Purification and characterization of an N-terminal acidic amino acid-specific aminopeptidase from soybean cotyledons (glycine max)

Article abstract of DOI:10.1271/bbb.90617

A novel enzyme that catalyzes the efficient hydrolysis of Glu-Glu was isolated from soybean cotyledons by ammonium sulfate fractionation and successive column chromatographies of Q-sepharose, Phenyl sepharose, and Superdex 200. The apparent molecular mass of this enzyme was found to be 56kDa and 510 kDa by SDS-polyacrylamide gel electrophoresis and Superdex 200 HR 10/30 column chromatography respectively. The enzyme had high activity against Glu-/7-nitroanilide (/NA) and Asp-pNA, whereas Leu-pNA, Phe-pNA, Ala-/;NA, and Pro-pNA were not hydrolyzed. The synthetic dipeptides Glu-Xxx and Asp-Xxx were hydrolyzed, but Xxx-Glu was not. The digestion of a Glu-rich oligopep-tide, chromogranin A (Glu-Glu-Glu-Glu-Glu-Met-Ala-Val-Val-Pro-Gln-Gly- Leu-Phe-Arg-Gly-NH₂) using this purified enzyme was also investigated. Glutamic acid residues were cleaved one by one from the N-terminus. These observations indicate that the enzyme removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. It is thought that it was an N-terminal acidic amino acid-specific aminopeptidase from a plant.

Full text of DOI:10.1271/bbb.90617

Biosci. Biotechnol. Biochem., 74 (1), 113–118, 2010
Purification and Characterization of an N-Terminal Acidic Amino
Acid-Specific Aminopeptidase from Soybean Cotyledons (Glycine max)
y
Minao ASANO, Nami NAKAMURA, Misako KAWAI, Tetsuya MIWA, and Noriki NIO
Institute of Life Sciences, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan
Received August 19, 2009; Accepted October 1, 2009; Online Publication, January 7, 2010
[doi:10.1271/bbb.90617]
A novel enzyme that catalyzes the efficient hydrolysis
of Glu-Glu was isolated from soybean cotyledons by
ammonium sulfate fractionation and successive column
chromatographies of Q-sepharose, Phenyl sepharose,
and Superdex 200. The apparent molecular mass of this
enzyme was found to be 56 kDa and 510 kDa by SDS-
polyacrylamide gel electrophoresis and Superdex 200
HR 10/30 column chromatography respectively. The
enzyme had high activity against Glu-p-nitroanilide
(pNA) and Asp-pNA, whereas Leu-pNA, Phe-pNA, Ala-
pNA, and Pro-pNA were not hydrolyzed. The synthetic
dipeptides Glu-Xxx and Asp-Xxx were hydrolyzed, but
Xxx-Glu was not. The digestion of a Glu-rich oligopep-
tide, chromogranin A (Glu-Glu-Glu-Glu-Glu-Met-Ala-
Val-Val-Pro-Gln-Gly-Leu-Phe-Arg-Gly-NH2) using this
purified enzyme was also investigated. Glutamic acid
residues were cleaved one by one from the N-terminus.
These observations indicate that the enzyme removes
glutamyl or aspartyl residues from N-terminal acidic
amino acid-containing peptides. It is thought that it was
an N-terminal acidic amino acid-specific aminopepti-
dase from a plant.
intermediary fragments of approximately 30 kDa, called
c30. These fragments were identified as being derived
from the degradation of ꢀ-conglycinin, a major storage
protein in soybeans.8) Using an assay system capable
of detecting hydrolytic activity toward c30, protease
D3 was purified from the germinating cotyledons of
soybeans.9) Characterization of this enzyme showed that
it was cysteine protease. Its specificities were examined
using peptide-4-methylcoumary-7-amide (MCAs) and
peptides hormones, and cathepsin L-like broad specific-
ity was observed at pH 4.0.9) These results indicate that
protease D3 contributes to protein turnover in germi-
nating soybean cotyledons, as do other cysteine pro-
teases in animals and plants.10,11) We also confirmed
that it was difficult to digest native soybean storage
proteins using commercially available proteases for
food processing. Therefore, protease D3 probably plays
a critical role in protein metabolism in soybean seeds
and has enzymatic activity hydrolyzing native soybean
storage proteins. Peptidases such as carboxypepti-
dase,12) prolidase,13) and leucine-aminopeptidase14) have
also been reported in soybean cotyledons. Glycinin and
ꢀ-conglycinin, major storage proteins of soybean, are
abundant in acidic amino acid sequences such as -Glu-
Glu- and -Glu-Asp-.15) Aminopeptidase, which liberates
glutamic acid or aspartic acid from the N-terminus of
peptide, is called glutamyl aminopeptidase16–18) or
aspartyl aminopeptidase.19,20) These enzymes have
been reported to be present in mammals, yeast, and
filamentous fungi,20–22) but have not been purified from
plants.
