The amino acid sequence of satyr tragopan lysozyme and its activity

Article abstract of DOI:10.1271/bbb.67.2621

The amino acid sequence of satyr tragopan lysozyme and its activity was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln) comparing with Temminck's tragopan lysozyme and five amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and 103 (Asn to Ser) with chicken lysozyme. The time course analysis using N-acetylglucosamine pentamer as a substrate showed a decrease of binding free energy change, 1.1 kcal/mol at subsite A and 0.2 kcal/mol at subsite B, between satyr tragopan and chicken lysozymes. This was assumed to be responsible for the amino acid substitutions at subsite A-B at position 101 (Asp to Gly), however another substitution at position 103 (Asn to Ser) considered not to affect the change of the substrate binding affinity by the observation of identical time course of satyr tragopan lysozyme with turkey and Temminck's tragopan lysozymes that carried the identical amino acids with chicken lysozyme at this position. These results indicate that the observed decrease of binding free energy change at subsites A-B of satyr tragopan lysozyme was responsible for the amino acid substitution at position 101 (Asp to Gly).

Full text of DOI:10.1271/bbb.67.2621

This article was downloaded by: [University of Saskatchewan Library]
On: 27 December 2014, At: 20:15
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Bioscience, Biotechnology, and Biochemistry
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tbbb20
The Amino Acid Sequence of Satyr Tragopan
Lysozyme and Its Activity
Tomohiro ARAKIa, Gen TOSHIMAa, Tomomi KUSAOa, Yuki CHIJIIWAa, Shunsuke KAWAMURAa
& Takao TORIKATAa
a Department of Bioscience, School of Agriculture, Kyushu Tokai UniversityAso,
Kumamoto 869-1404, Japan
Published online: 22 May 2014.
To cite this article: Tomohiro ARAKI, Gen TOSHIMA, Tomomi KUSAO, Yuki CHIJIIWA, Shunsuke KAWAMURA & Takao
TORIKATA (2003) The Amino Acid Sequence of Satyr Tragopan Lysozyme and Its Activity, Bioscience, Biotechnology, and
Biochemistry, 67:12, 2621-2626, DOI: 10.1271/bbb.67.2621
To link to this article: http://dx.doi.org/10.1271/bbb.67.2621
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Biosci. Biotechnol. Biochem., 67 (12), 2621–2626, 2003
The Amino Acid Sequence of Satyr Tragopan Lysozyme and Its Activity
Tomohiro ARAKI,† Gen TOSHIMA, Tomomi KUSAO, Yuki CHIJIIWA, Shunsuke KAWAMURA
,
and Takao TORIKATA
Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto 869-1404, Japan
Received July 14, 2003; Accepted September 12, 2003
The amino acid sequence of satyr tragopan lysozyme
and its activity was analyzed. Carboxymethylated
lysozyme was digested with trypsin and the resulting
peptides were sequenced. The established amino acid
sequence had three amino acid substitutions at positions
103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln)
comparing with Temminck's tragopan lysozyme and
ˆve amino acid substitutions at positions 3 (Phe to Tyr),
15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and
103 (Asn to Ser) with chicken lysozyme. The time course
analysis using N-acetylglucosamine pentamer as a
substrate showed a decrease of binding free energy
sequences of phasianid birds are known for 14
lysozymes and we established a rapid sequence
determination method for chicken-type lysozymes
from Galliformes.9–15) This method enables us to
detect the amino acid substitutions occurred in
closely related mutants by peptide map ˆtting and
quantitative amino acid analysis.
By the extensive study of chicken lysozyme, it is
revealed that this enzyme carries six substrate binding
sites called subsites A, B, C, D, E, and F. A substrate
bound at the subsites is cleaved between subsites
D and E through the conventional acid catalytic
reaction of Glu35 and Asp52. This enzyme also
has high transglycosylation activity in addition to
the hydrolysis. However the reaction mechanism
for transglycosylation on this molecule remains
unknown.
