Purification and characterization of a new type of alpha-glucosidase from Paecilomyces lilacinus that has transglucosylation activity to produce alpha-1,3- and alpha-1,2-linked oligosaccharides.

Article abstract of DOI:10.1271/bbb.67.29

A fungus producing an alpha-glucosidase that synthesizes alpha-1,3- and alpha-1,2-linked glucooligosaccharides by transglucosylation was isolated and identified as Paecilomyces lilacinus. The cell-bound enzyme responsible for the synthesis was extracted by suspension of mycelia with 0.1 M phosphate buffer (pH 8.0), and the extract was purified. The molecular weight and the isoelectric point were estimated to be 54,000 and 9.1, respectively. The enzyme was most active at pH 5.0 and 65 degres C. The enzyme hydrolyzed maltose, nigerose, and kojibiose. The enzyme also hydrolyzed soluble starch and amylose with the rate toward maltose. p-Nitro-phenyl alpha-glucoside and isomaltose were not good substrates. The enzyme had high transglucosylation activity to synthesize oligosaccharides containing alpha-1,3- and alpha-1,2-linkages. At an early stage of the reaction, considerable maltotriose, 4-O-alpha-nigerosyl-D-glucose, and 4-O-alpha-kojibiosyl-D-glucose were synthesized. Afterwards, nigerose and kojibiose were accumulated gradually with glucose as an acceptor.

Full text of DOI:10.1271/bbb.67.29

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Purification and Characterization of a New Type of
α-Glucosidase from Paecilomyces lilacinus That Has
Transglucosylation Activity to Produce α-1,3- and
α-1,2-Linked…
a
a
b
c
Isao KOBAYASHI , Masahiro TOKUDA , Hiroyuki HASHIMOTO , Takashi KONDA , Hirofumi
d
d
NAKANO & Sumio KITAHATA
a
Honen Corporation 1-2-3 Ohtemachi, Chiyoda-ku, Tokyo 100-8150, Japan
b
Faculty of Agriculture, Shinshu University 8304 Minamiminowa, Nagano 399-4598,
Japan
c
Faculty of Textile Science, Kyoto Institute of Technology Matsugasaki, Sakyo-ku, Kyoto
6
06-8585, Japan
d
Osaka Municipal Technical Research Institute 6-50 Morinomiya 1-chome, Joto-ku, Osaka
36-8553, Japan
5
Published online: 22 May 2014.
To cite this article: Isao KOBAYASHI, Masahiro TOKUDA, Hiroyuki HASHIMOTO, Takashi KONDA, Hirofumi NAKANO & Sumio
KITAHATA (2003) Purification and Characterization of a New Type of α-Glucosidase from Paecilomyces lilacinus That Has
Transglucosylation Activity to Produce α-1,3- and α-1,2-Linked…, Bioscience, Biotechnology, and Biochemistry, 67:1,
29-35, DOI: 10.1271/bbb.67.29
To link to this article: http://dx.doi.org/10.1271/bbb.67.29
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Biosci. Biotechnol. Biochem., 67 (1), 29–35, 2003
Puriˆcation and Characterization of a New Type of a-Glucosidase
from Paecilomyces lilacinus That Has Transglucosylation Activity
to Produce
a
-1,3- and
a
-1,2-Linked Oligosaccharides
Isao KOBAYASHI,1, Masahiro TOKUDA, Hiroyuki HASHIMOTO,2 Takashi KONDA,3
†
1
4
4
Hirofumi NAKANO, and Sumio KITAHATA
1
Honen Corporation, 1-2-3 Ohtemachi, Chiyoda-ku, Tokyo 100-8150, Japan
2
3
4
Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4598, Japan
Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
Osaka Municipal Technical Research Institute, 6-50 Morinomiya 1-chome, Joto-ku, Osaka 536-8553, Japan
Received April 26, 2002; Accepted August 26, 2002
2
)
A fungus producing an
a
-glucosidase that synthesizes
totriose from maltose. There are few studies of the
-glucosidases that synthesize oligosaccharides con-
taining -1,2-, or -1,3-glucosidic linkages (Fig. 1).
