Purification and characterization of a novel fungal α-glucosidase from Mortierella alliacea with high starch-hydrolytic activity

Article abstract of DOI:10.1271/bbb.66.2415

The fungal strain Mortierella alliacea YN-15 is an arachidonic acid producer that assimilates soluble starch despite having undetectable α-amylase activity. Here, a α-glucosidase responsible for the starch hydrolysis was purified from the culture broth through four-step column chromatography. Maltose and other oligosaccharides were less preferentially hydrolyzed and were used as a glucosyl donor for transglucosylation by the enzyme, demonstrating distinct substrate specificity as a fungal α-glucosidase. The purified enzyme consisted of two heterosubunits of 61 and 31 kDa that were not linked by a covalent bond but stably aggregated to each other even at a high salt concentration (0.5 M), and behaved like a single 92-kDa component in gel-filtration chromatography. The hydrolytic activity on maltose reached a maximum at 55°C and in a pH range of 5.0-6.0, and in the presence of ethanol, the transglucosylation reaction to form ethyl-α-D-glucoside was optimal at pH 5.0 and a temperature range of 45-50°C.

Full text of DOI:10.1271/bbb.66.2415

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Purification and Characterization of a Novel Fungal
α-Glucosidase from Mortierella alliacea with High
Starch-hydrolytic Activity
Yoshio TANAKAa, Tsunehiro AKIa, Yûki HIDAKAab, Yûji FURUYAab, Seiji KAWAMOTOa, Seiko
SHIGETAa, Kazuhisa ONOa & Osamu SUZUKIac
a Department of Molecular Biotechnology, Graduate School of Advanced Sciences of
Matter, Hiroshima University 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan
b Present address: Ikeda Tohka Industries Co., Ltd. 95-7 Minooki, Fukuyama, Hiroshima
721-8558, Japan
c Present address: Ikeura Patent Office Shibuya, Tokyo 151-0053, Japan
Published online: 22 May 2014.
To cite this article: Yoshio TANAKA, Tsunehiro AKI, Yûki HIDAKA, Yûji FURUYA, Seiji KAWAMOTO, Seiko SHIGETA, Kazuhisa
ONO & Osamu SUZUKI (2002) Purification and Characterization of a Novel Fungal α-Glucosidase from Mortierella alliacea
with High Starch-hydrolytic Activity, Bioscience, Biotechnology, and Biochemistry, 66:11, 2415-2423, DOI: 10.1271/
bbb.66.2415
To link to this article: http://dx.doi.org/10.1271/bbb.66.2415
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Biosci. Biotechnol. Biochem., 66 (11), 2415–2423, 2002
Puriˆcation and Characterization of a Novel Fungal a-Glucosidase
from Mortierella alliacea with High Starch-hydrolytic Activity
Yoshio TANAKA, Tsunehiro AKI,† Yu
Seiko SHIGETA, Kazuhisa ONO, and Osamu S
â
ki HIDAKA, Yu
â
ji FURUYA, Seiji KAWAMOTO
,
*
*
UZUKI**
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,
Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan
Received May 27, 2002; Accepted July 18, 2002
The fungal strain Mortierella alliacea YN-15 is an
arachidonic acid producer that assimilates soluble
medium. Moreover, undigested soluble starch
remained until the late phase of its culture, which was
accompanied by a decline in the oil content, suggest-
ing that the accumulated lipid was re-used as an
energy source.5) Throughout the fermentation, we
could not detect even a trace of endo-type amylolytic
enzyme activity, but did detect exo-type glucose-
forming activity. These ˆndings imply that the
glucose-forming enzyme is responsible for the diges-
tion of soluble starch and that the incomplete diges-
tion would be due to the formation of the limit dex-
trin by the enzyme. In fact, when the starch medium
starch despite having undetectable
a-amylase activity.
Here, a -glucosidase responsible for the starch hydrol-
a
ysis was puriˆed from the culture broth through
four-step column chromatography. Maltose and other
oligosaccharides were less preferentially hydrolyzed and
were used as a glucosyl donor for transglucosylation by
the enzyme, demonstrating distinct substrate speciˆcity
as a fungal a-glucosidase. The puriˆed enzyme consist-
ed of two heterosubunits of 61 and 31 kDa that were not
linked by a covalent bond but stably aggregated to each
other even at a high salt concentration (0.5
M
), and
was treated with a-amylase (EC 3.2.1.1) from
behaved like a single 92-kDa component in gel-ˆltration
chromatography. The hydrolytic activity on maltose
another origin, the isolate fully used the liqueˆed
starch and accumulated a level of lipids comparable
to when glucose was used as a carbon source. There-
fore, the enzyme(s) playing an essential role in releas-
ing glucose from starch in the YN-15 culture would
be a certain type of saccharogenic exo-type enzyme
deˆcient or weak in debranching and liquefying ac-
tivities.
reached a maximum at 55
5.0–6.0, and in the presence of ethanol, the trans-
glucosylation reaction to form ethyl- -glucoside was
optimal at pH 5.0 and a temperature range of 45–50 C.
