Novel aminopeptidase specific for glycine from Actinomucor elegans.

Article abstract of DOI:10.1271/bbb.67.83

Glycyl aminopeptidase was purified 600-fold from a cell extract of Actinomucor elegans by ammonium sulfate fractionation and sequential chromatography on DEAE-Toyopearl, Toyopearl HW65C, and FPLC-Superdex 200 HR, with recovery of 3.3% of the activity. The enzyme highly specifically hydrolyzed Gly-X (amino acid, peptide, or arylamide) bonds. The enzyme hydrolyzed other amino acid residues but at a rate of less than one fifth that with Gly. The order was Gly >> Ala >> Met > Arg > Ser > Leu. The Km value for glycyl-2-naphthylamide was 0.24 mM. The enzyme was most active at pH 8.0 with glycyl-2-naphthylamide as the substrate and its optimal temperature was 40 degrees C. The enzyme was inhibited by iodoacetic acid, and p-chloromercuribenzoate but not done by diisopropylfluorophosphate, o-phenanthroline, or EDTA. Magnesium and calcium had no effect on enzymic activity, but the activity was suppressed by cadmium, zinc, and copper ions. The molecular mass was estimated to be 320 kDa by gel filtration on FPLC-Superdex 200 HR and 56.5 kDa by SDS-PAGE, so the enzyme probably was a hexamer.