Key words: N-terminal acidic amino acid-specific ami-
nopeptidase; soybean; cotyledon; aspartyl
aminopeptidase
Shutov and Vaintraub suggested a role of proteases in
the mobilization of storage proteins at the cotyledon
during seed germination.1) In addition, many peptidases
in cotyledon tissue that play important roles in the
degradation process of storage proteins, have been
reported.2–5) Aminopeptidases catalyze the sequential
removal of amino acids from the unblocked N termini of
peptides and proteins. Various aminopeptidases with
different substrate specificities are widely distributed
in eukaryotes and prokaryotes.6) They are generally
classified in terms of their substrate specificities, the
preference for a neutral, acidic, or basic amino acid in
the P1 position. They play an important role in protein
and peptide metabolism.7)
We speculated that glutamyl aminopeptidase or
aspartyl aminopeptidase exists in the germinating
soybean cotyledone and plays an important role in
soybean germination. In this study, we looked for acidic
amino acid specific aminopeptidase in soybean cotyle-
dons with Glu-Glu as a substrate, and isolated a novel
aminopeptidase from the soybean cotyledon that re-
moves acidic amino acids sequentially from N-terminus
acidic amino acid-containing peptides.
We have been searching for proteases and peptidases
that can degrade storage proteins and their fragments to
oligopeptides or amino acids in germinating seeds of the
soybean Glycine max. First we investigated the protein
degradation process during germination in the protein
bodies of soybeans, and identified a group of key
Materials and Methods
Plant material. Soybean seeds, Glycine max [L.] Merrill cv. Kegon,
were purchased from Sakata-no-Tane (Yokohama, Japan). They were
sown on wet vermiculite (0 DAI) and allowed to germinate in a
phytotron maintained at 25 ꢀC under a 12 h-light/12 h-dark cycle.
y
To whom correspondence should be addressed. Tel: +81-44-223-4172; Fax: +81-44-246-6241; E-mail: noriki nio@ajinomoto.com
Abbreviations: AEBSF, 4-(2-aminoethyl)-benzensulfonyl fluoride; DAI, day-after imbibition; E-64, L-trans-epoxysuccinyl-leucylamide-(4-
guanidino)butane; MALDI, matrix assisted laser desorption-ionization; 2-ME, 2-mercaptoethanol; pNA, p-nitroanilide

114
M. ASANO et al.
Water was supplied once a day. On days 0–10, the cotyledons alone
were dissected from the other tissues, rinsed with distilled water, and
frozen at ꢁ80 ꢀC until use.
saturated with 65% saturation ammonium sulfate (564 g/3,400 ml). To
recover the precipitated proteins, the supernatant was stirred for 15 h at
4 ꢀC and then centrifuged at 32;000 ꢂ g for 30 min. The precipitates
were dissolved in 120 ml of 50 mM potassium phosphate buffer,
100 mM NaCl (pH 7.0, 2 mM NaN3) and dialyzed against the same
buffer (2 liters ꢂ 5, 16 h) at 7 ꢀC. After dialysis, the solution was
centrifuged at 32;000 ꢂ g for 20 min at 4 ꢀC and then filtered through a
0.2 mm filter (Nalgene Labware, Rochester, NY).