9
C and in a pH range of
a-
D
9
Key words: arachidonic acid; carbohydrate hydrolase;
-glucosidase; Mortierella alliacea; starch
a
a
-Glucosidase (EC 3.2.1.20) is an exo-type carbo-
hydrase with diverse speciˆcity that catalyzes the
Microbial oils rich in polyunsaturated fatty acids
may replace marine ˆsh oils as a food material be-
cause of certain advantages, which include ease of
processing and a stable supply. Several lower fungi,
algae, and marine diatoms are commercial sources of
eicosapentaenoic and docosahexaenoic acids.1) For
arachidonic acid production, as an alternative to
release of
terminal of
a
-
D
-glucose from the non-reducing
a
-glucosyl-linked substrates.6) The
a-
glucosidases from animals, plants, and fungi have
higher activity toward maltooligosaccharides than
heterogeneous substrates such as sucrose and
allylglucosides.7) In particular, the fungal enzymes
preferentially hydrolyze the shorter chain length of
previously reported strains such as Mortierella alpina
the saccharides substrate:
from Aspergillus oryzae8) and A. niger9) had slight
activity toward -glucans, but the enzymes puriˆed
from Mucor javanicus
a-glucosidases crystallized
2–4)
and M. elongata
,
we have isolated a ˆlamentous
fungus, M. alliacea YN-15.5) The isolate uses either
a
10)
11)
glucose or soluble starch even at a high concentration
,
Mu. racemosus
,
Paecilo-
12)
(
¿
15
z
) for the characteristic cell growth and
myces varioti,
and Penicillium spp.13,14) degraded
productivity of arachidonic acid. However, when
soluble starch was used as a sole carbon source, the
mycelial oil content was less than that in a glucose
soluble starch to some extent. Their substrate
speciˆcities are in contrast to those of fungal
glucoamylases (EC 3.2.1.3), the other exo-type en-
†
To whom correspondence should be addressed. Tel: +81-824-24-7755; Fax: +81-824-24-7754; E-mail: aki
＠
hiroshima-u.ac.jp
*
**
Present address: Ikeda Tohka Industries Co., Ltd., 95-7 Minooki, Fukuyama, Hiroshima 721-8558, Japan
Present address: Ikeura Patent O‹ce, Shibuya, Tokyo 151-0053, Japan
Abbreviations
:
a
-EG, ethyl
a
-glucoside; PNPG,
p
-nitrophenyl
a
-
D
-glucopyranoside; PNPM,
p
-nitrophenyl
a
-
D
-maltoside

2416
Y. TANAKA et al.
zymes that act more eŠectively on
a
-glucans such as
‰ow rate of 3 ml min. Enzyme activity fractions
W
starch and glycogen.15–18) In this paper, we describe
the puriˆcation and characterization of an exo-type
carbohydrate hydrolase from M. alliacea with un-
usual substrate speciˆcity.
(24 ml) were collected.
(iv) Step 4: Second cation exchange chromato-
graphy. In a similar manner to that described in Step
3, except that the ‰ow rate was 2 ml min, the pooled
W
fraction was put on, and resolved again, on a
Resource S column, and fractions (15 ml) containing
enzyme activity were collected.
Materials and Methods
Microorganism and culture conditions. The M.
alliacea strain YN-15 was maintained on an agar
(v) Step 5: Gel ˆltration chromatography. The
resulting enzyme solution was concentrated to 1 ml
and gel-ˆltered on a HiLoad Superdex 200 column
medium of 5
at 15 C. The ˆrst culture was prepared by incubation
of the strain at 28 C for 3 d in a medium (5 ml in a
15-ml test tube) containing 20 g soluble starch, 0.2 g
yeast extract, 5 g NaNO3, 5 g K2HPO4, 1 g MgSO4
z corn meal agar and 1.5z bacto agar
×
9
(2.6 60 cm; Amersham Bioscience) equilibrated
with a 0.05 sodium phosphate buŠer (pH 7.0) con-
taining 0.15
9
M
M
NaCl at a ‰ow rate of 0.5 ml min. En-
W
・
zyme activity fractions (21 ml) were pooled.
・
7H2O, 20 mg FeSO4 7H2O, 10 mg CaCl2, 10 mg
・
・
・
ZnSO4 7H2O, 2 mg MnCl2 4H2O, 0.2 mg CuSO4
Anomeric analysis. The enzyme solution was dia-
・
5H2O, 0.2 mg KI, 0.2 mg (NH4)6Mo7O24 4H2O, and
lyzed against a 0.01
M
sodium acetate buŠer (pH 5.0).
0.2 mg H3BO3 per liter, pH 7.0. Sixty 500-ml shaking
‰asks each containing 100 ml of the same medium,
except that NaNO3 was replaced by (NH4)2HPO4,
were inoculated with the ˆrst culture, and these cul-
A reaction mixture containing 5 m
-maltoside (PNPM; Nacalai Tesque, Kyoto,
Japan), 0.005 (w v) arabitol (as an internal stan-
dard), and the enzyme solution was incubated at
25 C for up to 6 min. Portions were immediately
M
p-nitrophenyl
a
-
D
z
W
tures were continued at 28
shaking.