Chiba et al reported that the buckwheat enzyme
produces nigerose ( -1,3-linked glucobiose) and
a
-1,3- and -1,2-linked glucooligosaccharides by
a
a
transglucosylation was isolated and identiˆed as
Paecilomyces lilacinus. The cell-bound enzyme respon-
sible for the synthesis was extracted by suspension of
a
a
3
)
.
a
mycelia with 0.1
M
phosphate buŠer (pH 8.0), and the
extract was puriˆed. The molecular weight and the
isoelectric point were estimated to be 54,000 and 9.1,
respectively. The enzyme was most active at pH 5.0 and
6
5
9
C. The enzyme hydrolyzed maltose, nigerose, and
kojibiose. The enzyme also hydrolyzed soluble starch
and amylose with the rate toward maltose. -Nitro-
phenyl -glucoside and isomaltose were not good sub-
strates. The enzyme had high transglucosylation activity
to synthesize oligosaccharides containing -1,3- and
,2-linkages. At an early stage of the reaction, consider-
able maltotriose, 4- -nigerosyl- -glucose, and 4-
-kojibiosyl- -glucose were synthesized. Afterwards,
p
a
a
a-
1
O
-
a
D
O-
a
D
nigerose and kojibiose were accumulated gradually with
glucose as an acceptor.
Key words:
a
-glucosidase; Paecilomyces lilacinus
kojibiose; nigerose; transglucosylation
;
a
-Glucosidases (EC 3.2.1.20,
hydrolase) are typical exo-type carbohydrolases,
which liberate -glucose from the nonreducing termi-
nals of substrates such as maltose, isomaltose, and
phenyl -glucoside. The enzymes transfer glucosyl
a- -glucoside gluco-
D
a
a
groups from the substrates onto acceptors, but the
proˆles of the products depend on the enzymes. An
enzyme from Aspergillus niger, for instance, yields
mostly
a-1,6-linked oligosaccharides such as isomal-
Fig. 1. Oligosaccharides Containing
a
-1,3- and
a
-1,2 Glucosidic
-glucose, and
tose, isomaltotriose, and panose when glucose or
Linkages.
1
)
maltose are the acceptors. A Penicillium purpuroge-
num enzyme synthesizes mostly
(
a) Kojibiose, (b) nigerose, (c) 4-
O
-
a
-nigerosyl-D
a
-1,4-linked mal-
(d) 4- -kojibiosyl- -glucose.
O
-
a
D
†
To whom correspondence should be addressed. Fax: +81-45-852-6357; Tel: +81-45-852-4014; E-mail: ikobayashi
-aminobenzoic acid ethyl ester; IEF, isoelectric focusing
＠
honen.co.jp
Abbreviations: ABEE,
p

3
0
I. KOBAYASHI et al.
1
1)
kojibiose (
with soluble starch. An enzyme from an Acremoni-
um sp. was found to synthesize nigerose and 4-
a
-1,2-linked glucobiose) during reaction
previous paper. 4-
O
-
a
-Kojibiosyl- -glucose was the
D
gift of the Hayashibara Biochemical Laboratories,
Inc. Glucoamylase (Glutase 6000) was kindly sup-
plied by Hankyu Bioindustry Co., Ltd. (Osaka).
Potato dextrose was purchased from (Nissui Phar-
maceutical Co., Ltd., Tokyo, Japan). All other
chemicals were of the highest grade commercially
available.
O
-
4
)
a
-nigerosyl-
D
-glucose from maltose. These two
-1,3-glucosidic linkages
oligosaccharides containing
a
have several practical uses: they make the salty taste
of foodstuŠs more mellow, and prevent anthocya-
nins, color substances in food materials, from
5
)
oxidating or discoloring. Immunopotentiating the
T-helper 1-like immune response may be their phys-
Screening of microorganisms. Microorganisms
were isolated from soil samples in agar medium
6
,7)
iological activity. Two oligosaccharides containing
an -1,2-glucosidic linkages, kojibiose and 4-
kojibiosyl- -glucose, synthesized by kojibiose phos-
a
O
-
a
-
plates containing 0.2
0.05 HPO , 0.01
1.5 agar (pH 7.0). The isolates were cultivated aer-
obically at 30 C for 72 h in a liquid medium (10 ml)
containing 1 maltose, 1 polypepton, 0.05
HPO , 0.01 KCl, and 0.01 MgSO (pH 7.0).
