Purification and characterization of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis)

Article abstract of DOI:10.1080/09168451.2015.1116926

Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an α-glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 °C. Furthermore, chloride ion altered PYG's substrate specificity. PYG exhibited the highest V_max/K_m value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.

Full text of DOI:10.1080/09168451.2015.1116926

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Purification and characterization of a chloride ion-
dependent α-glucosidase from the midgut gland of
Japanese scallop (Patinopecten yessoensis)
Yasushi Masuda, Masayuki Okuyama, Takahisa Iizuka, Hiroyuki Nakai,
Wataru Saburi, Taro Fukukawa, Janjira Maneesan, Takayoshi Tagami,
Tetsushi Naraoka, Haruhide Mori & Atsuo Kimura
To cite this article: Yasushi Masuda, Masayuki Okuyama, Takahisa Iizuka, Hiroyuki
Nakai, Wataru Saburi, Taro Fukukawa, Janjira Maneesan, Takayoshi Tagami, Tetsushi
Naraoka, Haruhide Mori & Atsuo Kimura (2015): Purification and characterization
of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese
scallop (Patinopecten yessoensis), Bioscience, Biotechnology, and Biochemistry, DOI:
10.1080/09168451.2015.1116926
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Date: 21 December 2015, At: 23:24

Bioscience, Biotechnology, and Biochemistry, 2015
Purification and characterization of a chloride ion-dependent α-glucosidase
from the midgut gland of Japanese scallop (Patinopecten yessoensis)
Yasushi Masuda1, Masayuki Okuyama1, Takahisa Iizuka1, Hiroyuki Nakai1, Wataru Saburi1,
Taro Fukukawa1, Janjira Maneesan1, Takayoshi Tagami1, Tetsushi Naraoka2, Haruhide Mori1 and
Atsuo Kimura1,*
2
1Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan; Aomori Industrial Research Center,
Aomori, Japan
Received August 5, 2015; accepted October 19, 2015
http://dx.doi.org/10.1080/09168451.2015.1116926
Marine glycoside hydrolases hold enormous
potential due to their habitat-related characteristics
such as salt tolerance, barophilicity, and cold toler-
ance. We purified an α-glucosidase (PYG) from the
midgut gland of the Japanese scallop (Patinopecten
yessoensis) and found that this enzyme has unique
characteristics. The use of acarbose affinity chro-
matography during the purification was particularly
effective, increasing the specific activity 570-fold.
PYG is an interesting chloride ion-dependent
enzyme. Chloride ion causes distinctive changes in
its enzymatic properties, increasing its hydrolysis
rate, changing the pH profile of its enzyme activity,
shifting the range of its pH stability to the alkaline
region, and raising its optimal temperature from 37
to 55 °C. Furthermore, chloride ion altered PYG’s
substrate specificity. PYG exhibited the highest
Vmax/Km value toward maltooctaose in the absence
of chloride ion and toward maltotriose in the pres-
ence of chloride ion.
the enzymes from land-dwelling creatures. Shelled inver-
tebrates in the marine environment have been shown to
possess various polysaccharide-degrading enzymes, such
as alginate lyase, cellulase, and mannanase, in their
digestive fluids.3–8) This is because they feed on seaweed
and need the ability to digest the abundant polysaccha-
rides, such as alginic acid, cellulose, laminarin, and man-
nan present in seaweed. Thus, studies on alginate lyase,
cellulase, laminarinase, and mannanase can often be
found in the literature, whereas there is little information
on enzymes that digest α-glucans.9)
α-Glucosidase (α-glucoside glucohydrolase, EC
3.2.1.20) catalyzes the liberation of α-glucose from the
non-reducing termini of substrates such as mal-
tooligosaccharides and α-glucan. In mammals,
α-glucosidase, existing in the small intestine as
sucrase–isomaltase and the maltase–glucoamylase com-
plex, is a terminal enzyme in the degradation of starch
and its related compounds. These carbohydrates are
digested into maltooligosaccharides by salivary and
pancreatic α-amylases, and maltooligosaccharides are
digested into glucose by α-glucosidases localized to the
small intestine. Thus, α-glucosidase is one of the most
important enzymes of α-glucan utilization in mammals.