In the previous studies, we found that limited
substitution at subsites aŠects the substrate binding
or reaction mechanisms such as hydration and trans-
glycosylation. The time-course analysis of the degra-
dation of a substrate, N-acetylglucosamine pentamer
(GlcNAc)5, with the HPLC measurement can be used
to evaluate the diŠerences of the binding free energy
change at each subsite and rate constants for hydra-
tion and transglycosylation by computer simulation.
Therefore we can directly estimate the contribution
of the amino acid that occurs in the active site
compared with the amino acid sequences of two
mutant enzymes, which carry amino acid substitu-
tions at the active site. The naturally occurring
lysozyme molecules genetically related closely to
chicken lysozyme which carry limited amino acid
substitutions at substrate binding site, are useful to
evaluate the reaction mechanisms of lysozyme such
as substrate binding and the lysozyme catalysis
mechanism, and the information obtained is applica-
ble to the mutagenesis study focused on the amino
acids that participate in the substrate binding and
catalysis.
change, 1.1 kcal mol at subsite A and 0.2 kcal mol at
W
W
subsite B, between satyr tragopan and chicken lyso-
zymes. This was assumed to be responsible for the ami-
no acid substitutions at subsite A-B at position 101 (Asp
to Gly), however another substitution at position 103
(Asn to Ser) considered not to aŠect the change of the
substrate binding a‹nity by the observation of identical
time course of satyr tragopan lysozyme with turkey and
Temminck's tragopan lysozymes that carried the identi-
cal amino acids with chicken lysozyme at this position.
These results indicate that the observed decrease of
binding free energy change at subsites A-B of satyr
tragopan lysozyme was responsible for the amino acid
substitution at position 101 (Asp to Gly).
Key words: lysozyme; amino acid sequence; egg white
Lysozyme is one of the best-characterized hydro-
lases, which cleave b-1,4 linkages of N-acetylglucosa-
mine (GlcNAc) homopolymer and GlcNAc-N-
acetylmuramic acid heteropolymer, which causes
lysis of the bacteria containing that polymer in their
cell walls. This enzyme is classiˆed into three types,
chicken type,1) goose type,2) and phage type.3) The
amino acid sequences of lysozymes have been report-
ed for many birds, mammals,4) ˆshes,5) and insects.6,7)
Only some birds have goose-type lysozyme.8) The
others have chicken-type ones.
One of the most intensive investigations for amino
acid sequences of chicken type lysozyme is for avian
egg white, especially for Galliformes. The amino acid
In this study we report the amino acid sequence of
satyr tragopan (Tragopan satyra) lysozyme and
compare its amino acid sequence and activity with
†
To whom correspondence should be addressed. Fax:
81-967-67-3929; E-mail: taraki ktmail.ktokai-u.ac.jp
＋ ＠
Data deposition: The sequence reported in this paper has been deposited in the PIR database (accession No. JC7908).

2622
T. ARAKI et al.
other lysozymes to evaluate the enzymatic properties
of this lysozyme.
evacuated sealed tubes at 110
9
C for 20 hr with
constant boiling HCl containing 0.05
z
b
-mercap-
toethanol. The resulting hydrolysates were analyzed
with an amino acid analyzer (Model 835, Hitachi
Co., Japan). The amino acids of tryptic peptides
were sequenced by a protein sequencer model PSQ1
(Shimadzu Co., Japan).
Materials and Methods
Puriˆcation of lysozyme. Freshly laid satyr
tragopan eggs were a kindly gift from Kobe Oji Zoo,
Kobe, Japan and Temminck's tragopan eggs were
from Kyoto Municipal Zoo, Kyoto, Japan. The egg
white (33 ml) was extracted with 66 ml of water and
treated with isoelectric precipitations at pHs 4.0 with
Enzyme action.
a) Lysis activity. The lytic activity was assayed
using the lyophilized cell wall of M. luteus as a
1
M
HCl and 7.0 with 1
M
NaOH and the clariˆed
substrate. Samples (10 to 100
ml) of the eluent were
solution after centrifuge at 12,000 rpm was then put
added to 3 ml of the substrate suspension in 0.1
M
×
on a CM-Toyopearl column (1.5 46 cm) equilibrat-
phosphate buŠer, pH 7.0, adjusted to OD 1.0 at
540 nm. One enzyme unit was deˆned as the amount
causing a decrease of 0.1 absorbance unit at 540 nm
ed with 0.03
M
phosphate buŠer (pH 7.0) with the
‰ow rate of 20 ml h and 5 ml each in volume was
W
collected. The column was then eluted stepwise with
in the reaction for 1 min at 259C.
