-101 of lysozyme could significantly weaken substrate
binding (Yamada et al. 1981)
Covalent Coupling through His-15
In contrast to the "shot gun" strategy that couples lysozyme to the solid
support randomly through the carboxyl groups on its surface, coupling through
the single histidine residue of the protein that is nonessential for its activity
seems much more appealing. Amino acid composition analysis by HPLC of
polystyrene-lysozyme beads obtained with the His-15 coupling protocol is given
in Table 11. No natural histidine residue was detected in the hydrolysate of the
lysozyme-coated polystyrene beads, whereas a newly formed 3carboxylmethylhistidine was found. The numbers of the other amino acid
residues matched very well to those of the control and the calculated values.
These results clearly indicated that binding did occur and only through the
alkylation of His-15 residues by the bromoacetylated 6-aminocaproic acid
groups at the end of the spacers.
73
Table 11. Amino acid compositions of lysozyme immobilized through His-15.
Native lysozyme
Immobilized lysozyme
Amino acid
Theory
Control
Asp
21
21.6
20.1
21.9
Thr
7
6.1
6.2
6.7
Ser
10
9.3
10.6
10.7
Glu
5
4.8
5.7
4.9
Pro
2
1.8
1.8
2.3
Gly
12
11.7
12.0
11.6
Ala
12
12.9
11.7
12.0
3-CM-Hisa
m
-
0.8
0.7
Val
6
6.0
6.1
6.1
Met
2
1.7
1.8
1.7
lie
6
5.7
5.4
5.5
Leu
8
8.0
8.0
7.9
Tyr
3
2.8
3.1
2.8
His
1
1.1
0.0
0.0
Lys
6
5.7
6.3
6.1
Arg
11
10.4
11.1
10.7
' 3-Carboxylmethylhistidine
1 AA His-15 2 AA His-15
74
The observation that no native histidine residues were detected also
indicated that the extensive post-coupling washing steps, especially exposure
to 6M urea for 30 min, worked well at removing the lysozyme molecules that
had been noncovalently adsorbed to the beads. Although urea is perhaps the
most commonly used denaturing agent for proteins, exposure to 6 M urea has
been used successfully for removing adsorbed enzyme without inactivating
coupled enzyme in several studies (Weetall, 1976). Lysozyme has been
reported to survive up to 7 M urea without any unfolding occurred (Leonis,
1956). This was confirmed in this study by observing that free lysozyme (20
mg/L) displayed no activity loss after immersion in 6 M urea for 30 min at room
temperature (data not shown).
Efficacy of the His-15 with Spacer Strategy
Compared to the random EDC binding no spacer control, coupling
through His-15 binding without spacer already produced significant
improvement in terms of total activity of the bound enzyme (Table 12). The test
displayed higher activity (188 vs. 158 U/g resin), although its protein load was
only half of that of the control's (0.59 vs. 1.24 mg/ resin). This further
suggested that there was a significant amount of non-productive coupling
occurred in the random EDC binding control.
75
Table 12. Characterization of polystyrene-lysozyme catalysts
Method
of
attachment
Activity
Retained activity
compared to free
enzyme*
Lysozyme
bound
(mg/g resin)
(U/g resin)
(%)
No spacer, EDC
1.24
158
1.4
No spacer, His-15
0.59
188
3.5
1 AA spacer, EDC
1.44
314
2.4
1 AA spacer, His-15
0.84
801
10.5
2 AA spacer, His-15
1.31
1631
13.7
3 AA spacer, His-15
2.21
2736
14.2
4 AA spacer, His-15
2.13
2536
13.1
Based on the specific activity of free lysozyme at 9.09 U/pg protein.
76
Conceivably, the His-15 binding caused much less change in the tertiary
structure of the enzyme. In the meantime, since the His-15 residue is on the
back of side of the activity center, it could be pictured that lysozyme molecules
bound to the matrix through this group would have their activity centers all
facing outwards, rendering much higher accessibility to substrate. A similar yet
more dramatic benefit of His-15 binding is illustrated in Table 12, when the total
and retained activity of 1 AA spacer, His-15 is compared to that of the 1 AA
spacer, EDC configuration.