The culture broth (50 l) was incubated with 50 l of
20 maltose containing 0.01 NaN at 40 C for
z
maltose, 0.05
z
MgSO
NH
4
NO
3
,
D
z
z
K
2
4
z
KCl, 0.01
z
4
, and
8
)
9)
phorylase, have low cariogenicity and digestibil-
1
0)
ity. In view of the practical usefulness of these rare
oligosaccharides and of the few reports on the
glucosidases that synthesize them, we started screen-
ing of microbes for the -glucosidase. The most
9
z
z
a
-
z
z
K
2
4
z
4
a
m
m
9
di‹cult problem was the separation and identiˆca-
tion of the oligosaccharides with diŠerent linkages in
the many samples obtained by the screening. To solve
the problem, we used a method described in our
z
z
3
72 h. After the mixtuere was boiled, the transglucosy-
lation products were checked by TLC and the ABEE
conversion as described below.
1
1)
preceding paper: the saccharides were reacted with
-aminobenzoic acid ethyl ester (ABEE), and the
p
Cultivation of Paecilomyces lilacinus. The selected
fungal isolate, Paecilomyces sp., was used to inocu-
late 5 ml of potato dextrose agar in a slant containing
potato extract, 2
and incubated at 30
derivatives were then analyzed by HPLC. Suitable
HPLC conditions were found for identifying a num-
ber of
containing
a
-linked glucooligosaccharides including ones
0.4
z
z glucose, and 1.5z agar,
1
1)
a
-1,2- and 1,3-glucosidic linkages. The
9
C for 2 days, by which time
method (termed ``ABEE conversion'' in this study)
allowed the simultaneous analysis of complex mix-
tures of reducing saccharides even in microbial cul-
ture broth or enzyme reaction mixtures. We used this
method to ˆnd microorganisms that produces
mycelia had grown. Sterilized water (15 ml) was
added to the slant and the test tube was vortexed
vigorously. A 0.5 ml portion of the suspension of
mycelia was withdrawn and added to 150 ml of a
medium containing 5
z
maltose, 1
z
peptone, 0.05
z
oligosaccharides with
ages. A fungus identiˆed later as Paecilomyces lilaci-
nus was obtained as a producer of an -glucosidase
that synthesized much oligosaccharides containing
,3-glucosidic linkage from maltose by a transfer
reaction at an early stage. In addition, the enzyme ac-
cumulated oligosaccharides containing -1,2-gluco-
a
-1,2- or
a
-1,3-glucosidic link-
K
2
HPO , 0.01 KCl, and 0.01
4
z
z
MgSO , adjusted
4
to pH 7.0, in 500 ml shaking ‰asks. After cultivation
for 3 days with reciprocating shaking at 80 rpm and
a
a
-
35 9C , the mycelia were harvested by being ˆltering
1
with Toyo Roshi ˆlter paper No. 2. About 500 g (wet
weight) of mycelia was obtained from 10-liter cul-
tures.
a
sidic linkage during the prolonged reaction. This
paper describes the puriˆcation and some properties
Analytical methods. TLC was done with a TLC
plate (Kiesel Gel 60, Merck, Darmstadt, Germany)
and a solvent system of ethyl acetate-acetic acid-
of this new a-glucosidase.
Materials and Methods
water (3:1:1, v v). The spot of sugars was detected by
W
spraying of the plate with 50
methanol and its being heated at 150
version was done by the method of Yasuno et al.
z
(w w) sulfuric acid in
W
Materials. Glucose, maltose, trehalose,
p
-nitro-
9C . ABEE con-
12)
phenyl
a
-glucoside, and soluble starch were pur-
chased from Nacalai Tesque Chemical, Inc. (Kyoto,
Japan). Kojibiose, nigerose, isomaltose, maltotriose,
maltotetraose, maltohexaose and glycogen (from
oyster) were obtained from Wako Pure Chemical
Ind., Ltd. (Osaka, Japan). Amylose, (Hayashibara
Biochemical Laboratories, Inc., Okayama, Japan),
with an ABEE labeling kit (Seikagaku Corporation,
Tokyo). ABEE derivatives were analyzed by HPLC
under conditions optimized for analysis of
a-linked
glucooligosaccharides as described in our previous
1
1)
paper with the temperature at 30
25 C. Native PAGE was done with an 7.5
mide gel in -alanine-acetic acid buŠer (pH 4.5) as
described by Reisfeld et al
9
C instead of
9
z
acryla-
maltopentaose and
Reˆning Co., Ltd., Yokohama, Japan) were ob-
tained from commercial sources. 4- -Nigerosyl-
glucose was prepared by a method described in a
a
-cyclodextrin (Ensuiko Sugar
b
1
3)
.