the same buŠer containing 0.3
M
NaCl. The lysozyme
b) Time course. The enzyme reaction was done
fraction was rechromatographed on the same column
with a gradient of 0.1 to 0.3 NaCl in the same
with the substrate GlcNAc pentamer. The reaction
M
M
mixture containing 0.1 m
substrate dissolved in 10 m
was incubated at 50 C. At a given reaction time,
500 l of the reaction mixture was withdrawn and
M
lysozyme and 1 m
sodium acetate, pH 5.0
M
of
buŠer and with the same conditions. Enzyme activity
was monitored by a lytic activity using the lyophilized
cell wall of Micrococcus luteus (Sigma Co., USA)
M
9
m
as a substrate. Lysozyme solution (10 to 100
was added to the substrate suspension adjusted to
OD 1.0 at 540 nm in 3.0 ml of 0.1 phosphate
m
l)
rapidly chilled in an ultra ˆltration ˆlter (Ultrafree
C3-LGC, Millipore Co.) containing 70 mg of Bio-
－
9
Rex 70 in a cool cup ( 80 C). The reaction mixture
M
buŠer, pH 7.0, and monitored the reduction of
absorbance at 540 nm.
was then centrifuged through a ˆlter membrane and
the resulting solution was lyophilized. The dried
sample was dissolved in 50 ml of water and 10 ml of
Carboxymethylation. Lysozyme was reduced and
the solution was put onto a gel ˆltration column
(TSKgel G-Oligo-PW, Tosoh Co.) using a JASCO
800-Series HPLC. Elution was done with distilled
water at room temperature. Each chitooligosaccha-
ride was monitored and measured at 220 nm using
the standard curve obtained from authentic saccha-
ride solutions.
c) Computer simulation. The rate equation and
binding free energy of the lysozyme-catalyzed
reaction on the initial substrate GlcNAc pentamer
was numerically solved to obtain the calculated time
courses by the method previously reported. The rate
constants, k＋1, k－1, and k＋2, used in the calculation
are shown in Fig. 1.17,18)
carboxymethylated for structural analysis.16) Namely,
10 mg of lysozyme was dissolved in 1.4
buŠer, pH 8.6, and then 1.2 g of urea, 100
EDTA, and 33 l of -mercaptoethanol were added.
The solution was left for 1 h at 37 C under N2 gas.
After the reduction, 89 mg of monoiodoacetic acid in
300 l of 1.0 NaOH was added and left for 1 h at
room temperature in the dark. The reaction mixture
M
tris-HCl
m
l of 5
z
m
b
9
m
M
×
was desalted through Sephadex G-50 column (1.7
46 cm) in 0.2
M
NH4OH and protein fraction was
lyophilized (Cm-Lysozyme).
Enzymatic Digestion. Cm-lysozyme (5 mg) was
dissolved in 1 ml of tris-HCl buŠer, pH 8.0, digested
with trypsin (1 50, w w, TR-TPCK, Cooper Bio-
W
W
Result and Discussion
9
medical Co., USA) at 37 C for 4 h. The digest was
directly separated by HPLC.
Satyr tragopan lysozyme was puriˆed by conven-
tional puriˆcation method. A water extract of egg
white was treated with acidic and neutral pHs to
precipitate acidic and neutral proteins and the clari-
ˆed solution was then chromatographed on a cation
exchange column, which yielded a single peak with
lytic activity (data not shown). The enzyme was then
carboxymethylated and digested with trypsin. The
digest analyzed by peptide mapping. The HPLC
peptide map of satyr tragopan lysozyme was
compared with that of chicken lysozyme (Fig. 2). The
map-ˆtting method indicates that the change of
Peptide Mapping and Sequence Analysis. The
tryptic peptides were separated with a reversed-phase
×
(RP) HPLC column (ODS 120A S5, 4.0 250 mm,
Yamamura Chemical Co., Japan) using the JASCO
800 series HPLC (Japan Spectroscopic Co., Japan).