Chromatography on Q sepharose HP. An anion exchange column,
HiLoad Q Sepharose HP 26/10 (GE Healthcare Japan, Tokyo) was
equilibrated with 50 mM potassium phosphate buffer and 100 mM NaCl
(pH 7.0, 2 mM NaN3). The enzyme solution (5,160 mg protein/190 ml)
was divided into eight aliquots, and each aliquot was subjected to the
column. The column was then washed with a 5-fold volume of the
buffer. After washing, the absorbed active fraction was eluted with an
increasing linear gradient of 100 mM to 250 mM NaCl in an 8-fold
column volume of the buffer. The active fractions were collected and
concentrated from 2,000 ml to 90 ml with an ultracentrifugation unit
(Minitan). After 11.88 g of ammonium sulfate was added to this
concentrate and dissolved, the solution was centrifuged at 32;000 ꢂ g
for 10 min to give the supernatant.
Reagents. Polyacrylamide gel, Multigel (15/25) (T ¼ 15{25%) was
purchased from Dai-Ichi Pure Chemical (Tokyo). A glutamic acid
assay kit was from Yamasa (Tokyo). Chromogranin A and E-64 were
from the Peptide Institute (Osaka, Japan). AEBSF was from Sigma-
Aldrich Japan (Tokyo), and pNA substrates for aminopeptidase were
from Bachem (Bubendorf, Switzerland). Synthetic dipeptides contain-
ing Glu and/or Asp were from the Peptide Institute and Kokusan
Chemicals (Tokyo). All other chemicals were of analytical grade.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–
PAGE). SDS–PAGE was performed under reducing conditions using
Multigel (15/25) (T ¼ 15{25%) by the method of Laemmli.23)
Protein assays. The protein concentration was measured with a Bio-
Rad Protein Assay Kit (Bio Rad, Hercules, CA) with bovine ꢁ-globulin
as the standard.
Phenyl sepharose HP hydrophobic chromatography. The super-
natant obtained at step 3 above was divided into two aliquots, and each
aliquot was applied to Phenyl Sepharose HP 26/10 (GE Healthcare
Japan, Tokyo). The column was equilibrated with 50 mM potassium
phosphate buffer containing ammonium sulfate (1.0 M). After the
addition of the sample, the column was washed with a 5-fold column
volume buffer. The absorbed active component was then fractionated
in a decreasing linear gradient of ammonium sulfate concentration
from 1.0 M to 0 M. The active fractions were collected and concentrated
from 180 ml to 9.8 ml with an ultracentrifugation unit (Minitan and
Centriprep 10 (Millipore)). The concentrate was centrifuged at
32;000 ꢂ g for 10 min to give the supernatant.
Enzyme assays. Enzyme activity was determined by quantitative
analysis of Glu released from Glu-Glu. L-ꢂ-Glu-Glu (0.02 ml of
50 mM), 0.02 ml of 100 mM HEPES buffer (pH 7.2), and 0.14 ml of
H2O were mixed and preincubated at 37 ꢀC for 5 min, and then 0.02 ml
of enzyme solution was added and the mixture was incubated at 37 ꢀC
for 20 min. The reaction was terminated by adding 0.05 ml of 50%
aqueous acetic acid. The quantity of glutamic acid was determined
with a glutamic acid assay kit and by conventional amino acid analysis
by the ninhydrin method. One unit of enzyme activity corresponded to
the release of 2 mmol of Glu from Glu-Glu per min at 37 ꢀC.
Other dipeptidase activity was measured by the method described
above, and conventional amino acid analysis was conducted by the
ninhydrin method, but replacing substrate Glu-Glu with another
dipeptide, Glu-Lys, Glu-Gly, Glu-Ala, Glu-Phe, Glu-Thr, Glu-Ser,
Glu-Asp, ꢁ-Glu-Glu, Pro-Glu, Lys-Glu, Phe-Glu, Ala-Glu, Ser-Glu,
Asp-"-N-Lys, Asp-Lys, Asp-Ala, Asp-Glu, Asp-Phe, Asp-Asp, or
ꢁ-Glu-Leu.
Superdex 200 gel filtration chromatography. The supernatant
obtained at step 4 above was fractionated by gel filtration through
HiLoad 26/60 Superdex 200 pg. The supernatant was divided into two
aliquots, and each aliquot was applied to the column which had been
equilibrated with 50 mM potassium phosphate buffer and 100 mM NaCl
(pH 7.0, 2 mM NaN3). Elution was performed with the same buffer.