SDS-PAGE was done in
O
-
a
D
-
7.5
0.1
z
z
(w v) polyacrylamide gel in the presence of
W
1
4)
SDS as described by Laemmli. Protein was

Puriˆcation of Paecilomyces lilacinus
a-Glucosidase
31
stained with Quick-CBB (Wako). Deglycosylation
was done by the incubation of -glycanase
Glycopeptidase F, Takara Shuzo Co., Ltd., Kyoto)
with the heat-denatured enzyme with 0.5 SDS at
C for 24 h.
The isoelectric point of the enzyme was estimated
by isoelectric focusing (IEF) on a Phastgel IEF 3-9
with the Phastsystem (Amersham-Pharmacia Biotech
K.K., Tokyo).
(500 g, wet weight) was solubilized by suspension of
mycelia in 400 ml of 0.1 phosphate buŠer, pH 8.0,
at 5 C for 24 h with gentle stirring. The suspension
was centrifuged at 20,200 and the supernatant
was collected. This procedure repeated two more
times and the extracts were combined. The solution
N
M
(
9
z
×
g
4
0
9
was dialyzed against 50 m
(pH 5.5).
M
sodium acetate buŠer
Step 2: SP-Toyopearl column chromatography.
The dialyzed enzyme solution (1,570 ml) was put on
Enzyme assay.
at 50 C in a reaction mixture (2.5 ml) containing
maltose and 50 m sodium acetate buŠer (pH
a
-Glucosidase activity was assayed
9
an SP-Toyopearl 550M column (3
×
25 cm, Tosoh
acetate buŠer
2
5
5
0 m
M
M
corporation) equilibrated with 50 m
M
.5). The optimum pH of the enzyme was at about
.0 in this experiment but the enzyme assay was done
(pH 5.5). The enzyme was eluted with a linear
gradient of NaCl concentrations from 0 to 0.75
(each, 600 ml). The active fractions (eluted at about
M
at 5.5 in this paper. The reaction was stopped by the
addition of 125 l of 0.2 NaOH and the sample
was neutralized with 125 l of 0.2 acetic acid. The
m
M
0.5 ) were collected (80 ml). The speciˆc activity of
M
m
M
this active fraction was 3.02 units mg protein, 62-
W
glucose liberated was measured with a Glucose B-test
Wako kit (Wako). One unit of enzyme activity was
deˆned as the amount of enzyme that hydrolyzed
fold that of the crude extract.
Step 3: Butyl-Toyopearl column chromatography.
1
m
mol of maltose (2
m
mol of glucose as product) per
Solid (NH
till a concentration of (NH
solution was put on a Butyl-Toyopearl 650M column
(1.8 25 cm, Tosoh) equilibrated with 20 m acetate
buŠer (pH 5.5) containing 1.5 (NH SO . The en-
zyme was eluted with a linear gradient of (NH SO
concentrations from 1.5 to 0 (each, 500 ml). The
) were collect-
4
)
2
SO
4
was added to the enzyme solution
minute under these conditions. Protein was measured
4
)
2
SO
4
become 1.5 . The
M
1
5)
by the method of Lowry et al
albumin as the standard.
.
with bovine serum
×
M
M
4
)
2
4
EŠects of pH and temperature. The eŠects of pH
on the activity were assayed at 50 C in a reaction
mixture containing maltose (20 m ), the enzyme
0.01 unit ml), and 100 m of Britton-Robinson
4
)
2
4
9
M
M
active fractions (eluted at about 0.75
M
(
M
ed (30 ml) and concentrated with polyethylene glycol
W
4
buŠer (pH 2.8–9.1). The pH stability of the enzyme
was analyzed with enzyme that had been incubated in
(mean molecular weight, 2
×
10 ). The speciˆc activi-
ty of this active fraction was 10.0 units mg protein.