The peptide elution was done with a linear gradient
elution system of 0.1
A) and 60 acetonitrile in solvent A (solvent B).
A gradient of 0 to 50 of solvent B was used
for 130 min. Tryptic peptides were hydrolyzed in
z tri‰uoroacetic acid (solvent
z
z
z

Satyr Tragopan Lysozyme
2623
Fig. 1. Reaction Scheme for the Lysozyme-catalyzed Reaction of GlcNAc Pentamer.
In this scheme, k＋1, k－1, and k＋ are the rate constants for cleavage of the glycosidic linkage, transglycosylation, and hydration,
2
respectively.
retention time of peptide peaks and the subsequent
amino acid analysis of their amino acid compositions
reveals the amino acid substitution, deletion, or
insertion in a tryptic peptide. Further, the insertion
or deletion between tryptic peptides including Lys or
Arg can be detected by the amino acid analysis
stoichiometrically as well as the identiˆcation a free
Lys or Arg residue derived from successive Lys-Arg
or Lys-Lys residues. In case of undetectable amino
acid substitution by amino acid analysis such as Asn
W
Asp or Gln Glu, it has been proved to be detectable
W
by the change of retention time. Therefore the amino
acid compositions of all peaks of satyr tragopan
lysozyme were analyzed. The peptides in each peak
of satyr tragopan lysozyme that have both the identi-
cal amino acid composition and the identical elution
position to chicken lysozyme were considered to have
the identical amino acid sequence as described above.
The peptides that have diŠerent amino acid composi-
tions and elution positions were analyzed for their
amino acid sequences, and substitutions were found
to occur on the peptides in peaks 3, 4, 5, 7, and 13 by
this analysis (indicated by vertical arrows in Fig. 2).
The result proved that these peptides have substitu-
tions at Phe3 to Tyr, His15 to Leu, Gln41 to His,
Asp101 to Gly and Asn103 to Ser when compared
with the corresponding tryptic peptide of chicken
lysozyme (Fig. 3).
Fig. 2. Comparison of Reversed-phase HPLC Pattern of Satyr
Tragopan Lysozyme with That of Chicken Lysozyme.
Peaks in satyr tragopan lysozyme that are indicated by arrows
are the peaks appearing in diŠerent positions and with diŠerent
amino acid compositions when compared with chicken lyso-
zyme. For detailed conditions of HPLC see the text. HEL,
chicken lysozyme; TSL, satyr tragopan lysozyme.
The peak correspond to T13 was obtained as a
single peak on the peptide map of satyr tragopan
lysozyme. On the map of chicken lysozyme, T13
normally shows the multiple peaks that are caused by
the deamidation of Asn103 in T13 peptide. This
result may be due to the amino acid substitution of

2624
T. ARAKI et al.
Asn103 in satyr tragopan lysozyme (Asn103 to Ser).
The amino acid sequence established for satyr
tragopan lysozyme was compared with chicken,
turkey, and Temminck's tragopan lysozymes15) and
the amino acid substitutions, compared to chicken
lysozyme, probed by sequence analysis of these
peptides, are boxed in on Fig. 3. This enzyme had 5,
4, and 3 amino acid substitutions when compared
with chicken, turkey, and Temminck's tragopan
lysozyme, respectively. Among these substitutions,
Gly101 and Ser103 were located at the loop region of
subsites A-C (Fig. 4). Asp101 is reported to interact
with the substrate (GlcNAc at subsites A and B) by
hydrogen bonding and the substitution by Gly at this
position may cause the loss of hydrogen bonding. On
Fig. 4, the possible conformation change of the side
chain of amino acids at position 101 and 103 on
chicken lysozyme were drawn by the energy minimi-
zation program on a Swiss PDB Viewer.20) The result
indicates that the change of the size and the location
of the side chain after mutation showed the possibili-
ty of the loss of hydrogen bonding at subsites A and
B (this simulation did not consider the main chain
movements). The substitution at position 101 is also
found in turkey and Temminck's tragopan lysozymes
and the eŠect of amino acid substitution for enzyme
activity is notable point. Further Ser103 was ˆrst
found in phasianid bird's lysozymes. Although the
importance of the contribution of the amino acid at
position 101 is well known, limited information is
available on the amino acid at position 103, on the
loop region at the substrate binding subsites A-B.