The nonessential role of the single histidine residue for the lytic function
of lysozyme has been previously confirmed by a series of studies. According to
Parsons et al. (1967), alkylation of the residue by three different reagents
(iodoacetic acid, iodoaceamide, and 4-bromoacetamide-2-nitrophenol) did not
have any effect of either the binding properties of the enzyme or its catalytic
efficiency. These results are consistent with the similar alkylation studies by
Kravchenko et al. (1964) and Goux and Allerhand (1979) employing iodoacetic
acid. Furthermore, X-ray structural analysis have demonstrated that the single
histidine residue of the enzyme occurs on the surface of the molecule remote
from the binding cleft (Blake et al., 1965,1967; Imoto et al., 1972)
13
C NMR spectroscopy was used by Goux and Allerhand (1979) to study
the reaction of iodoacetate with His-15 of hen egg white lysozyme. With the use
of various reaction conditions, His-15 was carboxymethylated in detectable
quantities only at NE2of the imidazole group. A comparison of the spectrum of
chromatographically pure [NE2-cqarboxymethylhistidine-15] lysozyme with that
77
of the intact protein indicated that the chemical modification did not affect the
conformation of the enzyme.
The introduction of spacer also significantly benefited the total and
retained activity of the bound enzyme, specifically comparing the result of 1 AA
spacer/His-15 to that of no spacer/His-15; and 1 AA spacer/ EDC to no spacer/
EDC, respectively. Presumably the benefit is a result of reduced steric
hindrances. A not fully expected side benefit, however, is that the increasing
length of the spacer significantly increased the protein load - bound lysozyme
which went from 0.59 mg/g resin with no spacer to 0.84 mg/g resin with 1 AA
spacer; and kept increasing with the length of the spacer to 2.21 mg/g resin
with 3 AA spacer. However, further spacer length increase from 3 AA to 4 AA
did not result in further improvement in binding capacity.
More importantly, with the increasing protein load, the specific activity of
the bound enzyme also increased and eventually stabilized at a level of about
14%. This is the opposite of the reciprocal relation between the protein load
and specific activity of the bound enzyme as observed by many previous
studies on lysozyme immobilization (Datta et al., 1973; Moser et al., 1988;
Crapisi et al., 1992)
It is suggested that under the given coupling conditions, as length of the
spacer increased the bromoacetylated amino groups at the ends of the spacers
became more accessible to the His-15 side chains of lysozyme molecules, thus
resulting in the higher and productive immobilization yields.
78
Activity against Soluble and Insoluble Substrates
The retained activity of free and bound lysozyme against the glycol chitin
- a soluble substrate and lyophilized M. lysodeikticus cells were compared in
Table 13. When soluble chitin was the substrate, lysozyme immobilized with
the His-15/spacer strategy showed very high retained activity - around 80%,
regardless the different spacer lengths. This again suggests the approach did
preserve the natural conformation and catalytic function for most of the bound
protein molecules. Nonetheless, when insoluble cells was the substrate, the
retained activity only ranged from 10.5 -14.2%, albeit 10 times higher than that
of the no spacer/EDC control. This drastic difference of retained activity
between the two substrates suggests that the diffusional limitation due to the
bulky insoluble substrate remains to be the major hurdle for further improving
the lytic efficiency of the immobilized Lysoyzme system.
Kinetics Study of the Immobilized System
The Eadie-Hofstee plot (v against v/[S]) is a linear transformer of the
Michaelis-Menton equation and is an appropriate graph to use when one is
interested in detecting deviations from Michaelis-Menton behavior. It is
especially valuable at diagnosing the presence of diffusion effects in
immobilized enzyme system. This method yields straight lines only when the
enzyme obeys Michaelis-Menton kinetics and diffusion effects are negligible
79
Table 13. Specific activity of lysozyme against two different substrates.
Retained activity
compared to free lysozyme (%)
Lysozyme type
Free enzyme
Soluble Chitin
M. lysodeikticus cells
100
100
No Spacer EDC
33
1.4
No Spacer His-15
72
3.5
1AAHis-15
80
10.5
2AAHis-15
83
13.7
3AAHis-15
79
14.2
4AAHis-15
81
13.1
-
80
or fully excluded. With increasing diffusion limitations, however, the curves
depart from straight lines. Internal diffusion resistance yields a sigmoidal curve,
whereas external diffusion limitations are manifested in arched curves
(Engasserand Horvath, 1976; Gemeiner, 1992).