Full text of DOI:10.1271/bbb.67.83

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Novel Aminopeptidase Specific for Glycine from
Actinomucor elegans
Kiyoshi ITOa, Xiaohang MAac, Nik AZMIa, Hua-Shan HUANGa, Mikio FUJIIb & Tadashi
YOSHIMOTOa
a School of Pharmaceutical Sciences, Nagasaki University 1-14 Bunkyo-machi, Nagasaki
852-8521, Japan
b Foods Technology Laboratory, Japan Tobacco Inc.
c Department of Bioscience, Zhejiang University Hangzhou, 310029, China
Published online: 22 May 2014.
To cite this article: Kiyoshi ITO, Xiaohang MA, Nik AZMI, Hua-Shan HUANG, Mikio FUJII & Tadashi YOSHIMOTO (2003) Novel
Aminopeptidase Specific for Glycine from Actinomucor elegans , Bioscience, Biotechnology, and Biochemistry, 67:1, 83-88,
DOI: 10.1271/bbb.67.83
To link to this article: http://dx.doi.org/10.1271/bbb.67.83
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Biosci. Biotechnol. Biochem., 67 (1), 83–88, 2003
Novel Aminopeptidase Speciˆc for Glycine from Actinomucor elegans
1,
2
Kiyoshi ITO,1 Xiaohang M
and Tadashi YOSHIMOTO
A
†
,
Nik AZMI,1 Hua-Shan HUANG,1 Mikio FUJII
,
*
1,
1School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
2Foods Technology Laboratory, Japan Tobacco Inc., 443-1 Shirayamado, Ohito-cho, Tagata-gun,
Shizuoka 410-2318, Japan
Received July 2, 2002; Accepted August 31, 2002
Glycyl aminopeptidase was puriˆed 600-fold from a
cell extract of Actinomucor elegans by ammonium sul-
fate fractionation and sequential chromatography on
DEAE-Toyopearl, Toyopearl HW65C, and FPLC-
Methionine aminopeptidase acts in the processing of
protein synthesis.12) Cystinyl aminopeptidase is also
named oxytocinase, and is involved in the control of
oxytocine during pregnancy.14)
Glycine is the smallest of the 20 most common
amino acids that make up proteins and the only one
that has no stereoisomer. A Gly-X bond is very
slowly hydrolyzed even by leucine aminopeptidase6)
or alanine aminopeptidases11) that have broad
speciˆcity. An aminopeptidase highly speciˆc for gly-
cine residues is needed for basic studies of substrate
recognition and for complete hydrolysis of peptides
to amino acids. By screening microorganisms, we
Superdex 200 HR, with recovery of 3.3% of the activity.
The enzyme highly speciˆcally hydrolyzed Gly-X (ami-
no acid, peptide, or arylamide) bonds. The enzyme
hydrolyzed other amino acid residues but at a rate of
less than one ˆfth that with Gly. The order was Gly
Ala Met Arg Ser Leu. The Km value for glycyl-2-
naphthylamide was 0.24 m . The enzyme was most
active at pH 8.0 with glycyl-2-naphthylamide as the
substrate and its optimal temperature was 40 C. The
enzyme was inhibited by iodoacetic acid, and
chloromercuribenzoate but not done by diisopropyl-
‰uorophosphate, -phenanthroline, or EDTA. Mag-
:
:
À
À
À
M
9
p
-
found glycyl aminopeptidase activity in A. elegans.
This is the ˆrst report of a glycyl aminopeptidase.
o
nesium and calcium had no eŠect on enzymic activity,
but the activity was suppressed by cadmium, zinc, and
copper ions. The molecular mass was estimated to be
320 kDa by gel ˆltration on FPLC-Superdex 200 HR
and 56.5 kDa by SDS-PAGE, so the enzyme probably
was a hexamer.
Materials and Methods
Materials. Arg-
Leu- NA, Met-
Trp- NA were obtained from Bachem (Bubendorf,
b
NA(2-naphthylamide), Gly-
bNA,
b
b
b
NA, Glu- NA, Ser- NA, and
º
b
b
Switzerland). The other substrates used were from
our laboratory stock. Actinomucor elegans IFO 6408
was obtained from the Institute of Fermentation,
Osaka, and was maintained on potato sucrose agar,
Key words: glycine; aminopeptidase; glycyl amino-
peptidase; Actinomuco; peptidase
at 24
the enzyme contained cotton seed protein, 2.0
soybean protein, 0.5 ; glucose, 0.5 ; NaCl, 0.3
K2HPO4, 0.1 ; CaCO3, 0.2 ; and Mg2SO4
0.5 . For glycyl aminopeptidase production, the A.
elegans was cultivated in a 5-liter culture in a jar-
fermentor at 24 C for 4 days. The rate of aeration
9
C. The medium for screening and producing
Of the aminopeptidases that have been studied,1,2)
most have been leucine aminopeptidases (EC
3.4.11.1)3–7) or membrane alanine aminopeptidases
(EC 3.4.11.2).8–11) These enzymes have a broad
substrate speciˆcity. However, some enzymes with
narrow speciˆcity, methionine aminopeptidase (EC
3.4.11.18),12) prolyl aminopeptidase (EC 3.4.11.5),13)
cystinyl aminopeptidase (EC 3.4.11.3),14) arginyl
aminopeptidase (EC 3.4.11.6),15) glutamyl aminopep-
tidase (EC 3.4.11.7),16) and lysyl aminopeptidase (EC
3.4.11.15)17) also have been reported. Some of these
enzymes have particular biological signiˆcance.
z
z
;
;
z
z
・
7H2O,
z
z
z
9
was from 1 to 1.5 liters per minute and the speed of
rotation was 100 rpm. After the cultivation, the
mycelia of A. elegans were collected by ˆltration
for enzyme puriˆcation. For screening of glycyl
aminopeptidase, Gly-bNA as substrate and proteins
in medium were used.
†
To whom correspondence should be addressed: Tel Fax: +81-95-843-2444; E-mail: t-yoshimoto
Present address: Department of Bioscience, Zhejiang University, Hangzhou, 310029, China
＠
cc.nagasaki-u.ac.jp
W
*
Abbreviations
aminoethyl ether)-NNN
chloromercuribenzoate; PMSF, phenylmethylsulfonyl ‰uoride; SDS, sodium dodecyl sulfate;
:
b
NA, 2-naphthylamide; Boc,
-tetraacetic acid; FPLC, fast protein liquid chromatography; NHMec, 7-(4-methyl)coumarylamide; PCMB,
-benzyloxycarbonyl
t
-Butyloxycabonyl; DFP, diisopropyl‰uorophosphate; EGTA, ethylene glycol-bis(
b
p
-
-
?
N
?
Z,N

84
K. ITO et al.
Activity assay. The method used to measure en-
9)
zyme activity was based on that of Yoshimoto et al
.
To 0.8 ml of 20 m
added 0.1 ml of Gly-
in the same buŠer), the mixture was ˆrst incubated at
37 C, 0.1 ml of enzyme solution was added, and in-
cubation was continued at 37 C for 10 min. The
reaction was stopped by the addition of 0.5 ml of
Fast Garnet GBC solution (1 mg ml) containing 10
M
Tris-HCl buŠer, pH 7.0, was
b
NA solution (3 m , dissolved
M
9
9
z
W
Triton X-100 in 1
M
acetate buŠer (pH 4.0). The reac-
C. The
tion mixture was left for a few minutes at 37
9
absorbance at 550 nm was measured. One unit of en-
zyme activity was deˆned as the amount that released
1 mmol of b-naphthylamine per minute under the
Fig. 1. Chromatogram of Glycyl Aminopeptidase from A. ele-
gans on a Column of FPLC-Superdex 200 HR.
Absorbance at 280 nm ( ), activity ( ). Each fraction was of
4 ml.
standard conditions.
Peptide hydrolyzing activity was assayed by the
ninhydrin method and products were checked by