The digestive glands of several bivalves, including
Pecten maximus, Placopecten magellanicus, and Perna
viridis, possess α-glucosidase as well as other carbohy-
drate metabolic enzymes such as amylase, laminarinase,
and cellulase.10–12) Accordingly, α-glucosidase likely
plays an important role in the degradation of α-glucan
in bivalves. However, to date, there has been no
detailed examination of α-glucosidases from bivalves.
In this study, we purified an α-glucosidase from the
midgut gland of the Japanese scallop Patinopecten
yessoensis (PYG) and characterized its enzymatic prop-
erties. Kinetic studies indicated that PYG is a chloride
ion-dependent enzyme. Chloride ion significantly
increases the catalytic activity and alters the substrate
specificity of PYG.
Key words: scallop α-glucosidase; chloride ion
dependent; substrate specificity
Marine organisms are attracting attention as a biolog-
ical resource because they have adapted to the diversity
of the oceanic environment, such as high salinity, high
pressure, and low temperature, which allows enzymes
from marine sources to hold enormous potential.1,2)
Knowledge of these enzymes is certainly beneficial
from a biotechnological perspective. Most of the useful
materials obtained from marine resources thus far have
been derived from microbes1); however, animals,
plants, and algae may have hidden potential.
Marine glycoside hydrolases can have just as much
potential as any other enzymes. However, studies of
the glycoside hydrolases of marine organisms have
been inadequate compared with the rigorous studies of
*Corresponding author. kimura@abs.agr.hokudai.ac.jp
Abbreviations: BN-PAGE, blue native-PAGE; HPA, human pancreatic α-amylase; MALDI-TOF-MS, matrix-assisted laser desorption ionization
time-of-flight mass spectrometry; PYG, α-glucosidase from midgut gland of Patinopecten yessoensis.
© 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
Y. Masuda et al.
10 mM sodium acetate buffer (pH 5.0) containing
Materials and methods
5 mM MgCl2. The active fractions were pooled and
subjected to acarbose affinity chromatography at room
temperature. An acarbose Sepharose 4 fast flow column
(1.5 cm × 11 cm) was equilibrated with 10 mM sodium
acetate buffer (pH 5.0) containing 5 mM MgCl2 and
200 mM NaCl. Bound enzyme was eluted with 100 mL
of 4 M Tris-HCl buffer (pH 7.0). The eluted fractions
were pooled and dialyzed against 10 mM sodium acet-
ate buffer (pH 5.0) containing 5 mM MgCl2. The dialy-
sate was concentrated using Amicon Ultracel-30 K
Ultra Centrifuge Filters (Merck Millipore, Billerica,
MA, USA). The concentrated solution was loaded onto
Materials. Midgut glands of scallop (P. yessoensis)
were collected in Mutsu Bay of Aomori prefecture,
Japan, in 2005. Maltose, maltotetraose, maltopentaose,
maltohexaose, maltoheptaose, and maltooctaose were
kindly gifted by Nihon Shokuhin Kako (Tokyo, Japan).
Maltotriose, kojibiose, and nigerose were purchased
from Wako Pure Chemical Industries (Osaka, Japan).
Other chemicals used were analytical grade and
obtained from Nacalai Tesque (Kyoto, Japan) if not
otherwise specified.
a
Sephacryl S-200HR (GE Healthcare) column
Enzyme activity assay. Reaction mixtures contain-
ing 0.2% (w/v) maltose, 60 mM sodium acetate buffer
(pH 5.0), 0.03% (v/v) Triton X-100, and 300 mM NaCl
were incubated at 37 °C. Reactions were terminated by
the addition of two volumes of 2 M Tris-HCl buffer
(pH 7.0). Glucose liberated from the substrate was
quantified using the glucose oxidase-peroxidase
method13) performed with the Wako Glucose CII Test
(Wako Pure Chemical Industries). One unit of maltase
activity was defined as the amount of enzyme
hydrolyzing 1 μmol of maltose per min under the
conditions described above.