The Eadie-Hofstee plot method was used in this study to further explore
the diffusion limitation effects suggested by the huge difference between the
retained activity of the immobilized lysozyme against whole cell and soluble
chitin substrates. When the initial velocity data of 3AA/His-15 for six substrate
concentrations (0.05-0.175 mg/ml, see Chapter two) were converted to
generate the Eadie-Hofstee plot, a highly convex curve was obtained (Figure
13), suggesting a strong external diffusion effects. That is, when insoluble cells
are used as substrate, the rate of the substrate breakdown by the bound
enzyme is faster than that of the substrate transfer. The rate of the enzyme
reaction depends on the diffusion of the cells from the bulk solution to the
enzyme. This was not unexpected given the double-heterogeneous nature of
the system and the size of the cells.
So far there have been no reported studies on kinetic behavior of
immobilized lysozyme against M. lysodeikticus cells, presumably because the
retained activities were too low to conduct Michaelis-Menton kinetics studies.
There have been reports, however, that free lysozyme obeyed MichaelisMenton kinetics when lyophilized M. lysodeikticus was used as substrate
(Neville and Eyring, 1972; Maurel and Douzou 1976; Ohno and Morrison 1989).
All three studies reached the conclusion based on the linear fit of their
81
Figure 13. Eadie-Hofstee plot of immobilized lysozyme (100 mg 3 AA,His-15)
against M. lysodeikticus (0.05-0.175 mg/ml)
82
data to the double-reciprocal or Lineweaver-Burk plot. It is worth pointing out
that while the Lineweaver-Burk plot does have the advantage that the values of
v for given values of [S] are easy to read from it, it has the big disadvantage of
compressing the data points at high substrate concentration into a small region
and over emphasizing the points at lower concentration. According to Dowd
and Riggs (1965), it tends to give a deceptively "good" fit, even with unreliable
data.
Figure 14. is the Lineweaver-Burk plot from the same set of raw data
that yielded the significantly arched curve in the Eadie-Hofstee plot in Figure
13. Clearly the fit is fairly linear (at least as good as the fit in the Ohno and
Morrison paper (1989), if not better). So if based on the data fit from Figure13,
it would have been concluded that the immobilized lysozyme system obeys
Michaelis-Menton kinetics when it hydrolyzes M. lysodeikticus, which is clearly
not the case as the data suggested in this study.
A comparison between the two plots generated with the same set of raw
data demonstrate that the data points in the Eadie-Hofstee plot are equally
weighed, whereas in the Lineweaver-Burk plot the points at high substrate
concentrations are highly compressed and the points at lower concentrations
are stretched out. In other words the data at lower concentrations have much
bigger impact on the overall shape of the curve in the Lineweaver-Burk plot
than the ones at higher concentrations. This disadvantage of the LineweaverBurk plot could be particularly problematic for lysozyme study using the turbidity
assay method. Because the assay tends to give less reliable data when
83
0.009
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0
10
15
20
25
1/[S]
Figure 14. Lineweaver-Burk plot from the same data set of Figure 13.
84
the substrate concentration is low. Therefore the studies on kinetic behavior
of lysozyme using the Lineweaver-Burk plot should be viewed with caution.
Using Eadie-Hofstee plot Green (1995) studied the kinetics of lysozyme
inhibition caused by wine acids, ethanol, and wine phenolics. It was concluded
that lysozyme did not follow Michaelis-Menton kinetics when M. lysodeikticus is
used as its substrate based on the arc shaped curve obtained in the EadieHofstee plot. The proposed explanations are: 1) Changes in absorption
(Mabs/min) may not linearly correspond to individual P(1-4) NAG-NAM bonds
broken; the degree of cell wall degradation corresponding to change in optical
density is unknown (Yamasaki et al., 1968), 2) The substrate is presented in an
immobilized status; polysaccharide sections suitable for lysing are not in
solution but bound in the cell wall matrix, 3) Non-productive binding is a
consideration; NAG-NAM polymer 3 units in length will reversibly bind to the
catalytic cleft but are not hydrolyzed.