As chicken-type lysozyme catalyzes not only
hydrolysis of sugar chains but also a transglycosyla-
tion reaction, the product of oligosaccharide shows
complicated patterns. However, the time course
analysis using GlcNAc pentamer allowed the evalua-
tion of the eŠects of amino acids substituted at the
subsites, by computer simulation analysis (Fig. 1). To
evaluate the contribution of amino acid substitutions
to substrate binding and lysozyme catalyzed reaction,
the time course of satyr tragopan lysozyme for the
GlcNAc pentamer was analyzed and is shown in
Fig. 5 with time courses of chicken, turkey, and
Temminck's tragopan lysozymes. In the pattern of
satyr tragopan lysozyme, the order of the concentra-
tion of product oligomer at 20 min of reaction was 2,
1, 3, 4, and 5 instead of 1, 2, 4, 3, and 5 for chicken
lysozyme. This pattern is found to be similar to that
of turkey and Temminck's tragopan lysozymes
(Fig. 5). These lysozymes have the same substitution
Fig. 3. Amino Acid Sequence of Satyr Tragopan Lysozyme and
Its Comparison with Chicken, Turkey, and Temminck's
Tragopan Lysozymes.
The peptides obtained are indicated by T numbers by Can-
ˆeld's nomenclature of tryptic peptides of chicken lysozyme.1)
Substituted amino acids are boxed in. Other positions that con-
tain no substituted amino acid are indicated by dashes. A substi-
tution that occurred in satyr tragopan on the substrate binding
site is highlighted. HEL, chicken lysozyme; TSL, satyr tragopan
lysozyme; TEL, turkey lysozyme; TTL, Temminck's tragopan
lysozyme.
Fig. 4. Tertiary Structure of the Region of Subsites A-C.
The loop region (Asp101 to Trp108) is drawn by a ribbon diagram and the substrate GlcNAc trimer is drawn by ball and stick model.
NAG1, NAG2, and NAG3 indicate the GlcNAc residues located at subsites A, B, and C, respectively. The model was prepared using the
PDB data of 1HEW19) with the program of Swiss PDB Viewer. A: chicken lysozyme, B: mutation model of Asn103Ser.

Satyr Tragopan Lysozyme
2625
Table 1. Estimated Reaction Parameter Values for HEL and
TSL
Binding free energy
Rate constant
1
(kcal
W
mol)
(s－
k＋ k－ k＋
2
)
A
B
C
D
E
F
1
1
HEL
TSL
－
－
2.0
0.9
－
－
3.0
2.7
－
－
5.0 4.5
5.0 4.5
－
－
2.5
2.5
－
－
1.5 0.93 40.0 0.30
1.5 0.93 40.0 0.30
k＋1, k－1, and k＋2 are the rate constants for cleavage of the glycosidic link-
age, transglycosylation, and hydration, respectively.
A and B for satyr tragopan lysozyme were observed
－
－
as 1.0 and 2.8 kcal mol, respectively. Whereas,
W
the values of subsites A and B for chicken lysozyme
were
－
－
3.0 kcal mol, respectively. The
2.0 and
W
amino acid substitutions at the loop region of sites
A-C would have a profound eŠect on the decrease in
the a‹nity of subsites A and B for the sugar residues.
As the Asp101 of chicken lysozyme forms hydrogen
bonds with GlcNAc at subsite A and GlcNAc at
subsite B, the substitution of Asp to Gly, which has
no side chain at this position, will cause the loss of
hydrogen bonding (Fig. 4). Therefore, the diŠerence
between the value of the binding free energy for
satyr tragopan lysozyme and chicken lysozyme
may be attributed to this substitution at subsite A
(Asp101Gly).
The contribution of Asp101 in chicken lysozyme to
the substrate binding at subsite A was reported to
Fig. 5. Experimental Time Course for Chicken, Satyl Tragopan,
Turkey, and Temminck's Tragopan Lysozymes.