W
Britton-Robinson buŠer (pH 1.5–10.2) at 4
9
C for
2
4 h. The eŠects of temperature were assayed at vari-
Step 4: Sephacryl S-100 column chromatography.
The concentrated sample (3.0 ml) was put on a
ous temperatures in 50 m
M
sodium acetate buŠer (pH
5
.5). The temperature stability of the enzyme was
Sephacryl S-100 column (3
Pharmacia) with 20 m
taining 0.1
×
100 cm, Amersham-
analyzed with the enzyme which had been incubated
at various temperatures for 20 min.
M
acetate buŠer (pH 5.5) con-
M
NaCl. The enzyme was eluted with the
same buŠer at a ‰ow rate of 0.2 ml min. The active
W
EŠects of various metal ions and chemicals. The
fractions (75 ml) were collected. The speciˆc activity
enzyme (100 mU ml) was incubated with various
of this active fraction was 12.2 units mg protein.
W
W
2
+
2+
2+
2+
2+
metal ions (Ca , Hg , Mg , Na , Mn , or
2
+
Zn , all of 1.0 m
M
) or EDTA (1.0 m
M
) at 40
9
C for
Estimation of molecular weight by gel ˆltration.
The enzyme solution (0.2 ml) was put on a Superdex
200 HR (Amersham-Pharmacia) column equilibrated
1
h. The mixtures were diluted 100 times with 50 m
M
acetate buŠer (pH 5.5) and the residual activity was
measured under the standard assay conditions.
with 50 m
M
acetate buŠer (pH 5.5) containing 0.1
M
NaCl, and eluted. The molecular weight of the
enzyme was estimated by comparison of the retention
time with the times for standard proteins, lysozyme
Analysis of transglucosylation products. Trans-
glucosylation products were measured in mixtures
(
2.5 ml) containing 30
z
(w v) maltose, and 50 m
M
(14,500),
chymotrypsin
(25,000),
ovalbumin
W
sodium acetate buŠer (pH 5.5) kept at 50
9
C for 24 h.
(45,000), and bovine serum albumin (67,000).
After such mixtures were boiled for 10 min, the
oligosaccharides in the reaction mixture were put
through ABEE labeling and analyzed by HPLC.
Results and Discussion
Screening and identiˆcation of the fungal isolate
that produces a-glucosidase
Of the microorganisms grown on the agar plate
containing maltose, about 500 strains were selected
Puriˆcation of
lilacinus.
Step 1: Extraction. The enzyme of the fungus
a
-glucosidase from Paecilomyces

3
2
I. KOBAYASHI et al.
on the basis of their production of transglucosylation
products from maltose. The products were further
analyzed by ABEE conversion. An isolate, HKS-124,
was found to synthesize considerable nigerose and 4-
Table 1.
lilacinus
a-Glucosidase Activity and Growth of Paecilomyces
Enzyme activity (units ml)
W
Cultivation
Wet weight of mycelia
(
days)
(mg ml)
Supernatant Cell extract*
W
O
-a
-nigerosyl-
grew well on 2
malt extract and 2
Czapek-yeast agar media (3
extract, 0.01 HPO , 1.5
concentrate, which contained NaNO
D
-glucose. The strain was a fungus and
potato dextrose, 2 malt extract
agar, pH 5.6), and 2
sucrose, 0.5 yeast
agar, 0.1 Czapek
(30 ), KCl
7H O (0.1 ),
O (0.05 ); pH
z
z
3
4
5
6
1.46
8.27
15.8
25.6
31.8
51.7
48.5
33.2
23.8
42.8
45.9
42.0
(
2
z
z
z
z
z
z
z
K
2
4
z
z
* Activity per milliliter of culture broth.