(1.6 cm × 55 cm) equilibrated with 10 mM sodium
acetate buffer (pH 5.0) containing 5.0 mM MgCl2 and
200 mM NaCl. The active fractions were pooled and
dialyzed against a 10 mM sodium acetate buffer (pH
5.0). Purification procedures, except acarbose affinity
chromatography, were performed at 4 °C. The protein
concentration of the purified enzyme was calculated
from its absorbance at 280 nm in a 1-cm cuvette using
an extinction coefficient of (1.0 g/L)−1 cm−1.
Polyacrylamide gel electrophoresis.
SDS-PAGE
and Blue Native-polyacrylamide gel electrophoresis
(BN-PAGE) were performed using a Mini-Protean III
cell apparatus (Bio-Rad, Richmond, CA, USA). SDS-
PAGE was performed as described by Laemmli14) and
BN-PAGE as described by Schagger and von Jagow.15)
Protein was stained with Coomassie Brilliant Blue
(CBB) R-250 using a Rapid CBB staining kit (Kanto
Chemical, Tokyo, Japan). Mark12 unstained protein
standards and Native mark unstained protein standard
(Invitrogen, Life Technologies, Carlsbad, CA, USA)
were used as molecular size markers in SDS-PAGE
and BN-PAGE, respectively. Activity staining after
BN-PAGE was done by incubating the gel in 100 mM
sodium acetate buffer (pH 5.0) containing 1 mg/ml
4-methylumbelliferyl α-D-glucopyranoside (Sigma-
Aldrich) and 500 mM NaCl at 37 °C for 10 min. Fluo-
rescence from the 4-methylumbelliferone produced by
hydrolysis was detected using a UV transilluminator.
Preparation of acarbose Sepharose 4 fast flow.
Acarbose (0.1 mmol) (Sigma-Aldrich, St. Louis, MO,
USA) was stirred overnight with 3.5 mmol 2-(4-amino-
phenyl)ethylamine (Tokyo Chemical Industry, Tokyo,
Japan). The resulting aminoethylated acarbose was
reduced with sodium borohydride (0.63 mmol) in etha-
nol (1.5 mL) for 5 h at room temperature. The mixture
was titrated with 1 M acetic acid to pH 5.6 and then
applied to a Sephadex G-25 column equilibrated with
1 M sodium acetate buffer (pH 5.0). Upon isocratic
elution with 1 M sodium acetate buffer (pH 5.0), the
fractions containing modified acarbose were identified
by using thin-layer chromatography on silica gel 60
(nitromethane/1-propanol/water, 4/10/3). The plates
were visualized by charring with a methanolic solution
of sulfuric acid containing 0.02% α-naphthol. The mod-
ified acarbose was coupled with NHS-activated Sephar-
ose 4 Fast Flow (GE Healthcare, Buckinghamshire,
England) by following the supplier’s instruction.
In-gel digestion with trypsin.
Purified PYG
(15 μg) was subjected to SDS-PAGE, and a CBB-
stained protein band was excised. The gel piece was
washed three times with 100 μL of 50 mM NH4HCO3
in 50% (v/v) acetonitrile for 20 min at 37 °C and then
dried using a vacuum centrifugation. The dried gel was
swollen in 5 μL trypsin solution (1 mg/mL) containing
1 mM HCl and 20 mM CaCl2 at 4 °C for 10 min. After
reswelling the gel, 15 μL of 50 mM NH4HCO3 was
added and digestion was continued for 24 h at 37 °C.
Peptides were extracted three times in 100 μL of 60%
(v/v) acetonitrile, lyophilized, and redissolved in 0.1%
trifluoroacetic acid containing 50% (v/v) acetonitrile.
Purification of PYG.