HEL, chicken lysozyme; TSL, satyr tragopan lysozyme; TEL,
turkey lysozyme; TTL, Temminck's tragopan lysozyme. The
numerals in the ˆgure indicate the polymerization degree of
GlcNAc.
correspond to a binding free energy of about 30
z
of
the total binding free energy of subsite A.21)
However, the substitution of Asp to Gly that causes
the loss of the hydrogen bonding proved the contri-
bution of Asp101 to be about 50
z
of the total bind-
ing free energy of subsite A. Further, the decreasing
value of binding free energy were found at subsites
A and B instead of subsites A and C as reported
for Asp101-Glucosamine or Asp101-ethanolamine
modiˆed lysozyme.21) The diŠerence of the binding
free energy for these subsites may due to the
introduced chemicals and the removal of the side
chain of this residue.
Another substitution at subsite B (Asn103Ser)
seemed not to aŠect the change of the substrate
binding a‹nity with the observation of similar time
courses of satyr tragopan lysozyme to turkey
lysozyme and Temminck's tragopan lysozymes,
which have the identical amino acid at position 103
with chicken lysozyme (Fig. 5). Asn103 is reported to
have hydrogen bonding with GlcNAc at subsite A.
The result showed that the loss of the hydrogen
bonding at position 103 caused by the substitution
from Asn to Ser did not aŠect the substrate binding.
It is understandable, therefore, that the contribution
of the amino acid at position 103 to the substrate
binding would be quite little. This is supported by the
observation of similar time courses for native and
at position 101 as satyr tragopan lysozyme (Fig. 3).
When compared with these lysozymes, satyr
tragopan lysozyme has additional amino acid substi-
tution (Asn103 to Ser) at the loop region at subsite
A-C (Fig. 4). Taking the above factors into consider-
ation, we estimate that the amino acid substitution at
position 101 aŠects the diŠerence of reaction patterns
between satyr tragopan and chicken lysozyme, but
the substitution found only on satyr tragopan
lysozyme at position 103 do not aŠect the substrate
binding and cleavage by the comparison of the time
course patterns.
To conˆrm this notion, experimental time courses
were then simulated by computer analysis. The
values of binding free energy and the rate constants,
k＋1, k－1, and k＋ were obtained by the calculated
2
time course ˆtted to the experimental ones (patterns
not shown). The values of binding free energy and
the rate constant for calculated time courses for these
lysozymes are listed in Table 1. The diŠerence of the
values of binding free energy were found at subsites
A and B. Namely, the binding free energy of subsites

2626
T. ARAKI et al.
cristatus) lysozyme and its comparison with lyso-
zymes from phasianoid birds. Agric. Biol. Chem., 53,
2955–2962 (1989).
deamidated chicken or reeve's pheasant lysozymes at
position 103 (Asn to Asp) frequently occurring for
storage of the lysozyme (data not shown).12) Combin-
ing these results, we conclude that the binding free
energy change of satyr tragopan lysozyme observed
at subsites A and B is caused by the amino acid
substitution of Asp to Gly at position 101 on the loop
region.
10) Araki, T., Kuramoto, M., and Torikata, T., The
amino acid sequence of Lady Amherst's pheasant
(
(
Chrysolophus amherstiae) and golden pheasant
Chrysolophus pictus) egg-white lysozymes. Agric.
Biol. Chem., 54, 2299–2308 (1990).
11) Araki, T., Kudo, K., Kuramoto, M., and Torikata,
T., The amino acid sequence of lysozyme from
kalij pheasant egg-white. Agric. Biol. Chem., 55,
1701–1706 (1991).
Acknowledgments
12) Araki, T., Kuramoto, M., and Torikata, T., The
amino acid sequence of reeves' pheasant lysozyme.
Agric. Biol. Chem., 55, 1707–1713 (1991).
13) Araki, T., Kuramoto, M., and Torikata, T., The
amino acid sequence of copper pheasant lysozyme.
Biosci. Biotechnol. Biochem., 58, 794–795 (1994).
14) Araki, T., Matsumoto, T., and Torikata, T., The
amino acid sequence of monal pheasant lysozyme
and its activity. Biosci. Biotechnol. Biochem., 62,
1988–1994 (1998).