9
C for 7 d with reciprocal
9
frozen by immersion of the tube in liquid N2 and
lyophilized. The freeze-dried sample was converted
into trimethylsilyl derivatives by incubation with
Enzyme puriˆcation. An exo-type starch hydrolyt-
ic enzyme was puriˆed from the supernatant of the
M. alliacea YN-15 culture pursuant to the following
100
m
l of a trimethylsilylation reagent, TMSI-H (GL
C for 10 min, and was
Science, Tokyo, Japan), at 80
9
procedures at 4
measurement of
9
C, unless otherwise stated, by
analyzed quantitatively on a gas-liquid chromato-
graph (GC-8A; Shimadzu, Kyoto, Japan) equipped
p
-nitrophenyl -glucopyranoside
a
-
D
×
(PNPG) hydrolytic activity as described below.
(i) Step 1: Ethanol precipitation. The culture
broth (6 l) was ˆltered to remove mycelia. After the
addition of 11.2 l of cold ethanol, the suspension was
with a glass column (0.26 200 cm) packed with 3
z
Silicone SE-30 on Chromosorb WAW-DMCS (60–80
mesh; GL Science) at 170
carrier.19)
9
C with N2 gas as a
kept in
a refrigerator for 1 h. The resulting
precipitate was collected by centrifugation at 12,000
Enzyme assays.
ured by the release of
strate PNPG. An assay mixture (0.12 ml) consisting
of a 0.1 phosphate buŠer (pH 5.0), 5 m PNPG,
and enzyme solution was incubated at 37 C for
15 min. The reaction was stopped by the addition of
Na2CO3 at a concentration of 0.1 , and absorbance
a
-Glucosidase activity was meas-
×
M
g
for 15 min and dissolved in a 0.05
phosphate buŠer (pH 7.0) containing 2 ammonium
sulfate. The insoluble materials were removed by fur-
sodium
p
-nitrophenol from the sub-
M
M
M
ther centrifugation at 20,000
×
g
for 30 min.
9
(ii) Step 2: Hydrophobic interaction chromato-
graphy. The supernatant (1 l) was put on a HiLoad
M
Phenyl Sepharose column (2.6
×
10 cm; Amersham
of the reaction mixture was measured at 400 nm. One
unit of the enzyme activity was deˆned as the amount
Bioscience, Uppsala, Sweden) equilibrated with a
0.05 sodium phosphate buŠer (pH 7.0) containing
ammonium sulfate. After an extensive wash of
M
of enzyme liberating 1
m
W
mol of
p-nitrophenol per
＝
minute (e
2
M
18,400 l mol cm).
W
the column with the buŠer, the adsorbed proteins
were eluted with ammonium sulfate solution (600 ml)
The puriˆed enzyme was examined for its hydroly-
sis and transglucosylation activities toward various
substrates: maltose, trehalose, isomaltose, maltitol,
by a linear gradient from 2 to 0
M
at a ‰ow rate of
＝
10 ml min, and enzyme activity fractions (120 ml)
amylose EX-I (DP 18), pullulan (kindly provided
by Hayashibara Biochemical Laboratories,
Okayama, Japan), kojibiose, ethyl -glucoside (
W
were collected.
(iii) Step 3: First cation exchange chromato-
graphy. The pooled fraction was dialyzed against a
a
a-
EG), nigerose (Wako Pure Chemical, Osaka, Japan),
sucrose, soluble starch (Katayama Chemical, Osaka,
Japan), PNPG (Sigma, St. Louis, MO), glycogen,
amylopectin (Nacalai Tesque), and dextran
(Seikagaku Kogyo, Tokyo, Japan). For the hydroly-
0.02
M
sodium acetate buŠer (pH 5.0) and put on a
Resource S column (6 ml; Amersham Bioscience)
equilibrated with the same buŠer. After removal of
the non-adsorbed protein, the elution was done by a
120-ml linear gradient from 0 to 0.25
M
NaCl at a
sis analysis, a reaction mixture (100 ml) composed of

Novel
a
-Glucosidase of Mortierella alliacea
2417
8.2
sodium acetate buŠer (pH 5.0) was incubated at 37
for 30 min. The reaction was stopped by the addition
of two volumes of 2
Tris-HCl buŠer (pH 7.0),20)
and the release of glucose was measured by a Glucose
C-II Test Kit Wako (Wako Pure Chemical). The
hydrolytic activity was represented as the release of
glucose only from the non-reducing terminal of the
substrate, where the amount of glucose from the
reducing terminal was excluded. For the trans-
m
g ml enzyme, 0.4
z
(w v) substrate, and 40 m
M
the mixture composed of 0.82 mg ml enzyme and
W
W
W
9C
0.4
adjusted at the indicated pH was incubated at 37
for 15 min. For -EG-forming reactions, 20 l of
enzyme (0.82 g), 40 l of ethanol, and 40 l of 1
(w v) maltose in a 0.1 citrate-phosphate buŠer ad-
z (w v) maltose in the Britton-Robinson buŠer
W
9
C
M
a
m
m
m
M
m
z
W
justed at the indicated pH was incubated at 45
30 min.