The active fractions were concentrated to give a purified enzyme. The
purity of the enzyme was analyzed by SDS–PAGE and an analytical
grade gel filtration column, Superdex 200 HR 10/30 with an AKTA
FPLC system.
Aminopeptidase activities were measured using amino acid-pNA
(Glu, Asp, Leu, Phe, Gly, Ala, and Pro). The enzyme reaction was
initiated by mixing 0.05 ml of enzyme solution, 0.02 ml of 50 mM
amino acid-pNA substrate, 0.02 ml of 100 mM HEPES buffer (pH 7.2),
and 0.11 ml of H2O, and the mixture was incubated at 37 ꢀC for 20 min.
The reaction was terminated by adding 0.05 ml of 50% aqueous acetic
acid. The absorbance of the reaction mixture was measured at 410 nm
to evaluate the releasing activity of pNA from amino acid-pNA. One
unit of enzyme activity corresponded to the release of 1 mmol of amino
acid from amino acid-pNA per min at 37 ꢀC.
Effects of pH and temperature. The pH dependency of the enzyme
activity was determined in the following manner.
The enzyme reaction buffers used were sodium acetate buffer
(pH 4.0, 4.5, 5.0, 5.5, 6.0), potassium phosphate (pH 6.0, 7.0), Tris–
HCl buffer (pH 7.0, 7.8, 8.3, 8.8), and sodium carbonate buffer
(pH 9.0, 9.5, 10.0). The purified enzyme (180 ml, 3.5 mU) was prepared
in 50 mM buffer at each pH value and pre-incubated for 5 min at 30 ꢀC.
The substrate, Glu-Glu (20 ml, 5 mM for reaction), was added to each
sample. The reaction mixture was stirred and incubated for 20 min at
30 ꢀC. The reaction was terminated by adding 20 ml of 50% aqueous
acetic acid. The quantity of glutamic acid was determined with a
glutamic acid assay kit.
Search for Glu-Glu hydrolyzing peptidase. We looked for Glu-Glu
hydrolyzing peptidase from soybean cotyledons. Each soybean
cotyledon obtained from 0–10 DAI was homogenized in ice-cold
buffer (20 mM potassium phosphate buffer, pH 7.0, 200 mM NaCl,
10 mM 2-ME, 2 mM NaN3) at a fresh-weight: buffer ratio of 1 g: 5 ml.
The filtrates, filtered through gauze, were centrifuged at 32;000 g for
30 min. The supernatants were filtered with filter paper (Whatman
No. 2, Whatman Japan, Tokyo) to give the crude extract.
Measurement of the optimum temperature of the purified enzyme
was carried out as follows: The substrate, Glu-Glu (20 ml, 5 mM for
reaction) was added to 50 mM sodium acetate buffer (pH 6.0), and
180 ml of the reaction solution obtained (substrate solution) was pre-
incubated for 5 min at each temperature (25 ꢀC, 30 ꢀC, 37 ꢀC, 42 ꢀC,
50 ꢀC, 60 ꢀC, and 70 ꢀC). The purified enzyme (7 mU) was then added.
The reaction mixture was stirred and incubated for 20 min at each
temperature. The reaction was terminated by adding 20 ml of 50%
aqueous acetic acid. Enzyme activity was determined by the method
described above.
Purification of peptidase.
Extraction. Purification procedures were performed as described
below. All were performed below 10 ꢀC.
Cotyledons (600 g) of 7 DAI were homogenized and extracted with
3 liters of 20 mM potassium phosphate buffer (pH 7.0), 200 mM NaCl,
0.1 mM AEBSF, 10 mM E-64, and 10 mM 2-ME. The extract was filtered
with gauze, and then centrifuged (32;000 ꢂ g, 30 min). The supernatant
was collected and filtered through two layers of filter paper (No. 514A,
Advantec, Tokyo). The supernatant (19,780 mg of proteins) was
concentrated about 20 times with a concentrator with a polyethersul-
fone membrane (Minitan, Millipore, Billerica, MA).