Midgut glands (52 g) were
stirred in 10 mM sodium acetate buffer (pH 5.0) con-
taining 5 mM MgCl2 and 500 mM NaCl overnight at
4 °C to extract protein. The supernatant (crude enzyme)
was recovered by centrifugation at 18,700 × g for
20 min at 4 °C. Ammonium sulfate was added to the
crude enzyme until its concentration reached 70% satu-
ration, and the suspension was left overnight at 4 °C.
The precipitate was collected by centrifugation at
18,700 × g for 20 min. The precipitate was dissolved in
10 mM sodium acetate buffer (pH 5.0) containing
5 mM MgCl2 and 30% saturated ammonium sulfate
and then loaded onto a TOYOPEAL Butyl-650 M
(Tosoh, Tokyo, Japan) column equilibrated with the
same buffer. Absorbed proteins were eluted with a lin-
ear gradient of 30%–0% saturated ammonium sulfate in
Matrix-assisted laser desorption ionization time-
of-flight mass spectrometric analysis.
The tryptic
peptides were desalted using a Zip-Tip C-18 (Merck

Chloride ion-dependent α-glucosidase from scallop
3
Millipore, Billerica, MA, USA) according to the manu-
facturer’s instructions. The eluate (2 μL) was mixed
with 0.8 μⅬ of matrix solution [10 mg/mⅬ α-cyano-4-
hydroxycinnamic acid (Sigma-Aldrich) in 0.1% trifluo-
roacetic acid/50% (v/v) acetonitrile], and dried at room
temperature on the sample plate. Mass spectra were
recorded with a matrix-assisted laser desorption ioniza-
tion time-of-flight mass spectrometer (MALDI-TOF-
MS)(Voyager-DE STR, Applied Biosystems, Foster
City, CA, USA) with delayed ion extraction technol-
ogy. All mass spectra were obtained in reflector mode
and calibrated with GRAMS software (Applied Biosys-
tems) using angiotensin II as an external mass standard.
We used the MALDI-TOF-MS in the facilities of the
Research Faculty of Agriculture, Hokkaido University.
maltopentaose, maltohexaose, maltoheptaose, maltooc-
taose, trehalose, kojibiose, and nigerose) were mea-
sured in the absence or presence of 300 mM NaCl
under standard assay conditions. Enzyme concentra-
tions used were 0.102 to 40.6 μg/mL. The initial veloc-
ity was expressed as the number of μmol of glucose
released from the non-reducing end of the substrate per
min per mg protein. When determining the hydrolysis
rate for disaccharides, the concentration of liberated
glucose was divided by two.
Kinetic parameters. The kinetic parameters (Vmax
and Km) for kojibiose, nigerose, and maltooligosaccha-
rides in the absence or presence of 300 mM NaCl were
calculated using the initial velocities of reactions con-
taining six to eight different substrate concentrations
(0.2–32 mM). These initial rates were fitted to the
Michaelis–Menten equation using the computer pro-
gram Kaleidagraph 3.6. The initial velocities were mea-
sured in the standard assay conditions except for the
NaCl concentration. The enzyme concentration used
was 127 ng/mL.
Effect of various salts on enzyme activity.
The
enzyme reactions were performed in 60 mM sodium
acetate buffer (pH 5.0) containing 0.03% (v/v) Triton
X-100, 0.2% maltose, and diluted enzyme (102 ng/mL)
at 37 °C while varying the concentrations of various
salts (0 mM–400 mM MgSO4, NaCl, KCl, MgCl2 or
CaCl2). The liberated glucose was measured as
described above.
Results and discussion
Purification of PYG
Effects of pH and temperature. The effect of pH
on maltose hydrolysis activity was determined using the
standard assay conditions with the exception that
60 mM sodium acetate (pH 3.6–pH 5.6), 60 mM sodium
phosphate (pH 5.4–pH 8.2), or Britton–Robinson
(pH 2.2–pH 6.9) buffers were used. Enzyme concentra-
tions used were 406 ng/mL (in the absence of NaCl) and
81.2 ng/mL (in the presence of 300 mM NaCl).