3
(
5.0
z
), MgSO
4
・
7H
2
O (5.0z
), FeSO
4
・
2
z
ZnSO
4
・
7H O (0.1
2
z
), and CuSO
4
・
H
2
z
6
.2), forming colonies at least 60 mm across during 2
Table 2. Puriˆcation of Intracellular
Paecilomyces lilacinus
a
-Glucosidase from
weeks of culture at 25 C. The color of the colonies
9
was white in the ˆrst several days and gradually
became purple about 1 week of cultivation on potato
dextrose. The fungus had typical penicillus structure,
stretching lemon-shaped conidia from slender
necking sterigmata. The strain was identiˆed as
Paecilomyces lilacinus on the basis of detailed
morphological observations and analysis of the
Speciˆc
activity
Total
Total
Puriˆ-
cation
(fold)
Yield
Procedure
protein activity
(
units mg
(z)
W
(mg)
(units)
protein)
Extraction
5830
57.9
8.32
4.85
284
175
83.0
60.1
0.0487 100
3.02
9.98
1.0
62.0
29.2 205
21.2 254
SP-Toyopearl
Butyl-Toyopearl
Sephacryl S-100
61.6
12.4
2
8S-rDNA sequence by NCIMB Japan (Shizuoka,
Japan).
Cultivation
z
more times, and 80 of the activity was recovered
Paecilomyces lilacinus HKS-124 grew well in liquid
media containing glucose, maltose, sucrose, lactose,
dextrin, or starch as carbon sources. Among the sac-
compared with the solubilization with sonication.
BuŠers of lower pHs than 5 were not eŠective for the
extraction and those of higher pHs than 9 slightly in-
activated the enzyme. Thus the extraction was done
at about pH 8. The speciˆc activity of the enzyme
preparation thus prepared was almost twice that ob-
tained by sonic disintegration of the mycelia.
charides, the addition of maltose at 5
the most -glucosidase activity (not shown). Peptone
1.0
z (w v) gave
W
a
(
z
) also was eŠective as a nitrogen source in
terms of enzyme production.
The activities in the broth supernatant and cell ex-
tract of the mycelia which was prepared by sonic dis-
ruption, are shown in Table 1. The enzyme activity in
the extract reached a maximum at the 4th day of cul-
tivation and then decreased. On the other hand, the
enzyme activity in the supernatant had risen each
time it was assayed. The enzyme seemed to leak from
the mycelia into the culture broth as cultivation con-
tinued, and the enzyme could easily be extracted
from mycelia so the enzyme seemed to be cell-bound
and was probably liberated by autolysis.
From the extract, the enzyme was puriˆed through
three steps of column chromatography and the
speciˆc activity was increased 255-fold with a yield of
21
z
(Table 2). The puriˆed enzyme migrated as a
single protein band in native PAGE (Fig. 2(A)).
However, in SDS-PAGE, the enzyme gave two major
protein bands (molecular weights, 60,000 and 54,000)
and one minor band (52,000). When the enzyme was
treated with
N-glycanase, these heterogeneous bands
disappeared and a major protein band appeared at
the molecular weight of 52,000 (Fig. 2(B)). By gel
ˆltration on a Superdex 200 HR column, the molecu-
lar weight was estimated to be 54,000. These results
indicated that the enzyme was a glycoprotein without
subunits and the heterogeneity may have arisen from
a diŠerence in molecular weights of carbohydrate
chains. The isoelectric point of the Paecilomyces en-
zyme was about 9.1 by IEF. The optimum pH and
temperature of the enzyme were about pH 5.0 and
During the cultivation of the fungus on maltose,
transglucosylation products including nigerose and 4-
O
-
a
-nigerosyl- -glucose had accumulated in the
D
medium by day 3. This results suggested that not only
the cell-free enzyme but also the mycelia could be
used for the production of oligosaccharides with a-
1
,3-glucosidic linkages.
Puriˆcation of the cell-bound
Paecilomyces lilacinus
a
-glucosidase from
65
temperature was between 3 to at least 9 and below
60 C. The enzyme retained 70 of activity at 65 C.
The enzyme was inactivated by Hg
(residual activity; 19 and 48 , respectively).