TLC. After the enzyme was incubated with 3 m
substrate in 20 m Tris-HCl, pH 7.0, at 37 C, 10 ml
of the reaction mixture was assayed by the ninhydrin
method. Another 10- l portion of the reaction
mixture was removed for identiˆcation of a newly
formed amino acids by TLC with -BuOH–
M
M
9
1.0 mg ml. The puriˆed enzyme concentration was
W
m
measured by the method of Bradford18) with bovine
serum albumin as the standard.
n
AcOH–H2O (4:1:2) as the solvent. Comparison was
done with authentic markers.
Measurment of molecular weight. The molecular
weight of glycyl aminopeptidase was measured by
SDS-PAGE. A 12.5
z
gel was used. The standard
Puriˆcation of glycyl aminopeptidase. All proce-
proteins used in the experiment were produced by
Amersham Biosciences. The molecular mass of the
native enzyme was measured by gel ˆltration on
FPLC-Superdex 200 HR.
9
dures for enzyme puriˆcation were done at 4 C.
After fermentation, mycerria were suspended in the
same volume of 20 m
glycerol added to 10
M
Tris-HCl buŠer, pH 8.0, with
and broken by being blended
z
for 5 minute with an Ace Homogenizer. The mixture
was centrifuged at 12,000 for 10 min. The
supernatant, which contained most of the glycyl
aminopeptidase activity, was dialyzed against 20 m
Tris-HCl buŠer, pH 7.0, and put on a column of
DEAE-Toyopearl equilibrated with the same buŠer.
The enzyme was eluted with a linear gradient of NaCl
Results
×
g
Screening for and puriˆcation of glycyl aminopep-
tidase
M
After screening several microorganisms, we found
glycyl aminopeptidase activity in A. elegans. IFO
6408. The mycelia (590 grams, wet weight) were sus-
from 0 to 1.0
dialyzed against 20 m
ammonium sulfate was added to 50
M
. The active fractions were collected,
Tris-HCl, pH 7.0, and
saturation.
pended in 590 ml of 20 m
M
Tris-HCl buŠer, pH 7.0,
M
and disrupted with a blender. The debris of the cell
×
g
z
walls was separated by centrifugation at 12,000
The enzyme solution was then put on a column of
Toyopearl HW 65C previously equilibrated with the
for 10 min. More than 95 of the activity of glycyl
z
aminopeptidase remained in the supernatant and the
supernatant was puriˆed further.
same buŠer containing 50
Then the column was washed with 150 ml of the same
buŠer containing 50 ammonium sulfate, and the
enzyme was eluted with 1,200 ml of buŠer with a
linear gradient of ammonium sulfate from 50 to
. The fractions containing glycyl aminopeptidase
z ammonium sulfate.
The crude enzyme was ˆrst put on a DEAE-
×
Toyopearl column (4 15 cm) and was puriˆed 20-
z
fold with a yield of 27
z. Then, the enzyme was puri-
z
ˆed by hydrophobic interaction chromatography on
×
0
z
the Toyopearl HW65C column (4 15 cm). The en-
were pooled and put on a Superdex 200 HR column
(Amersham Biosciences), and washed out with
zyme was eluted in fractions 45 to 60, with the peak
at 54. Fractions 52–56 were collected and pooled.
The enzyme was desalted by dialysis put on a Super-
dex 200 HR column, and washed out with the same
buŠer (Fig. 1). The active fractions were collected
and pooled as the puriˆed enzyme for further experi-
ment. The speciˆc activity of the puriˆed enzyme was
20 m
M
Tris-HCl buŠer, pH 7.0. The active fractions
were pooled for later experiments.
Protein assay. Absorbance at 280 nm was meas-
ured to estimate the protein concentration through-
out the puriˆcation. Absorbance at 280 nm of 1.0
was assumed to indicate a protein concentration of
14.2 units mg protein (18.5 units mg protein con-
W
W
centration by the method of Bradford). The steps for