For pH–stability experiments, the purified enzyme
(203 μg/mL) diluted with 40 mM Briton–Robinson
buffer (pH 2.0–pH 11.0) containing 0.05% (v/v) Triton
X-100 was kept at 4 °C for 24 h in either the absence or
presence of 300 mM NaCl. The remaining activity was
measured under the standard assay conditions except
that the remaining activity of samples treated in the
absence of NaCl was measured using the NaCl-free buf-
fer. The optimal temperatures for the hydrolytic activity
of the enzyme in the absence of NaCl or in the presence
of 300 mM NaCl were measured under the standard
assay condition except that the temperature was varied
from 25 to 65 °C. Enzyme concentrations used were
406 ng/mL (in the absence of NaCl) and 102 ng/mL (in
the presence of 300 mM NaCl). For thermal stability
experiments, the purified enzyme (508 ng/mL in the
absence of NaCl and 102 ng/mL in the presence of
300 mM NaCl) in 10 mM sodium acetate buffer (pH
5.0) containing 0.05% (v/v) Triton X-100 was incubated
at 30 to 65 °C for 15 min. The residual activity was
assayed under the standard assay conditions except that
the remaining activity treated without NaCl was mea-
sured using NaCl-free buffer.
PYG was purified using a combination of ammonium
sulfate precipitation and several column chromato-
graphic steps, including acarbose affinity chromatogra-
phy, which was performed using acarbose covalently
cross-linked to a Sepharose 4 fast flow column. The
enzyme preparation was subjected to electrophoresis to
confirm the purity of the enzyme. The enzyme migrated
as a single protein band with a molecular mass of about
66 kDa on BN-PAGE, stained by CBB R-250 or visual-
ized by activity staining (Fig. 1(A)). However, on SDS-
PAGE, the enzyme separated into two components
(Fig. 1(B)). The molecular masses of the major and
minor components were 51 and 40 kDa, respectively.
The results of PAGEs imply that these components do
not dimerize, and exist as a monomer. The components
could not be separated using further purification proce-
dures including ion-exchange, gel filtration, and
(B)
kDa
200
(A)
M
1
2
M
1
kDa
146
116
97.4
66.3
55.4
66.0
36.5
31.0
20.0
21.5
Fig. 1. Electrophoretic analysis of PYG.
Hydrolysis of maltooligosaccharides and various dis-
Notes: A: BN-PAGE of PYG. Lane M, marker proteins; lanes 1
and 2, PYG (4 μg protein for each lane) visualized by protein stain-
ing and activity staining, respectively. B: SDS-PAGE of PYG. Lane
M, marker proteins; lane 1, 5.5 μg PYG.
accharides.
The initial velocities of the hydrolysis
reactions performed using 2 mM concentrations of
various substrates (maltose, maltotriose, maltotetraose,

4
Y. Masuda et al.
affinity chromatography was the most effective step,
increasing the specific activity 570-fold.
(A)
100
80
60
40
20
0
Effect of various salts on the hydrolysis rate
The effect of MgCl2 and MgSO4 on PYG activity
was investigated by measuring the rate of maltose
hydrolysis (Fig. 3). The hydrolysis rate in 60 mM
sodium acetate buffer without any salts was 13.1 μmol/
min/mg. MgSO4 doubled the hydrolysis rate: the
hydrolysis rate in the presence of 400 mM MgSO4 was
26.4 μmol/min/mg. MgCl2 increased more efficiently
than the sulfate ion: the hydrolysis rate in the presence
of 400 mM MgCl2 was 124 μmol/min/mg. The effect
of MgCl2, NaCl, KCl, and CaCl2 on PYG activity was
investigated (Fig. 3). Every salt increased the enzyme
activity in the same manner and the activity was satu-
rated at the concentration of 40 mM. The hydrolysis
rates in the presence of sufficient concentration
(400 mM) of salts were 124 (MgCl2), 122 (NaCl), 123
(KCl), and 128 (CaCl2) μmol/min/mg, respectively.