Among the three explanations, the lack of direct correlation between the
turbidity decreasing rate and the number of glycosidic bonds split is the most
fundamental one. In fact the initial decreasing rate of turbidity may very likely
underestimate the actual activity of lysozyme in the system and thus cause the
nonconformity of the Michaelis-Menton if the following scenario is true:
In the lysis of cells by lysozyme the first step must be assumed to be the
absorption of the lysozyme molecules onto the very large surface of the cell
wall (Diameter ceii/Diameter lysozyme = 200 /1, (Datta et al., 1973), which
electrostatic forces play a large part in the binding process (Price and Pethig,
85
1986). Assume all cells are identical and the (3-1,4 bond are evenly distributed
over the surface of the cells; and after a certain amount of the P-1,4 linkage on
the surface of a cell is the broken, say 10,000, the whole cell bursts, or
collapses, into pieces of debris or fragments. All enzyme molecules start
hydrolyzing cell walls simultaneously, because substrate is in great excess and
the system is being thoroughly agitated.
The generated fragments, which still have many of the intact glycosidic
bonds, would become more accessible substrate for the enzyme than the intact
cells, because they would diffuse faster to the activity site of the enzyme. Then,
only a portion of the original enzyme molecules will move on to the next intact
cells, while a significant amount of the enzyme will keep on hydrolyzing the cell
wall fragments from the first round attack. Therefore the latter portion of the
enzyme would contribute significantly less to the further decrease of turbidity,
since according to Prasad and Litwack (1963) the whole cells would produce a
greater turbimetric change when they collapse than that produced by
fragmented cell walls being continuously lysed.
When lysozyme is immobilized the underestimation of activity or
nonconformity of Michaelis-Menton becomes only worse because of the
diffusion limitation caused by the immobilization. Shortly after the turbidity starts
decreasing, two different groups of substrates are generated for the bound
enzyme: the small cell wall pieces still with the intact glycosidic bonds and the
big whole cells yet to be broken. Naturally the former group would diffuse much
faster to the bound enzyme than the latter and become the favored substrate in
86
the system. Consequently, as the hydrolysis proceeds, more and more of the
enzyme reactions become less contributing to the reduction in turbidity of the
cell suspension. Thus a severe deviation from Michaelis-Mention was
observed.
Effect ofpH on Activity of the Immobilized Lysozyme
There was no significant shift of the pH optimum between the free
enzyme and the bound enzyme (3AA/His-15) (Figure 15). Both showed
maximum activity at about pH 6.4. But the curve for bound enzyme is slightly
constricted compared to the control. The second pH optimum around 9 was
not observed in this experiment presumably due to the high ionic strength of the
buffer used.
The bell-shaped pH-optimum curve can be explained on the basis of the
charge status of the critical carboxyl groups of Glu-35 and Asp-52 residues. It
can be assumed that at pH 6.2 in the given buffer system, the -COOH groups
in Glu-35 is un-ionized and Asp-52 is ionized, which is the requirement for the
enzyme to be fully active (Lipscomb, 1971; Imoto et al., 1972). Then the
decrease on the alkaline side is the result of the ionization of Glu-35, whereas
the decrease on the acid side reflects the gradual protonation of Asp-52.
87
Figure15. Effect of pH on the lytic activity of free and bound lysozyme
(3AA,His-15)
88
The same pH optimum observed for both the free and bound enzyme
would indicate that there is little or no charge-charge interaction involved in the
immobilized system that would affect the charge status of Glu-35 and Asp-52 at
the optimum pH of free enzyme.
Hydrophobicity of the Polystyrene Support- Study on Inhibition Effect of
Pentane on Lysozyme Immobilized on Polystyrene Beads
It is observed that, in general, the level of activity of an immobilized
hydrolase, which lysozyme is, depends on the degree of hydration of the
polymer matrix (Manecke, 1962). Therefore the inherent hydrophobicity of the
polystyrene matrix used in this study could actually be a disadvantage. It could
also adversely affect the coupling yields of the protein. Nevertheless, since the
surface of the matrix is heavily derivatized with chloro groups, about 4.3 mmol/g
resin, which would form hydrogen bonds with the water molecules around them,
it is possible that the highly hydrophilic chloro groups would offset the
hydrophobicity of the polystyrene.