Glycyl Aminopeptidase from A. elegans
Table 1. Steps and Results of Puriˆcation of Glycyl Aminopepti-
85
dase
Speciˆc
activity
W
Puriˆ-
cation
(fold)
Protein Activity
Yield
Step
(mg)
(units)
(z)
(U mg)
Crude enzyme
DEAE-Toyopearl
Toyopearl HW65C
FPLC-Superdex
200 HR
25000
329
600
160.4
39.4
0.024 100
1.0
20.4
0.49
3.94
27
6.6 164
10
1.4
19.9
14.2
3.3 592
Table 2. Substrate Speciˆcity of the Glycyl Aminopeptidase
Relative
activity
value
Activity
Km
(m
kcat
Substrate
(
m
mol min)
M
)
(sec„1
)
W
(
z
)
Gly-
Ala-
Met-
Arg-
b
b
NA
NA
b
14.35
2.60
0.91
0.90
0.50
0.37
100
0.24
0.66
24.2
1.4
Fig. 2. SDS-PAGE of Glycyl Aminopeptidase from A. elegans
M, marker proteins; S, sample; arrow, enzyme.
.
18.1
6.3
6.3
3.5
2.6
NA
NA
NA
b
Ser-
b
Leu-bNA
The following compounds were not hydrolyzed: Trp-
Asp- NA, Lys- NA, Val- NA, Cys-di- NA, Glu- NA, Thr-
Glu- NA.
b
NA, Pro-
bNA,
b
b
b
b
b
b
NA, or
º
b
the puriˆcation of glycyl aminopeptidase and the
results are summarized in Table 1.
By SDS-PAGE, there was only one band and the
molecular weight of the enzyme was estimated to be
56,500 (Fig. 2). The molecular weight was estimated
to be 320,000 by gel ˆltration on FPLC-Superdex 200
HR.
EŠects of pH and temperature on activity and
stability of glycyl aminopeptidase
The optimum pH of the enzyme was 8.0 using Gly-
b
NA as the substrate (Fig. 3(A)). The enzyme
retained more than 90
tween pH 6.0 and 10.0 after incubation at 30
30 min (not shown). The optimum temperature was
40 C for a 10 min reaction and 50 of the initial
activity was observed after incubation for 15 min at
30 C at pH 7.0 (Fig. 3(B)).
z
of its original activity be-
9
C for
9
z
9
Substrate speciˆcity of glycyl aminopeptidase
Gly- NA (Km of 0.24 m
and kcat of 24.2 sec„1
was the best substrate, and Ala- NA ( m of 0.66 m
and kcat of 1.1 sec„1 ), Met-
NA, Arg- NA, Ser-
NA, and Leu- NA also were hydrolyzed (Table
2). However, Trp- NA, Pro- NA, Asp- NA, Lys-
NA, Val- NA, Cys-di- NA, Glu- NA, Thr- NA,
and Glu- NA were not hydrolyzed at all. The en-
zyme also released the glycine from peptides such as
b
M
)
b
K
M
b
b
b
b
b
b
b
Fig. 3. A: pH Activity (A) and Thermal Activity (B) of Glycyl
Aminopeptidase from A. elegans
(A) The enzyme activity was assayed by the standard method
at 37 C with a 10-minute reaction. (B) The enzyme acitivity was
assayed at pH 7.0.
b
b
b
b
b
b
.
º
9
Gly-Gly-Gly, Gly-Phe (0.71
Gly-Glu.
mmol min mg), and
W
W