These results indicate that the chloride ion increased
the hydrolytic activity efficiently, but the identity of the
cation had little effect on the activity.
1000
1300
1600
1900
2200
2500
m/z
(B)
100
80
60
40
20
0
1000
1300
1600
1900
2200
2500
m/z
Fig. 2. MALDI-TOF-MS analysis of PYG tryptic peptides.
Notes: A: MALDI-TOF-MS spectra of tryptic peptides obtained
from 51-kDa component. B: MALDI-TOF-MS spectra of tryptic
peptides obtained from 40-kDa component.
pH and thermal properties of PYG
The optimal pH of maltose hydrolytic activity and
pH stability of PYG in the presence or absence of
NaCl were examined. We used 300 mM NaCl, in
which the activity was adequately enhanced, as the
salt-existence condition. The optimal pH values in the
absence and presence of 300 mM NaCl were 3.9 and
4.0, respectively (Fig. 4(A)). The pH–activity profiles
showed a bell-shaped curve with or without NaCl. The
enzyme was incubated at various pHs in the presence
or absence of NaCl at 4 °C for 24 h, and its residual
activity was measured. The stable range was defined as
the pH range exhibiting more than 90% residual activ-
ity. NaCl shifted the pH–stability range to the alkaline
region. PYG without NaCl was stable in a pH range
from 3.2 to 6.3, whereas PYG with NaCl was stable in
a pH range from 4.1 to 7.3 (Fig. 4(B)). In addition,
when the enzyme was treated at pH 10.0, the residual
activity was 50% in the presence of NaCl, whereas the
enzyme was almost inactive in the absence of NaCl.
The optimal temperature and thermal stability in the
presence or absence of NaCl were examined. The opti-
mal temperature increased from 37 to 55 °C with the
addition of NaCl (Fig. 4(C)). The thermal stability of
the enzyme in the presence or absence was investigated
based on the measurement of the residual activity after
heat treatment for 15 min. The stable region was
defined as the temperature range exhibiting more than
90% residual activity. PYG was stable up to 40 °C
under both NaCl conditions. However, the residual
activity at 45 °C was different in the presence or
absence of NaCl. The enzyme exhibited 80% residual
activity in the presence of NaCl, whereas the residual
activity was 10% in the absence of NaCl. (Fig. 4(D)).
The increase in pH and thermal stabilities with the
addition of NaCl might be explained by facilitating ener-
getic interactions within the protein structure and by the
stabilization of surface-exposed residues. Alteromonas
140
120
100
80
140
120
100
80
60
60
40
40
20
20
0
0
0
100
200
300
400
0
1
2
3
4
5
Salt concentration (mM)
Salt concentration (mM)
Fig. 3. Effect of various salts on enzyme activity.
Notes: Maltose hydrolysis rates measured in the presence of
0–400 mM MgCl2 (■), CaCl2 (□), KCl (●), NaCl (○), and MgSO4
(▲) represent in panel at left. Panel at right shows an enlarged view
at low concentration of salts (0–5 mM).
hydrophobic interaction chromatographies. MALDI-
TOF-MS of the mixture of tryptic fragments generated
by in-gel digestion of the 51- and 40-kDa components
indicated that nine peptides derived from each
component were identical (Fig. 2). It was likely that the
51- and 40-kDa components were derived from the same
peptide, which underwent different proteolysis. The mid-
gut gland contains proteases since it is a digestive tract
with functions similar to those of the liver and pancreas
in mammals.16) It is possible that PYG undergoes a
proteolytic process that splits it into two components.
The enzyme preparation was regarded as the purified
enzyme (PYG) and was thus used for further analysis.
Peptide masses determined by MALDI-TOF-MS were
used in database searches by Mascot; however, no gly-
coside hydrolase was identified as a significant match.