Thermal stability. Determination of the thermal stability of the
purified enzyme was carried out as follows: The purified enzyme in
50 mM sodium acetate buffer (pH 6.0) was incubated for 80 min at
25 ꢀC, 42 ꢀC, 50 ꢀC, 60 ꢀC, and 70 ꢀC. The treated enzyme solution
(7 mU) was then added to 180 ml of Glu-Glu at a final concentration of
5 mM, followed by incubation for 20 min at 30 ꢀC. The reaction was
Ammonium sulfate precipitation. The pH of the extract was adjusted
to pH 7.0 with NaOH, and a 40% saturation was made with ammonium
sulfate (763 g/3,150 ml). The solution was stirred for 6 h and
centrifuged at 32;000 ꢂ g for 30 min at 4 ꢀC. The supernatant was

N-Terminal Acidic Amino Acid-Specific Aminopeptidase from Soybean
690 440 158 kDa
115
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
A
140
120
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
10
0
0
5
10
15
20
Day-after imbibition (DAI)
Elution volume (ml)
Fig. 1. Course of Changes in the Enzyme Activity of Glu-Glu
Hydrolyzing Peptidase.
1
2
B
The cotyledons (1 g) were homogenized and extracted with 5 ml
of 50 mM sodium phosphate buffer (pH 7.0), 200 mM NaCl, and
10 mM 2-ME. The extract was filtered with gauze and then
centrifuged (16;000 ꢂ g, 30 min). The supernatant was collected
and filtered through two layers of filter paper. The enzyme activities
(units/h/mg of protein) of Glu-Glu hydrolyzing peptidase were
measured in each extract of cotyledons from 0 DAI to 10 DAI.
Results are the means for two independent experiments.
kDa
97.4
66.2
45.0
31.0
21.5
14.5
terminated by adding 20 ml of 50% aqueous acetic acid. Residual
activity was determined by the method described above.
Detection of hydrolysis of chromogranin A by the purified enzyme.
Hydrolysis of chromogranin A, an oligopeptide containing five
glutamic acids at the N-terminal, was examined by the purified
enzyme, as follows: 500 mM ammonium carbonate buffer (0.02 ml),
0.05 ml of 5 mM chromogranin A, 0.08 ml of H2O, and 0.05 ml of the
purified enzyme (6 mU) were incubated at 37 ꢀC. A 0.02-ml portion of
the reaction solution was sampled at 0, 1, and 15 h. The reaction was
stopped with 0.02 ml of 50% aqueous acetic acid. This reaction
solution was analyzed for molecular weight by MALDI-TOF mass
spectra obtained on a Kompakt Maldi III (Shimazu-Kratos, Kyoto,
Japan) mass spectrometer using 2,5-dihydroxybenzoic acid (DHBA)
and 2-(4-hydroxyphenylazo)benzonic acid (HABA) as a matrix.
Fig. 2. Separation of Purified Glu-Glu Hydrolyzing Peptidase by
Chromatography.
A, Analytical gel filtration of purified Glu-Glu hydrolyzing
peptidase. The purity of the enzyme was analyzed with an analytical
grade gel filtration column, Superdex 200 HR 10/30 with an AKTA
FPLC system. Thyroglobulin (690 kDa), ferritin (440 kDa), and
aldorase (158 kDa) were used as molecular size markers. B, Results
of SDS–PAGE analysis of the purified Glu-Glu hydrolyzing
peptidase. Lane 1, marker proteins; lane 2, the purified enzyme.
Rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin
(66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soy-
bean trypsin inhibitor (21.5 kDa), and lysozyme (14.5 kDa) were
used as molecular size markers. The arrow indicates the position of
the purified Glu-Glu hydrolyzing peptidase.
Results and Discussion
Enzyme activity of soybean cotyledons each day after
imbibition
The new enzyme was detected by measuring its
activity with Glu-Glu as substrate. L-ꢂ-glutamylglutamic
acid (Glu-Glu) hydrolyzing activity was present in each
cotyledon extract from 0 DAI to 10 DAI (Fig. 1). In a
previous study, we found that the total amounts of
proteins, major storage proteins, and their fragments
decreased as time passed during soybean germination
and seedling growth. Finally, the proteins in the
cotyledon disappeared by 10 DAI.8) In contrast, the
enzyme activity (mmole/h/mg of protein) of this
peptidase was higher after 6 DAI (Fig. 1). Hence we
purified this enzyme from the cotyledons of 7 DAI in
view of the results from the enzyme activity of
cotyledons obtained each day after imbibition, as
described above. In addition, the influence of various
protease inhibitors was investigated. We found that
extraction efficiency increased very much with the
addition of AEBSF and E-64 (data not shown). Hence
the soybean cotyledon homogenate for enzyme purifi-
cation was prepared with the buffer solution containing
them.