9C for
(iv) EŠects of temperature on maltose hydrolytic
and -EG-forming activities. For maltose hydrolytic
reactions, a mixture composed of 0.04 sodium
acetate (pH 5.0), 0.4 (w v) maltose, and enzyme
(0.82 mg ml) was incubated at various temperatures
a
glucosylation assay, a reaction mixture (100
composed of 8.2 g ml enzyme, 40 ethanol, 0.4
(w v) substrate, and 40 m sodium acetate buŠer
C for 30 min. To stop
the reaction, the sample was heated in a boiling-water
bath for 10 min. After the sample (100 l) was mixed
with rhamnitol as an internal standard and dried, the
acetylation was done by the addition of 200 l each
of acetic anhydride and pyridine then heated at
100 C for 1 h. The solvent was evaporated, and the
sample was dissolved in ethanol. The acetylated
EG was analyzed on a gas-liquid chromatograph
m
l)
M
m
z
z
z
W
W
M
W
W
(pH 5.0) was incubated at 45
9
for 15 min. For the
a-EG-forming reaction,
8.2 g ml enzyme was used and the reaction time was
30 min. Ethanol was added at 40
forming activity was measured.
m
W
m
z
when the a-EG-
m
Protein analysis. Protein concentration was
measured with a DC Protein Assay Kit (Bio-Rad,
Hercules, CA) with bovine serum albumin as the
9
a
-
standard. Electrophoresis on a 7.5z non-denaturing
×
(GC-8A) equipped with a glass column (0.32
polyacrylamide slab gel (Native PAGE) at pH 8.8
was done as described by Davis.22) SDS-PAGE was
done as described by Laemmli23) in the presence or
100 cm) packed with 10 LAC 4R-886 on Chro-
z
mosorb WAW (100–120 mesh; GL Science) at 200
with N2 gas as a carrier.
9
C
absence of b-mercaptoethanol. For two-dimensional
(2D)-PAGE, an Immobiline Dry Strip 4–7 (Amer-
sham Bioscience) was used to resolve proteins in the
Subsite analysis. Maltose, maltotriose, malto-
tetraose, maltopentaose, maltohexaose, and malto-
ˆrst dimension, and SDS-12.5z-PAGE was done in
heptaose (
À
96
z
purity) were kindly provided by
the second dimension. The proteins in the gels were
stained by 2D-Silver Stain-II ``Daiichi'' (Daiichi
Kagaku, Tokyo, Japan) according to the manufac-
turer's instructions.
Hayashibara Biochemical Laboratories and used as
substrates for measurement of the kinetic parameters
for their hydrolysis. The Michaelis constant,
maximum velocity, , and the molecular activity, k0
V e0), were calculated from Lineweaver-Burk plots
Km, the
V
(
Amino-terminal peptide sequencing. The amino-
terminal sequence of the enzyme that was separated
by SDS-PAGE and transfered to a polyvinylidene
‰uoride membrane was analyzed by Edman degrada-
tion on a protein sequencer (Model 492; Applied
Biosystems, Tokyo, Japan).
W
and the molecular concentration of the enzyme, e0
.
The subsite a‹nities, Ai (i, subsite number), and the
intrinsic rate constant,
k
int, were evaluated from the
21)
parameters by the theory of Hiromi et al
.
Measurement of physical properties. Glucose and
-EG generated in the following reactions were meas-
a
Results
ured as described above.
(i) pH stability. Enzyme solutions (8.2
m
g ml) in
Puriˆcation of enzyme
W
a Britton-Robinson buŠer (5 m
M
citrate, 5 m
diethylbarbituric acid),
which has a wide pH range, were adjusted to various
pH and kept at 4 C for 24 hr. Two volumes each of
1.0 sodium acetate buŠer (pH 5.0) and 1 (w v)
maltose solution were added, and the mixture was
incubated at 37 C for 15 min.
(ii) Heat stability. After 20
M
In a preliminary study, the PNPG hydrolytic activ-
ity reached its maximum at 7 d in a 9-d cultivation of
KH2PO4, 5 m
M
H3BO3, 5 m
M
M. alliacea on a medium containing 2
z soluble
9
starch. The activity was puriˆed from the culture su-
pernatant through ethanol precipitation and four-
step column chromatography at 33-fold with a yield
M
z
W
9
z
of 2.3 , and the speciˆc activity of the puriˆed en-
m
l of the enzyme solu-
g) was kept at various temperatures for
l each of 0.1 sodium acetate buŠer (pH
z (w v) maltose were added, and the mix-
zyme was 3.2 U mg (Table 1). The gel ˆltration chro-
W
tion (0.82
15 min, 40
m
matography demonstrated that the puriˆed enzyme
showed a single peak with a substantial PNPG-
hydrolytic activity (Fig. 1(A)).