It has been reported that pentane inhibited lysozyme activity against M.
lysodeikticus by 56% when added at 5% (v/v) to the reaction (Wantanabe and
Takesue, 1974). Pentane was used in this study to probe the level of
hydrophobicity of surface of the polystyrene matrix based on the hypothesis
that if the surface is highly hydrophobic the concentration of pentane near the
surface of the matrix would be higher than that in the bulk due to hydrophobic
interaction between the two, therefore pentane would exhibit stronger inhibition
89
to the immobilized lysozyme than to the free enzyme even though the
concentration of pentane in the bulk for the two systems was the same.
This proved to be the case (Table 14). When 4% (v/v) pentane was
added to M. lysodeikticus substrate solution (0.2mg/ml in 66mM phosphate
buffer, pH 6.24), the activity of the free enzyme (2ppm) reduced by 32%, while
that of the bound enzyme (3AA, His-15) reduced by 51%. This suggests that
the concentration of pentane in the microenvironment of the immobilized
lysozyme was significantly higher than that in the bulk due to the hydrophobicity
of the polystyrene matrix. The surface of the support remains hydrophobic
despite the heavy chloro-derivatization.
Stability of the Immobilized Lysozyme
The operational stability of an immobilized lysozyme system is important
for its practical use. Figure 16 shows the result of the stability study of the
immobilized Lysoyzme (3AA/His15). Activities of free and two bound enzymes
against lyophilized M lysodeikticus were monitored daily with the methods
describe earlier. After each assay, the bound enzymes were rinsed with 500 ml
of 66mM phosphate buffer and stored at 40C along with the free enzyme stock
(2ppm). After 14 days the 3AA/His-15 sample retained 90% of its original
activity, demonstrating a good stability that rivals that of the free enzyme. The
control of no-spacer/EDC sample, however, retained only 45 percent of its
activity after 14 days. For both bound enzymes, no protein loss
90
Table 14. The inhibition effect of 4%(v/v) pentane on the activity of free and
immobilized lysozyme against M. lysodeikticus.
Lysozyme
Activity Lost
(%)
Free (2ppm)
32
Immobilized (0.5 g resin; 3AA,His-15)
51
91
-♦— EDC no spacer
-■—3AA,l-lis15
Free lysozyme
20
10
0 *
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Time (day)
Figure 16. Stability of bound and free lysozymes over a period of 14 days
when stored at 4°C.
92
was detected in the wash done each time after the activity assay, indicating a
stable support and bond linkage.
The result again suggests that the His-15/spacer strategy minimized the
conformational change to the lysozyme molecule to an extent that the bound
enzyme had practically the same stability of the free enzyme. However, with
the control the stress caused by multiple bonding between the protein and
support eventually took a toll on its conformation and consequently the stability
over time.
93
CHAPTER 4
CONCLUSION
The three key issues in the covalent attachment of a biological active
protein to an insoluble support are: 1.) The protein should be attached to the
matrix by the fewest possible bonds to minimize conformational change; 2.)
The binding site(s) on the enzyme to the supports should be located as far as
practical from its active center and be nonessential for its tertiary structure; 3.)
The binding method should minimize the steric interference between the
support and the immobilized enzyme.
The fortuitous facts that the 15th amino acid residue in lysozyme
molecule is the only histidine (His-15) that is located on the opposite side of its
lytic activity center and nonessential for its tertiary structure allowed us to apply
the rules above rather successfully in this study. By coupling through the sole
histidine residue on the enzyme to minimize conformational change and
introducing spacers to reduce steric hindrance, the total and retained activity of
the preparation were 15 and 9 folds higher than those of the control prepared
with random covalent coupling protocol using the same polystyrene support. In
addition to reducing steric hindrance, the spacer also significantly increased the
immobilization yield.
94
The minimal conformation change of the immobilized lysozyme was also
supported by the observations from this study that its optimum pH and
operational stability were virtually identical to those of the free enzyme.