Purification of the enzyme
A novel N-terminal acidic amino acid specific
aminopeptidase was purified from soybean cotyledons
of 7 DAI. The soybean cotyledon homogenate was
filtrated and centrifuged, and it was precipitated with
65% ammonium sulfate. It was successively fractio-
nated by chromatographies on columns of Q-sepharose
and Phenyl sepharose. The active fraction collected
from Phenyl sepharose was applied to a Superdex 200
column, and its active fractions were combined and
used as the purified enzyme. The purification steps for
the enzyme are summarized in Table 1. The specific
activity of the purified enzyme was determined to be
66.3 units/mg with a yield of 35% when Glu-Glu was
used as substrate.

116
M. ASANO et al.
Table 1. Purification of N-Terminal Amino Acid-Specific Amino-
peptidase
100
90
80
70
60
50
40
30
20
10
0
A
Total
protein activity activity
Total
Specific
Yield Purification
(%)
Step
(-fold)
(mg)
(U)
(U/mg)
Extract
20,000
150 7:6 ꢂ 10ꢁ3 100
200 3:9 ꢂ 10ꢁ2 130
1.0
5.1
Ammonium sulfate 5,200
precipitation
Q-sepharose
Phenyl sepharose
Superdex 200
710
9.2
0.8
150 2:2 ꢂ 10ꢁ1 100
29
1000
8700
72
53
7.8
66
47
35
3
4
5
6
7
8
9
10
11
pH
U, One unit of enzyme activity corresponded to the release of 2 mmol of Glu
from Glu-Glu per min at 37 ꢀC.
100
90
80
70
60
50
40
30
20
10
0
B
Table 2. Effects of Potential Inhibitors on the Activity of the Purified
Enzyme
Concentration
(mM)
Relative activity
(%)
Chemical
None
ZnCl2
100
68.3
107
0.4
0.4
0.4
0.4
5.0
20
MnCl2
.
CoCl2 6H2O
97.1
106
20
30
40
50
60
70
80
.
CaCl2 2H2O
EDTA
Temperature (°C)
83.5
68.1
32.0
47.1
EDTA
DTT
o-Phenanthroline
100
90
0.1
2.0
C
80
70
60
50
40
30
20
10
0
Activity was expressed as the percentage of activity remaining after a 10-
min incubation of the enzyme in the presence of the potential inhibitor.
Activity was measured with substrate Glu-Glu. Results are the means for
three independent experiments.
Table 3. Substrate Specificity toward Amino Acid-pNA
X-pNA
Specific activity (U/mg)
20
30
40
50
60
70
80
Glu-pNA
Asp-pNA
Leu-pNA
Phe-pNA
Gly-pNA
Ala-pNA
Pro-pNA
319
132
0
Temperature (°C)
Fig. 3. Characteristics of the Purified Glu-Glu Hydrolyzing Pepti-
dase.
0
A, pH dependency of hydrolysis activity toward Glu-Glu at 30 ꢀC.
B, Optimal reaction temperature for the hydrolysis of Glu-Glu at
pH 6.0. C, Thermal stability of the purified Glu-Glu hydrolyzing
peptidase. The purified enzyme was incubated at temperatures for
80 min. Results are the means for two independent experiments.
0
0
0
U, One unit of enzyme activity corresponded to the release of 1 mmol of
amino acid from amino acid-pNA per min at 37 ꢀC. Results are the means
for two independent experiments.
it is thought that metal influences its activity. Various
amiopeptidases with various substrate specificities are
distributed widely in prokaryotes and eukaryotes. Most
aminopeptidases are metalloproteases, and are inhibited
by chelating agents.24) However, DTT also clearly
inhibited the activity. The primary structure of the
enzyme is currently under investigation. Molecular
cloning and characterization of the gene are also in
progress. We intend to come to a conclusion as to the
enzyme type after confirming the homology of the
enzyme and various aminopeptidaes.