m
M
5.0) and 1
ture was incubated at 37
(iii) EŠects of pH on maltose hydrolytic and
forming activities. For maltose hydrolytic reactions,
W
9
C for 15 min.
a
-EG
Anomeric conˆguration of hydrolyzed product
The puriˆed enzyme released glucose, but not mal-

2418
Y. TANAKA et al.
Table 1. Summary of Puriˆcation of
a
-Glucosidase from M. alliacea YN-15
Volume
(ml)
Protein
(mg)
PNPG hydrolytic
activity (U)
Speciˆc activity
Yield
Puriˆcation
(fold)
Puriˆcation step
(U mg)
(z)
W
Crude extract
5600
1000
120
24
15
21
1200
140
18
120
29
10
7.0
6.4
2.8
0.10
0.21
0.56
1.2
1.5
3.2
100
24
8.3
5.8
5.3
2.3
1.0
2.1
5.5
12
15
33
Ethanol fractionation
Phenyl Sepharose
1st Resource S
2nd Resource S
Superdex 200
5.7
4.3
0.88
Fig. 1. Puriˆcation of
(A) The PNPG hydrolytic activity eluted from the 2nd Resource S column was put on a Superdex 200 column (2.6
ed with 0.05
mass was estimated by gel ˆltration on a Superdex 200 PC 3.2 30 column (Amersham Bioscience) equilibrated with 0.05
a-Glucosidase from M. alliacea YN-15.
×
60 cm) equilibrat-
M
phosphate-buŠered saline. Fractions containing high activity (elution volume 206–227 ml) were collected. (B) The relative
sodium phos-
V0), where V0 is the void volume of the column, VR
M
W
phate buŠer, pH 7.0, containing 0.5
M NaCl. The Kav was deˆned as (VR„V0) (VC„
W
is the elution volume of the protein, and VC is the geometric bed volume. Calibration standards used were as follows: 1, ribonuclease A
(13.7 kDa); 2, chymotrypsinogen A (25 kDa); 3, ovalbumin (43 kDa); 4, bovine serum albumin (67 kDa); 5, aldolase (158 kDa); 6, cata-
lase (232 kDa); and 7, ferritin (440 kDa). M, a-glucosidase from M. alliacea YN-15 (estimated at 92 kDa).
tooligosaccharides, as the sole product during diges-
tion of soluble starch (not shown), suggesting that
ous substrates is summarized in Table 2. Homogene-
ous substrates such as maltose and soluble starch
were acted on more than heterogeneous ones such as
sucrose and PNPG. Among the former substrates,
the enzyme acted preferentially on the glucosyl link-
the action pattern of the enzyme is of the exo-type. a-
Glucosidase and glucoamylase are typical exo-type
carbohydrases and distinguished in the stereochemi-
cal course of the hydrolysis reaction that occurs with
retention and inversion, respectively, of the anomeric
conˆguration.24) Accordingly, the change in the con-
ˆguration at the anomeric carbon of released glucose
was examined in the initial reaction of the puriˆed
age with an anomeric conˆguration of
tose, starch, amylose, amylopectin, and glycogen),
-1,3 (nigerose), and -1,2 (kojibiose) to some extent
but scarcely on -1,6 (isomaltose, pullulan, and dex-
a-1,4 (mal-
a
a
a
tran). Above all, a higher activity for hydrolysis was
obtained on soluble starch rather than maltose. This
enzyme to PNPM. As shown in Fig. 2,
quickly increased with a minor level of
a
b
-glucose
-glucose
property is unique among fungal
a-glucosidases,
unavoidably generated through
a
spontaneous
most of which have a tendency to hydrolyze mal-
tooligosaccharides more rapidly than polysaccha-
rides.7)
mutarotation of some of the liberated
a
-glucose.19)
Therefore, the puriˆed enzyme could be deˆned as
glucosidase.
a-
Although fungal glucoamylases also hydrolyze
polysaccharides preferentially to maltooligosaccha-
rides, these enzymes are rationally not capable of
catalyzing transglucosylation reactions.6) To test this
Substrate speciˆcity
The hydrolytic activity of the enzyme toward vari-

Novel
a
-Glucosidase of Mortierella alliacea
Table 2. Substrate Speciˆcity of M. alliacea
2419
-Glucosidase
a
Hydrolysisa,b
a
-EG formationb
(mg -EG mg
protein min)
(
m
mol of glucose from
Substate
a
W
non-reducing end mg
W
W
protein min)
W
Maltose (
a
-1,4)
-1,1)
-1,2)
-1,3)
Isomaltose ( -1,6)
-2)
29.8
0.3
7.0
26.1
1.0
0.9
5.3
3.4
0.3
42.9
8.3
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
1.7
0.1
0.3
0.7
0.1
0.1
0.1
0.2
0.1
5.2
0.4
1.9
2.9
0.1
0.1
0.91
±
0.05
Trehalose (
Kojibiose (
Nigerose (
a, a
NDc
a
0.16
0.28
±
±
0.03
a
0.04
a
NDc
Sucrose (
Maltitol
PNPG
a
-1,
b
NDc
0.06
0.02
±
±
—
±
—
—
±
0.01
0.01
a
-EG
Soluble starch
1.19
0.08
Amylose (DP
Amylopectin
Glycogen
Pullulan
Dextran
＝
18)
34.3
30.2
0.9
1.56
0.06
NDc
NDc
Fig. 2. Changes with Time in
through PNPM Hydrolysis by M. alliacea
The amount of glucose relative to arabitol was calculated
from their peak areas on the chromatogram. Closed circles
glucose; open circles -glucose.