However, the drastic difference of retained activity between the soluble
substrate glycol chitin and insoluble substrate lyophilized M. lysodeikticus cells
(80% vs. 14%) suggested that the collision and diffusion limitations remained
the major hurdle for further improving the lytic efficiency of the immobilized
lysozyme system. Kinetics study of the system using the Eadie-Hofstee plot
confirmed that there was strong external diffusion effect occurring when
lyophilized M. lysodeikticus cells was used as substrate. This was not
unexpected given the double-heterogeneous nature of the system and the
bulky size of the insoluble substrate.
For future research, it would be interesting to test different hydrophilic
supports, which may improve the yield and lytic activity of the system. In the
meantime, other reagents that react to the imidazole group of His-15 are worth
exploring, such as, diazonium salts and formaldehyde.
95
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Blake, C.C.F., Johnson, L.N., Mair, G.A., North, A.C.T., Philips, D.C. and
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Blake, C.C.F., Cassels, R., Dobson, CM., Poulsen, F.M., Williams, R.J.P. and
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APPENDICES
APPENDIX A
On the Initial OD Rise in the First Few Seconds in the Turbidity Assay of
Lysozyme - the Puff Theory
The rule of thumb of enzyme kinetics study is always to measure the
initial rate of a reaction. When initial rates are used, complicating factors such
as the reverse reaction, product inhibition and inactivation of the enzyme can
be avoided and a much simpler rate equation can be used.
However, an interesting observation from this study when meaning
lysozyme activity with the turbidity method is that, instead of decreasing
immediately, the OD of M. lysodeikticus suspension often rises during the first 5
seconds or so right after the enzyme is injected into the cuvette (Figure A.1). To
avoid this interference, the activity of lysozyme samples was actually
determined from the slope of the curve between 20 and 40 seconds, NOT 0 to
40 seconds. Otherwise the computer calculated activities would be significantly
less or even negative!
A plausible explanation is that in that the initial attack of lysozyme on the
cell walls caused the cells to expand, or puff up, before they burst into pieces
due to the further hydrolysis from lysozyme. It is the initial puffing of the cells
made the OD rise.
108
Data supported the hypotheses: When extremely small amount of
lysozyme was added - 0.001 ppm, the OD did not decline after the initial rise,
because the enzyme was only enough to make limited damages on the
0.560,
l-t ft*
/
0.55$
'*
fr^-.S
* * ^ ,''^* .'- ^..*t
in
0>\f? /A
■ ■
i-',-----ri !> •'•'.«■ ;,-i.:,V
/■iA4,s:. :.y^
;
it*
0.00
40.00
Ti.!ns! (sec?
Figure A 1. The OD rise observed during the first few seconds of turbidity assay
of lysozyme activity of samples with three different concentrations: top line 2.5 ppm, middle line - 1 ppm , bottom line - 0.1 ppm.
109
cells that caused them to expand, i.e. OD increase, but not enough to make the
cells disintegrate (Figure A.2). On the other hand, when the assay was done for
10 ppm lysozyme sample, the initial OD rise was almost not noticeable (data
not shown here). The cells started to be broken apart immediately after
exposed to the overwhelming amount of lysozyme.
This hypothesis could be easily tested by the following two experiments:
1.
Simply look for visual evidence of the puffing under microscope
during the first a few seconds of the hydrolysis reaction. A real-time
imaging device would be helpful;
2.
Use cell walls instead of whole cells as substrate for the assay. If the
hypothesis is correct, the OD rising should be not be observed. If
the cell walls is not commercially available, it could be prepared by
mechanically grinding the whole cells in a mortar with sand.
110
O.SiO.
8^^v'^sn/,"-- ■ ^ '
*<*
<"' ,.„^,-. ^>' V ". f^-:
O.505-
O.SOOi 0.00
20.00
40.00
sa.os
Figure A 2. The initial OD rise of a lysozyme sample at extremely low
concentration - 0.001 ppm.
Ill
APPENDIX B
HPLC Assay of Lysozyme in Wine and Beer
Objectives
Fordras HPLC method was optimized and compared to the classical
turbidity method to:
I. Test the heat stability of lysozyme samples treated at different
temperatures: room temperature, 750C, and 950C;
II. Detect lysozyme in red wine and beer samples treated with the
enzyme;
I. Monitor lysozyme released from lysozyme-tannin participation in red
wine due to addition of NaCI.