The purification process yielded 0.427 mg of purified
PYG, whose specific activity was 122 U/mg, from 52 g
of midgut glands with a yield of 39.8%. The PYG purifi-
cation procedures are summarized in Table 1. Acarbose

Chloride ion-dependent α-glucosidase from scallop
5
Table 1. PYG purification summary.
Procedure
Total protein (mg)a) Total activity (U) Specific activity (U/mg) Purification (-fold) Recovery (%)
Crude extract
11,900
3,350
636
0.921
0.426
131
124
150
135
52.1
0.0110
0.0370
0.236
134
1.00
3.36
21.4
12,100
11,100
100
94.6
115
93.9
39.8
Ammonium sulfate 60–30% fractionation
TOYOPEARL Butyl-650 M
Acarbose Sepharose 4 Fast Flow
Sephacryl S-200
122
a
1%
280nm;1cm
calculated under the assumption that E
¼ 10:0.
(A)
(B)
(D)
100
100
80
60
40
20
0
80
60
40
20
0
2
4
6
8
10
2
4
6
8
10 12 14
pH
pH
(C)
100
100
80
60
40
20
0
80
60
40
20
0
20
30
40
50
60
70
20
30
40
50
60
70
Temperature (°C)
Temperature (°C)
Fig. 4. Effect of NaCl on the pH–activity profile (A), the pH–stability (B), the temperature–activity profile (C), and the thermal stability (D) of
PYG in the absence (○) and presence (●) of 300 mM NaCl.
Notes: A: Enzyme activity was measured at various pH values and 37 °C. B: Enzyme was incubated at various pH values at 4 °C for 24 h, and
the residual activity was measured at pH 5.0 and 37 °C. C: Enzyme activity was measured at the indicated temperature and pH 5.0. D: Enzyme
was incubated at various temperatures at pH 5.0 for 15 min, and residual activity was measured at pH 5.0 and 37 °C.
haloplanctis α-amylase is stabilized by facilitating
energetic interactions between the protein structure and
the chloride ion.17) Furthermore, monovalent cation,
including potassium and sodium ions, enhances the over-
all thermal stability of several enzymes by stabilization
of surface-exposed residues.18) The thermal stabilization
of PYG might be caused by the similar manner as these
enzymes.
length; the highest Vmax/Km (57.1 μmol/min/mg/mM)
was observed for the hydrolysis of maltooctaose. On
the other hand, in the presence of NaCl, PYG exhibited
the highest Vmax/Km with maltotriose (370 μmol/min/
mg/mM). Vmax/Km values for maltooligosaccharides
longer than maltotriose were similar. On the other
hand, the Vmax and Km values with maltohexaose were
lower than those with other maltooligosaccharides.
Although it is hard to give a feasible reason for the
lower Vmax and Km, the lower Km seems to decrease
the Vmax value. As mentioned above, the Vmax/Km
value for maltohexaose is similar to that for other mal-
tooligosaccharides. It suggests that the activation free
energies for the reactions of the free enzyme and these
maltooligosaccharide substrates may be similar, and the
transition state of the enzyme–maltohexaose may be
stabilized equally to other maltooligosaccharides.
Meanwhile, the lower Km makes the reaction fall into a
thermodynamic pit, and causes a rise in a free-energy
barrier from the Michaelis complex to the transition
state.
Substrate specificity of PYG
The kinetic parameters for the hydrolysis of mal-
tooligosaccharides, kojibiose, and nigerose in the pres-
ence or absence of 300 mM NaCl were determined to
evaluate substrate specificity (Table 2). PYG readily
hydrolyzed maltooligosaccharides, nigerose, and koji-
biose, but hardly hydrolyzed trehalose and isomaltose.