a
-
and
b
-Glucose Produced
0.2
a
-Glucosidase.
a
Glucose from reducing terminal of substrate was not taken into ac-
count.
Values are from at least three independent experiments and represented
, a-
,
b
b
c
as the average
Not detected (less than 0.0005 mg
±
SE.
a
-EG mg-protein min).
W W
Table 3. Rate Parameters of
a-Glucosidase from M. alliacea
YN-15a
Km
(m
k0
k0 Km
W
„1 „1
b
Substrate
Vmax
(s„1
)
(m
M
s
)
・
M
)
Maltose
Maltotriose
Maltotetraose
Maltopentaose
Maltohexaose
Maltoheptaose
3.5
2.7
2.4
2.8
2.9
4.9
6.88
6.66
6.68
5.56
6.19
6.70
58.6
56.8
56.9
47.3
52.7
57.1
16.6
21.4
23.9
16.7
18.3
11.8
a
Reaction, 37
9
C, pH 5.0; Molecular mass, 92,000.
b
Glucose (mg) liberated from the non-reducing terminal of substrate
W
mg-protein min.
W
speciˆcity of this enzyme, the rate parameters for the
hydrolysis of maltooligosaccharides with various
chain lengths were measured. The molecular activity
Fig. 3. Subsite A‹nity of M. alliacea a-Glucosidase.
An intrinsic rate constant (kint) of 65 s„1 was found. The ar-
(
k
0) was independent of the polymerization degree of
the substrate (Table 3). This property is essentially
similar to those of other -glucosidases and clearly
distinct from any reported glucoamylases.6) On the
other hand, log (1 m) values, which can be regarded
row indicates the position of the catalytic site.
a
on the
a
-glucosidase from M. alliacea, a series of
K
W
hydrolysis reactions on the various substrates was
done in the presence of ethanol. Our enzyme could
use glycogen and soluble starch rather than maltose
to eŠectively transfer a glycosyl residue to ethanol
(Table 2). The substrate speciˆcity on the trans-
as the a‹nity of the enzyme for each substrate, were
at a maximum at maltotetraose ( 4) and decreased
n
＝
with an increase of the polymerization degree of the
substrate. Thus, a measure of the eŠectiveness of the
enzymic reaction, log (k0
Km), was the highest for
W
glucosylation reaction indicated that the puriˆed
a
-
this substrate.
glucosidase is responsible at least for eŠective hydrol-
ysis of soluble starch and glycogen, demonstrating a
Based on these rate parameters, a subsite analysis
was done by the method of Hiromi et al
.
21) As shown
in Fig. 3, the second subsite (A2) had the largest a‹n-
novel property for a fungal
a
-glucosidase.
ity (4.91 kcal mol; the same value was obtained by
W
Hydrolysis rate parameters and subsite structure
To further clarify the diŠerence in substrate
methods I and II21)), and the a‹nity of the ˆrst sub-
site (A1; 0.98 kcal mol) was greater than that of the
W

2420
Y. TANAKA et al.
Table 4. Comparison between Observed and Calculated Rate Parameters for M. alliacea
a
-Glucosidase
„1
Km (m
M
)
k0 (s„1
)
k0 Km (m
M
・
s„1
)
W
Substrate
na
Calcd.b
O Cc
Calcd.b
O Cc
Calcd.b
O Cc
W
W
W
Maltose
2
3
4
5
6
3.1
2.5
2.4
3.2
3.1
1.13
1.08
1.00
0.88
0.94
51.5
52.9
56.9
53.1
57.5
1.14
1.07
1.00
0.89
0.92
16.6
21.2
23.7
16.6
18.5
1.00
1.01
1.01
1.01
0.99
Maltotriose
Maltotetraose
Maltopentaose
Maltohexaose
a
Degree of polymerization of substrate in glucose units.
Calculated by using the values of Ai and kint obtained by method of Hiromi et al
The ratio of observed values in Table 3 to calculated ones.
b
c
21)
.