Observations and Discussions
I. Heat Stability
Lysozyme samples were treated at different temperatures as described below
and analyzed at room temperature with the HPLC and turbidity methods.
A. 500ppm, 250ppm and 125ppm prepared in distilled water (Milli-Q).
At room temperature highly consistent and linear results were obtained
for the two methods (Figure B.1)
B. 500 ppm in distilled water and heated to 75 'C for 1, 2, 3, and 4 hours.
112
As showed in the figure B.2, significantly different results were given by
the two methods. While the HPLC method showed a moderate decline of
detected concentration as the treatment time increased, the turbidity method
indicated significant increase of activity, especially for samples treated for 2 and
3 hours.
C. 500ppm in distilled water and heated to 95 V for 1, 2, 3, and 4 hours.
At this higher temperature, both methods showed decline of detected
concentration. However, the turbidity method revealed the remarkable heat
stability of the enzyme (Figure B.3).
The HPLC method separates lysozyme from background and detects it
as protein with UV281. An important aspect that need to be explored is that
how sensitive this method is to the tertiary and secondary structure change of
the enzyme. It is noticed that there is a small peak that always eludes about 20
seconds before the main lysozyme peak, and the two peaks are very poorly
separated (chromatograms not shown). The small peak is suspected to
represent a lysozyme protein with a minor different structure. Interestingly, as
the heating time increased, the main peak shrunk while the small peak grew
significantly. At room temperature, the small peak's area is about 5% of that of
the main peak. After being kept at 950C for 4 hours, the two peaks had about
the same area. Obviously, more protein was transformed into the different
structure. It would be interesting to see whether the transformation will
113
eventually complete if the heating process keeps going longer, and to examine
the activity with the turbidity method.
II. Lysozyme in Red Wine and Beer Samples
Red wine (Pinor Noir) and Pilsner style beer were spiked with 500 ppm
lysozyme and assayed for lysozyme concentration 48 hours later. The
separation of the lysozyme peaks were not very satisfactory. Attempts to
improve the separation by changing gradient have not been successful.
Data indicated that lysozyme content decreased quickly in the red wine,
while fairly stable in beer. Again different results were obtained from the two
different assay methods for red wine. This time more protein was detected with
less activity (Table B.1).
It has been proposed that the significant loss of lysozyme in red wine
was due its conjugation with the phenolic compounds through electrostatic
interaction and raising ionic strength of system could reverse the combining
process.
A. Releasing Lysozyme from Lysozyme-Tannin Complex in Red Wine with
Addition of NaCI
The same red wine sample was spiked with 500ppm lysozyme. Two days
later 2% and 8% NaCI were added to the samples and analyzed with the HPLC
114
method 24 hours later. Results showed that with 8% NaCI the absorbed
lysozyme was completely released (Table B.2)
o
HHPLC
rl'
■ Turbidity
125
Prepared Lysozyme Concentration
(ppm)
Figure B.1 Detected lysozyme concentration of lysozyme samples without heat
treatment from the HPLC and Turbidity methods.
hV''
115
HHPLC
■ Turbidity
Time (hr)
Figure B.2. Detected lysozyme concentration of lysozyme samples treated at
750C for 1,2,3, and 4 hours.
116
I
•
Figure B.3. Detected lysozyme concentration of lysozyme samples treated at
95C for 1,2,3, and 4 hours.
117
Table B 1. Comparison of Lysozyme activity in red wine and beer samples
detected with HPLC and turbidity methods, respectively.
Sample
HPLC Cone.
(ppm)
Turbidity Cone.
(ppm)
SOOppmStd
500
516
R. Wine w/500 ppm
212
176
Widmer Beer w/500 ppm
489
118
Table B. 2 Releasing lysozyme from lysozyme-tannin complex in red wine with
addition of NaCI at 2% and 8% levels.
Sample
HPLC Cone,
(ppm)
Percentage
of original
500 ppm Std
500
100
R. Wine w/500 ppm
234
47
R. Wine w/500 ppm + 2% NaCI
396
80
R. Wine w/500 ppm + 8% NaCI
500
100