Enzyme characterization
The apparent molecular mass of the purified enzyme
was estimated to be 510 kDa by Superdex 200 HR 10/30
column chromatography (Fig. 2A), and 56 kDa by SDS–
PAGE (Fig. 2B). This indicates that the enzyme was a
multimeric homopolymer.
Figure 3 shows the enzymatic properties of the
purified enzyme. It exhibited more than 80% activity
from pH 7 to 9 at 30 ꢀC (Fig. 3A). The optimum
reaction temperature was approximately 45 ꢀC at pH 6.0
(Fig. 3B). The enzyme was stable at temperatures below
50 ꢀC over an 80-min incubation at pH 6.0 (Fig. 3C).
To test the influence of several reagents on the
purified enzyme, enzyme activity towards Glu-Glu was
measured following a 10-min pre-incubation with
several reagents (Table 2). The enzyme was inhibited
by Zn2þ, EDTA, o-phenanthroline, and DTT. Therefore,
Substrate specificity
The hydrolytic activity of the purified enzyme on
various synthetic peptides was examined. First, the
substrate specificity of the enzyme was determined using
seven sets of pNA substrate. It had high activity against
Glu-pNA and Asp-pNA, whereas Leu-pNA, Phe-pNA,
Gly-pNA, Ala-pNA, and Pro-pNA were not hydrolyzed

N-Terminal Acidic Amino Acid-Specific Aminopeptidase from Soybean
117
Glu-Glu
Glu-Asp
Glu-Ser
Glu-Thr
Glu-Phe
Glu-Ala
Glu-Gly
Glu-Lys
γ-Glu-Glu
Ser-Glu
Ala-Glu
Phe-Glu
Lys-Glu
Pro-Glu
5E
100
A
50
0
800
1000
1200
1400
1600
1800
2000
2200
2400
Asp-Asp
Asp-Phe
Asp-Glu
5E
B
100
50
0
4E
Asp-Ala
Asp-Lys
Asp-ε-N-Lys
γ-Glu-Leu
Gln-Gly
3E
Gln-Gln
0
50
100
150
200
Relative activity (%)
800
1000
1200
1400
1600
1800
2000
2200
2400
Fig. 4. Activities of the Purified Enzyme toward Various Peptide
Substrates.
The specific activity toward Glu-Glu was defined as 100%.
Results are the means for two independent experiments.
0E
100
C
(Table 3). Secondly, 21 synthetic dipeptides containing
Glu and/or Asp were evaluated for the reactivity of the
enzyme. Glu-Xxx and Asp-Xxx were hydrolyzed, but
Xxx-Glu was not (Fig. 4). This suggests that the enzyme
exhibits substrate specificity for unblocked N-terminal
acidic amino-acid residues. Next we investigated the
reaction of a Glu-rich oligopeptide, chromogranin A
(Glu-Glu-Glu-Glu-Glu-Met-Ala-Val-Val-Pro-Gln-Gly-
Leu-Phe-Arg-Gly-NH2) as substrate. The resulting pep-
tides were detected by MALDI-TOF MS analysis
(Fig. 5). We detected the production of Glu-Glu-Glu-
Glu-Met-Ala-Val-Val-Pro-Gln-Gly-Leu-Phe-Arg-Gly-
NH2 and Glu-Glu-Glu-Met-Ala-Val-Val-Pro-Gln-Gly-
Leu-Phe-Arg-Gly-NH2 after 1 h. After 15 h, Glu-Glu-
Met-Ala-Val-Val-Pro-Gln-Gly-Leu-Phe-Arg-Gly-NH2,
Glu-Met-Ala-Val-Val-Pro-Gln-Gly-Leu-Phe-Arg-Gly-
NH2 and Met-Ala-Val-Val-Pro-Gln-Gly-Leu-Phe-Arg-
Gly-NH2 were also generated. It is thought that the
enzyme cleaved to the first N-terminal acidic amino-acid
residue of the peptides. These results indicate that the
enzyme is the N-terminal acidic amino acid-specific
aminopeptidase from the germinating soybean cotyle-
don. Its substrate specificity was similar to aspartyl
aminopeptidase (EC 3.4.11.21) and to glutamyl amino-
peptidase (EC 3.4.11.7). In particular, the behavior of
the enzyme showed similarity to those of the aspartyl
aminopeptidases from yeast and Aspergillus oryzae in its
enzymatic characterization.20–22) Aspartyl aminopepti-
dase has been reported to be present in mammals, yeast,
and filamentous fungi, but has not previously been
purified from plants.20–22) To confirm that the enzyme
was aspartyl aminopeptidase, we intend to determine the
amino acid sequence of the enzyme and to compare
them of the known aspartyl aminopeptidases.