86
K. ITO et al.
Table 3. EŠect of Chemicals on Glycyl Aminopeptidase
The enzyme was incubated with an additive at 309C at pH 7.0
for 5 min, and the residual activity of the reaction mixture was as-
sayed.
contrast, the aminopeptidase from Aspergillus sojae
is 220 kDa.19)
A leucine aminopeptidase from
humans is 360 kDa5) and cattle lens leucine
aminopepitdase is 324 kDa.20) These are homohex-
amers. The crystal structure of the cattle lens enzyme
is hexameric with a three-fold molecular axis.21)
Glycyl aminopeptidase may have a hexameric struc-
ture similar to leucine aminopeptidase.
Concentration
(m
Relative activity
Inhibitor
M
)
(z)
None
DFP
Iodoacetic acid
PCMB
—
100
1.0
1.0
0.1
1.0
1.0
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
103.3
41.7
12.5
105
78.8
0
18.5
1.3
61.9
0.3
80.1
58.0
100.1
101.1
The enzyme hydrolyzed Gly-X bonds. There are
two endopeptidases speciˆc for the glycine residue.
One is lysostaphin (EC 3.4.24.75),22) which hydro-
lyzes a Gly-Gly bond in the pentaglycine subunit of
the cell wall peptidoglycan.23) The enzyme is a zinc
endopeptidase and has a molecular mass of 27 kDa.
The enzyme hydrolyzes not only polyglycine but also
glycine-rich proteins such as insoluble elastin.
Another is glycyl endopeptidase (EC 3.4.22.25) from
Carica papaya latex.24) This enzyme is a sulfhydryl
enzyme.25) It is interesting that the enzyme is able to
hydrolyze Ala-X bonds. Substitution of Ala for Gly
in the Boc-Ala-Ala-Gly-NHMec substrate at position
P1 decrease the kcat 60-fold. In its amino acid se-
quence, it was similar to other cysteine endopepti-
o
-Phenanthroline
EDTA
Cd2+
Ni2+
Zn2+
Fe2+
Cu2+
Mn2+
Co2+
Mg2+
Ca2+
EŠects of chemicals on glycyl aminopeptidase
activity
Glycyl aminopeptidase was incubated with inhibi-
tors at concentrations from 0.1 to 1.0 m at room
dases from papaya latex, being 81 identical to cari-
z
M
cain, 70 to chymopapain, and 67 to papain. The
z
z
temperature for 20 min. Then the activity was as-
sayed by the standard method. Glycyl aminopepti-
dase was not inhibited by the chelating agents
crystal structure conˆrms the overall similarity to
papain, but with a much-altered S1 subsite, as the
side chains of Glu23 and Arg65 form a barrier across
the binding pocket and sterically exclude residues
with large side chains, making a small S1 pocket for
the glycine residue.25)
o
-phenanthroline or EDTA (Table 3). The serine en-
zyme inhibitor DFP had no eŠect, but the thiol inhi-
bitors iodoacetic acid and PCMB greatly decreased
the activity, indicating that there is a thiol group in
the reactive center of glycyl aminopeptidase.
Since glycyl aminopeptidase was inhibited by
PCMB and iodoacetic acid, it seemed to be a sul-
fhydryl enzyme like cysteine endopeptidases from
papaya. There are few examples of thiol aminopepti-
The enzyme was incubated with divalent cations at
the concentration of 1 m
M
in 20 m
M
Tris-HCl buŠer,
pH 7.0, at room temperature for 30 min. Then the
substrate was added and the activity was measured.
Magnesium and calcium had no eŠect on enzymic ac-
tivity, but the activity was suppressed by cadmium,
zinc, and copper ions.
dases: aminopeptidase C (pepC) from Lactococcus
26)
lactis
,
which resembles bleomycin hydrolase, and
pyroglutamyl peptidase, which releases pyroglutamic
acid at the amino terminal.27) However, most of the
aminopeptidases reported so far are metallo-en-
zymes.
Proline and glycine have unusual structures and
characteristics for amino acids. Proline is unusual in
that it is cyclic. Several kinds of proline-speciˆc pep-
tidases have been studied.28,29) It is interesting that
glycine-speciˆc peptidases are present in living
things.
Discussion
A
glycyl aminopeptidase was puriˆed to
homogeneity from A. elegans IFO 6408. Since glycyl
aminopeptidase is present inside cells, the enzyme
was extracted from the mycelia with a homogenizer.
However, it was not necessary to use detergent as
We found the enzyme in an A. elegans which is
used for the production of sufu, a traditional soybe-
an cheese in China. Many peptidases from Lactococ-
cus species produce a special taste in cheese. There
are reports that A. elegans produces an acid pro-
tease,30) and glycyl peptidase may aŠect the taste of
for membrane-binding enzymes, for example the
aminopeptidase N from E. coli
9)
.
The molecular mass of the enzyme was estimated
to be 320 kDa by gel ˆltration on FPLC-Superdex
200 HR and 56.5 kDa by SDS-PAGE, suggesting that
the enzyme was a hexamer. Aminopeptidases are of
two varieties, monomers and oligomers, which usu-
ally exist in the membrane or periplasmic space. The
aminopeptidases from Aspergillus oryzae and E. coli
are monomers, 26.56) and 80 kDa,9) respectively. In
sufu
.

Glycyl Aminopeptidase from A. elegans
87
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