The hydrolytic rates of 2 mM isomaltose and 2 mM
trehalose in the presence of 300 mM NaCl were
1.38 × 10−2 and 4.15 × 10−2 μmol/min/mg, respec-
tively. NaCl affected Km values more effectively than
Vmax values. The Vmax/Km values for disaccharides fol-
lowed the order, maltose > nigerose > kojibiose in the
presence or absence of NaCl. However, NaCl had an
effect on the preference for maltooligosaccharide chain
length. In the absence of NaCl, Vmax/Km for mal-
tooligosaccharides increased with increasing chain
Possible mechanism for chloride ion dependence of
PYG
The hydrolytic rate of PYG was significantly
increased by chloride ion, suggesting PYG is chloride

6
Y. Masuda et al.
Table 2. PYG kinetic parameters in the absence and presence of 300 mM NaCl.
Absence of NaCl
Presence of NaCl
Vmax (μmol/min/
Vmax/Km (μmol/min/mg/
Vmax (μmol/min/
Vmax/Km (μmol/min/mg/
Substrate
mg)
Km (mM)
mM)
mg)
Km (mM)
mM)
Maltose
Maltotriose
41.3 0.9
34.3 1.4
11.1 0.9
7.47 0.5
4.48
0.02
4.43
0.55
7.02
0.44
7.68
0.32
1.39
0.32
17.9
0.41
13.1
3.78
4.56
6.07
143
126
156
1
2
4
0.871 0.069
0.342 0.006
0.547 0.030
164
370
286
Maltotetraose 32.9 0.1
Maltopentaose 27.9 0.9
Maltohexaose 32.2 1.8
Maltoheptaose 67.4 2.9
6.36
4.59
8.80
57.1
0.508
1.42
144
1
0.533 0.046
0.189 0.012
0.671 0.048
0.637 0.028
2.15 0.10
271
287
218
233
9.02
22.9
54.0 1.0
146
148
2
2
Maltooctaose
Kojibiose
79.7 0.04
8.96 0.14
18.6 0.39
19.4 1.4
29.0 1.4
Kojibiose
1.27 0.09
0.33
ion-dependent enzyme. A few chloride ion-dependent
glycoside hydrolases have been reported thus far.19–21)
Human pancreatic α-amylase (HPA), a well-studied
chloride ion-dependent enzyme,22–24) catalyzes the
same α-glucoside hydrolysis as PYG and could furnish
information on the mechanism of the chloride ion
dependency of PYG. Biochemical and structural studies
of HPA have indicated that chloride ion makes the ori-
entation of an acid/base catalyst accurate, thereby
increasing its pKa and enhancing activity. The enhance-
ment of PYG catalytic activity by chloride ion was
associated with a small change in the bell-shaped pH-
rate profile of PYG (Fig. 4(A)); thus, chloride ion
might be connected to a catalytic residue as in HPA.
PYG underwent not only an increase in catalytic activ-
ity but also a change of substrate specificity toward mal-
tooligosaccharides in the presence of chloride ion. In the
case of HPA, chloride ion causes the conformational
change of substrate-binding loop. It is possible that chlo-
ride ion also causes a conformational change in PYG
and that the change in specificity is caused by a
conformational change in the substrate-binding site.
In conclusion, we succeeded in finding a unique α-glu-
cosidase from a marine source, purifying PYG from the
midgut gland of the scallop, and determining the unique
properties of this enzyme. The catalytic activity of PYG
was evidently enhanced by chloride ion. Furthermore,
substrate specificity was altered by the chloride ion. To
the best of our knowledge, this is the first report of a
chloride ion-dependent α-glucosidase. Unfortunately, we
could not clarify the molecular mechanism of the chlo-
ride ion dependency at this stage because no structural
information exists. It will be necessary to determine the
structure of PYG to elucidate the molecular mechanism
responsible for the chloride ion dependency.
H. Nakai, and W. Saburi. Analyzed the data: Y.
Masuda and M. Okuyama. Contributed reagents/materi-
als/analysis tools: T. Naraoka, T. Tagami, and J. Man-
eesan. Wrote the paper: M. Okuyama, Y. Masuda, and
A. Kimura.
Acknowledgment
We thank Mr K. Nishioka of Faculty of Agriculture,
Hokkaido University for preliminary experiments for
the purification of PYG.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the JSPS KAKENHI [grant number
26292049].
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