3E
4E
50
5E
2E
1E
0
800
1000
1200
1400
1600
1800
2000
2200
2400
Mass/Charge
Fig. 5. Time Course of the Reaction of Chromogranin A.
The change over time of chromogranin A (Glu-Glu-Glu-Glu-Glu-
Met-Ala-Val-Val-Pro-Gln-Gly-Leu-Phe-Arg-Gly-NH2) in the reac-
tion mixture was analyzed. The change in its molecular weight was
analyzed by MALDI TOF-MS analysis. Reaction times were as
follows: A, 0 h; B, 1 h; C, 15 h. The products of the digestion of
chromogranin A (EEEEEMAVVPQGLFRG-NH2, 5E) with the
purified enzyme are labeled 4E, 3E, 2E, 1E, and 0E. The products
and their molecular weights were as follows: 5E, EEEEEMAVV-
PQGLFRG-NH2, ½M þ Hꢃþ 1,821; 4E, EEEEMAVVPQGLFRG-
NH2, ½M þ Hꢃþ 1,692; 3E, EEEMAVVPQGLFRG-NH2, ½M þ Hꢃþ
1,563; 2E, EEMAVVPQGLFRG-NH2, ½M þ Hꢃþ 1,434; 1E, EMA-
VVPQGLFRG-NH2, ½M þ Hꢃþ 1,305; 0E, AVVPQGLFRG-NH2,
½M þ Hꢃþ 1,175. These molecular weights are in agreement with the
theoretical values.
The proteolysis of seed storage proteins is a critical
step in the process of germination and early growth. In
soybean seedling cotyledons, proteolysis of storage
proteins is also initiated by proteolytic enzymes with
narrow substrate specificity. One such initiating enzyme
is protease C1 in soybean.25) Protease C1 cleaves
wherever an exposed portion of the polypeptide chain
bears a string of acidic amino acid residues.26) Cleavage
occurred between two acidic amino acid residues in a

118
M. ASANO et al.
7) Gonzales T and Robert-Baudroy J, FEMS Microbiol. Rev., 18,
319–344 (1996).
region of the polypeptide that bears many other acidic
amino acids residues. There are acidic amino acid-rich
regions in two main storage proteins of soybean, the
ꢀ-conglycinin N-terminal and the C-terminal of the
glycinin acidic chain.15) It is thought that acidic amino
acid-specific aminopeptidase played an important role in
germinating soybean cotyledone. In this study, we
searched for aminopeptidase in germinating soybean
cotyledons with Glu-Glu as substrate, and isolated a
novel N-terminal acidic amino acid-specific amino-
peptidase. This aminopeptidase removed acidic amino
acids sequentially from N-terminus acidic amino acid-
containing peptides. Furthermore, 13 acidic dipeptides
(Glu-Glu, Glu-Asp, Glu-Ser, Glu-Thr, Glu-Phe, Glu-
Ala, Glu-Gly, Glu-Lys, Asp-Asp, Asp-Phe, Asp-Glu,
Asp-Ala, and Asp-Lys) hydrolyzed by this enzyme were
peptides isolated from a soy sauce.27) Therefore, it is
thought that the enzyme contributes to the improvement
of the hydrolysis rate of soybean protein. We believe it
might be possible to use the enzyme in a variety of food
processing applications.
8) Kawai M, Suzuki S, Asano M, Miwa T, and Shibai H, Biosci.
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Cysteine Proteases,’’ Oxford University Press, Oxford, pp. 8–
18 (1998).
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14371–14377 (1984).
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Here we report on the characterization of a novel
N-terminal acidic amino acid-specific aminopeptidase
from germinating soybean cotyledone. Studies of its
genetic properties and structural analysis are necessary,
and are underway.
20) Yokoyama R, Kawasaki H, and Hirano H, FEBS J., 273, 192–
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