15) Araki, T., Seki, S., and Torikata, T., The amino acid
sequence of Temminck's tragopan lysozyme and
evaluation of the in‰uence of amino acid substitution
for enzymatic activity against chtin oligomer. Chitin
and Chitosan Res., 6, 217–225 (2000).
16) Crestˆeld, A. M., Moore, S., and Stein, W. H., The
preparation and enzymatic hydrolysis of reduced and
S-carboxymethylated proteins. J. Biol. Chem., 238,
622–627 (1963).
We thank Dr. Kouichi Murata and Dr. Akiko
Yamada, Kobe Oji Zoo, Kobe Japan, and Dr.
Kaname Okahashi, Kyoto Municipal Zoo, Kyoto
Japan for preparing eggs.
References
1) Canˆeld, R. E., The amino acid sequence of egg
white lysozyme. J. Biol. Chem., 238, 2698–2707
(1963).
2) Simpson, R. J., Begg, G. S., Dorow, D. S., and
Morgan, F. J., Complete amino acid sequence of the
goose-type lysozyme from the egg white of the black
swan. Biochemistry, 19, 1814–1819 (1980).
3) Inoue, M., Imada, M., and Tsugita, A., The amino
acid sequence of T4 phage lysozyme. IV. Dilute acid
hydrolysis and the order of tryptic peptides. J. Biol.
Chem., 245, 3479–3484 (1970).
4) Canˆeld, R. E., Kammerman, S., Sobel, H. H., and
Morgan, F. J., Primary structure of lysozymes from
man and goose. Nature, 232, 16–17 (1971).
5) Dautigny, A., Prager, E. M., Pham-Dinh, D., Jolles,
J., Pakdel, F., Grinde, B., and Jolles, P., cDNA and
amino acid sequences of rainbow trout (Oncorhyn-
chus mykiss) lysozymes and their implications for
the evolution of lysozyme and lactalbumin. J. Mol.
Evol., 32, 187–198 (1991).
6) DaŠre, S., Kylsten, P., Samakovlis, C., and
Hultmark, D., The lysozyme locus in Drosophila
melanogaster: an expanded gene family adapted for
17) Kuhara, S., Ezaki, E., Fukamizo, T., and Hayashi,
K., Estimation of the free energy change of substrate
binding lysozyme-catalyzed reactions. J. Biochem.
92, 121–127 (1982).
,
18) Masaki, A., Fukamizo, T., Otakara, A., Torikata,
T., Hayashi, K., and Imoto, T., Estimation of
rate constants in lysozyme-catalyzed reaction of
chitooligosaccharides. J. Biochem., 90, 1167–1175
(1981).
19) Cheetham, J. C., Artymiuk, P. J., and Phillips, D.
C., Reˆnement of an enzyme complex with inhibitor
bound at partial occupancy. Hen egg-white lysozyme
and tri-N-acetylchitotriose at 1.75 Å resolution. J.
Mol. Biol., 224, 613–628 (1992).
20) Guex, N., and Peitsch, M. C., SWISS-MODEL and
the Swiss-Pdb Viewer: An environment for compara-
tive protein modeling. Electrophoresis, 18, 2714–2723
(1997).
21) Fukamizo, T., Hayashi, K., and Goto, S., The role of
binding subsite A in reactions catalyzed by hen egg-
white lysozyme. Eur. J. Biochem., 158, 463–467
(1986).
expression in the digestive tract. Mol. Gen. Genet.
242, 152–162 (1994).
,
7) Engstrom, A., Xanthopoulos, K. G., Boman, H. G.,
and Bennich, H., Amino acid and cDNA sequence of
lysozyme from Hyalophora cecropia. EMBO J., 4,
2119–2122 (1985).
8) Prager, E. M., Wilson, A. C., and Arnheim, N.,
Widespread distribution of lysozyme g in egg white
of birds. J. Biol. Chem., 249, 7295–7297 (1974).
9) Araki, T., Kudo, K., Kuramoto, M., and Torikata,
T., The amino acid sequence of Indian peafowl (Pavo