Fig. 4. Native PAGE (A), SDS-PAGE (B), and 2D-PAGE Analyses (C) of
Native PAGE was done on a 7.5 polyacrylamide gel at pH 8.8. Lanes 1 and 2 in panel B indicate the absence and presence of
captoethanol in the sample buŠer, respectively. Amounts of analyzed protein were 0.42 g (A and B) and 2.4 g (C). Molecular mass
(94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and trypsin inhibitor
a-Glucosidase from M. alliacea YN-15.
z
b-mer-
m
m
markers: phosphorylase
(20.1 kDa).
b
b
third one (
A
3; 0.16 kcal mol). When the rate
Physical properties
W
parameters, Km and
k
0, were calculated from the Ai
The molecular mass of the enzyme was estimated
at about 92 kDa on gel chromatography with the
and kint values, there seemed to be good agreement
between the calculated values and the experimental
ones (in Table 3) with a maximum deviation of about
running buŠer containing 0.5
M
NaCl (Fig. 1(B)).
Interestingly, the enzyme that gave a single band on
the native PAGE (Fig. 4(A)) had been separated into
two components of 61 kDa and 31 kDa on the SDS-
14
(Table 4). The subsite pattern (
to those of other fungal and plant
but was the reverse of glucoamylases of which A3 is
z
, indicating the validity of the assessment
1À 3) was similar
-glucosidases6)
A
A
a
PAGE, independent of the reduction by b-mercap-
toethanol (Fig. 4(B)). The two bands were also
resolved on the 2D-PAGE at the column of pI 6.3
(Fig. 4(C)). The amino-terminal peptide sequences of
the 61- and 31-kDa subunits were DDMXSNV-
QTRKDXGYL and ASTSIKYSINNAGRQAPL,
respectively, which had little similarity to each other
and to the sequences of any other proteins presented
21)
greater than A1
a‹nities at position 2 or more of glucoamylases have
been positive numbers,21) but those of
-glucosidases
.
Furthermore, the reported subsite
a
including ours were not necessarily so, re‰ecting the
diŠerence in mode of the enzyme-substrate complex.
These results do not contradict the identiˆcation of
our enzyme as an
a
-glucosidase.
in databases. These results indicate that the a-
glucosidase from M. alliacea is composed of heter-
odimers that are not covalently linked but stably ag-

Novel
a
-Glucosidase of Mortierella alliacea
2421
Fig. 5. Physical Properties of M. alliacea
(A) EŠects of pH on maltose hydrolytic and
mol glucose mg-protein min and 1.2 mg of a-EG mg-protein min, respectively. Closed circles, maltose hydrolytic activity; open
W W
a
-Glucosidase.
a
-EG forming activities. Maltose hydrolytic and
a-EG forming activities at pH 5.0 were
93
circles
-EG forming activity at 45
activity at pH 7.0 was 73
protein min.
m
W
W
,
a
-EG forming activity. (B) EŠects of temperature. Maltose hydrolytic activity at 55
C was 0.67 mg of -EG mg-protein min. Symbols are the same as in panel
mol glucose mg-protein min. (D) Heat stability. Hydrolytic activity at 40
9C was 153 mmol glucose mg-protein min.
W W
a
9
a
A
. (C) pH stability. Hydrolytic
C was 57 mol glucose mg-
W
W
W
m
9
m
W
W
W
gregated to each other with a salt-resistant nature.
The optimum pH and temperature were the pH
glucosidase from Mu. javanicus on soluble starch
was less than half of that on maltose.10) In this
range of 5.0–6.0 and 55
hydrolysis, and pH 5.0 and the range of 45–50
transglucosylation forming -EG (Figs. 5(A) and
9
C, respectively, for maltose
regard, the M. alliacea
polysaccharides better
(Table 2), could be extraordinary. Primarily,
a
-glucosidase, which degrades
9
C for
than oligosaccharides
a
a-
5(B)). The enzyme stability at various pH or tempera-
ture was measured by its maltose hydrolytic activity.
The enzyme was stable after a 24-h incubation in
buŠer with pH from 4.0 to 10.0 (Fig. 5(C)). As for
glucosidase and glucoamylase, as the typical exo-type
carbohydrate hydrolases, are distinguished from
each other by the anomeric conˆguration of their
products:
a
-glucosidase gives
a-glucose and glu-
temperature, retention of the enzyme at 50
9
C for 15
coamylase provides b
-glucose.19,24) The study on the
min had practically no eŠect on the activity, while
conˆguration of the anomeric carbon on the hydro-
lyzed product conclusively demonstrated that the
puriˆed enzyme was the former one (Fig. 2). The
high speciˆcity of the enzyme for soluble starch and
glycogen was also observed in the transglucosylation
reaction in the presence of ethanol (Table 2). This
could further rule out the possibility that the enzyme
is a glucoamylase that lacks transglucosylation activi-
ty.19) Moreover, the independence of the molecular
heating at 60
(Fig. 5(D)).
9 z
C resulted in an ¿80 reduction
Discussion
Glucoamylases from the fungi A. niger, A. oryzae,
Penicillium oxalicum, and Mu. rouxianus can hydro-
lyze soluble starch much faster than maltose at the in-
itial velocity ratio of 4–7:1.15–18) In contrast, fungal
a
-
activity (
tooligosaccharide substrates supported the identiˆca-
tion of the enzyme as -glucosidase (Table 3).
k0) on the polymerization degree of the mal-
glucosidases show lower activity toward soluble
starch. For example, the hydrolytic activity of
a
-
a

2422
Y. TANAKA et al.
Indeed, the k0 values for glucoamylases show strong
dependence on the saccharide chain length.21,25)
Therefore, it could be concluded that the M. alliacea
the post-translational modiˆcation remains un-
clear.29,30) We have obtained preliminary data indicat-
ing that the two subunits of the M. alliacea enzyme
are encoded by one gene. It is therefore interesting to
examine whether or not an unprocessed version of
the enzyme, over-expressed in Escherichia coli for in-
stance, retains the substrate binding and catalytic ac-
tivities, and to elucidate the meaning of the polypep-
tide processing.
enzyme is a member of a novel fungal
group.
a-glucosidase
a
-Glucosidases from various origins have been
classiˆed into two groups in terms of their conserved
amino acid sequences and substrate speciˆcities.6)
The ˆrst group contains bacterial and insect enzymes
that have a high hydrolytic activity toward heter-
ogeneous substrates. On the other hand, plant and
fungal enzymes included in the second group hydro-
lyze homogeneous substrates such as maltooligosac-
charides, soluble starch, and glycogen more rapidly.
However, for the latter group, the enzymes acting on
oligosaccharides and the ones on polysaccharides
should be discriminated based on the ˆne structure of
The original aim of our study was to investigate a
mechanism underlying the partial starch degradation
as a possible limiting step in arachidonic acid fermen-
tation by the strain YN-15. Since no activity of the
endo-type enzyme could be detected in the culture
broth when soluble starch was used as a carbon
source, it is feasible that the a-glucosidase of interest
plays an indispensable role in assimilation of the
starch medium. As far as debranching activity is ab-
sent in the culture, it is naturally expected that the
limit dextrin is accumulated in accordance with
depletion of the non-reducing terminal of the sub-
the enzyme and or other criteria. Our current data
W
could not explain the diŠerence between M. alliacea
and plant a-glucosidases, both of which showed high
speciˆcities for polysaccharides. For instance, on ki-
netic parameters, the m of our enzyme (Table 3) and
that of barley seed
-glucosidase26) on oligosaccha-
rides (DP 4) increased with an increase of the poly-
merization degree, but those of -glucosidases from
K
strate accessible by a-glucosidase. This idea is sup-
a
ported at least in part by the fact that the puriˆed
enzyme did not eŠectively attack the glucosyl linkage
Æ
a
of
of polysaccharides. Treatment of starch with
-amylase debranching enzymes (e.g. pullulanase) or
a-1,6 (Table 2), comprising the branched structure
sugar beet and rice decreased.26) Since amylopectin
was preferentially hydrolyzed by the M. alliacea en-
zyme rather than amylose (Table 2), the branched
structure of the substrate might be responsible for
rapid hydrolysis, and, therefore, the oligosaccharides
with a straight chain structure, especially of a high
polymerization degree, were not eŠectively hydro-
lyzed. The elucidation of the higher order structure
of our enzyme and its relation to the substrate recog-
nition will be necessary to illustrate in depth the
diŠerences from other enzymes.
a
W
introduction of isoamylase genes may be pragmatic
as a countermeasure.
Finally, the M. alliacea a-glucosidase with a strong
activity degrading polysaccharides might be useful
for other industrial applications. The transglucosyla-
tion reaction in the presence of ethanol as an accep-
tor (Table 2) resulted in the formation of a-EG, a
sweetening and ‰avoring agent that has attracted
interest owing to its skin barrier eŠect31) and noncar-
The M. alliacea
a
-glucosidase was expressed by
iogenicity.32) The
a-EG has been produced on an in-
arachidonic acid fermentation in a starch medium
and puriˆed from the culture supernatant. We do not
believe that this enzyme was leaked from cells by the
disruption of mycelia or by release from cell mem-
dustrial scale by a transglucosylation reaction using
maltose as a glucosyl donor.33) In case that glycogen
or soluble starch is used as a raw material, ordinary
a
-glucosidase must supply maltose with the help of
isoamylase and -amylase or -amylase. The im-
provement of debranching activity on the -glucosi-
branes during the cultivation and or puriˆcation
b
a
W
process because the cell extracts (with 4
M
urea) did
a
not contain signiˆcant levels of corresponding activi-
ty (data not shown). This is in contrast to that of the
dase reported in this study could provide an advan-
tage in the development of a one-step reaction system
for mass production of a-EG from polysaccharides.
a
-glucosidases from other Zygomycetes fungi includ-
ing Mu. javanicus27) and Mu. racemosus 28) which are
,
cell-bound types that could be recovered by the urea
treatment. Moreover, these Mucor enzymes consist
of single polypeptides. This characteristic was also
distinct from ours, which is composed of two subu-
nits of 61 kDa and 31 kDa (Fig. 4). In Ascomycetes
Acknowledgments
We thank Dr. S. Chiba for his helpful discussion
on subsite analysis.
fungi, such heterodimeric types of
a-glucosidases
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