9C . The stability of the enzyme forward pH and
The enzyme bound to the mycelia of Paecilomyces
lilacinus was easily extracted by the following proce-
dure: washed mycelia (500 g) were incubated with
9
z
9
and Zn
2
+
2+
z
z
0
.1
M
phosphate buŠer (pH 8.0) at 5
9
C for 24 h with
EDTA and metal ions tested had neither inhibition
nor activation eŠects.
occasional mixing. This procedure was repeated two

Puriˆcation of Paecilomyces lilacinus
a-Glucosidase
33
Table 3. Substrate Speciˆcity of Paecilomyces lilacinus
Glucosidase
a-
1
Relative2
K
m
M
V
max
min)
W
Substrate
activity
(
m
)
(m
M
(
z)
Maltose
100
0.163
n.d.
n.d.
n.d.
51.3
Maltotriose
Maltotetraose
Maltopentaose
Maltohexaose
Kojibiose
Nigerose
Isomaltose
Trehalose
26.1
11.3
30.1
46.5
85.4
183
0.9
0
0
0.35
69.9
80.5
9.60
1.44
0.14
0.86
n.d.
n.d.
n.d.
56.2
54.6
0.4
Sucrose
p
-Nitrophenyl
a
-glucoside
4
5
Soluble starch
Amylose
Glycogen
2.8
813
n.d.
n.d.
1
Enzyme activity was assayed at pH 5.5 but the optimum pH of the en-
zyme was at about 5.0.
2
The enzyme (0.01 U) was incubated with an oligosaccharide (10 m
M
),
Fig. 2. Native and SDS-PAGE of the Paecilomyces lilacinus a-
Glucosidase.
starch, amylose, glycogen (0.4
9
side (1 m ) at 50 C for 10 min. The liberated glucose was
z), and p-nitrophenyl a-gluco-
M
(
A) Native PAGE of the puriˆed enzyme. (B) SDS-PAGE of
the deglycosylated enzyme. Lane 1, marker proteins (from top):
phosphorylase (molecular weights: 94,000), bovine serum
albumin (67,000), ovalbumin (43,000), carbonic anhydrase
measured by the glucose oxidase method as described in
Materials and Methods. The following were inert as substrates:
b
dextran, cellobiose, a-cyclodextrin, pullulan, inullin.
3
n.d., ``not determined''.
4
5
mg ml.
(
(
30,000), soybean trypsin inhibitor (20,100), and
14,400). Lane 2, deglycosylated enzyme.
a-lactoalbumin
W
mg ml
・
min.
W
The enzyme from an Acremonium sp., an enzyme
that produces oligosaccharides containing -1,3-
the enzyme acted not only on homogeneous oligosac-
charides but also on glucans such as starch and glyco-
a
6
)
linkages (``nigerooligosaccharides'' ), has a larger
molecular weight (240,000 by gel ˆltration) than our
Paecilomyces enzyme, and a ternary structure com-
posed of subunits of two sizes (128,000 and 53,000 by
gen. The fungal a-glucosidases from Aspergillus
1) 17)
niger
,
Aspergillus oryzae
,
and an Acremonium
1
6)
sp. had low or no activity toward glucans. An en-
zyme obtained from the mycelia of Mucor javani-
1
6)
18)
SDS-PAGE). Moreover, the isoelectric point of our
cus has as much activity as the Paecilomyces en-
enzyme was diŠerent from that of the Acremonium
zyme although it produced no oligosaccharides con-
1
6)
enzyme (pI 2.7). On the basis of these properties,
we expected the protein structures of the Paecilo-
myces and Acremonium enzymes to be quite diŠer-
ent.
taining
Of the
were hydrolyzed as fast as maltose. Isomaltose was
decomposed much more slowly than maltose. for
maltose, kojibiose, nigerose, and isomaltose were
0.16, 1.44, 0.14, and 0.86 m respectively. Mucor
javanicus -glucosidase has similar speciˆcity; rela-
tive activities toward 1,2-, 1,3-, 1,4-, and 1,6-linked
a-1,2- or a-1,3-linkages.
a
-linked glucobioses, kojibiose and nigerose
K
m
Substrate speciˆcities of the enzyme
M
The
a
-glucosidase was reacted with various sub-
a
strates and the hydrolysis velocities relative to the ve-
locity on maltose were compared (Table 3). Enzyme
activity was assayed at pH 5.5 but the optimum pH
of the enzyme was near 5.0. The enzyme preferably
hydrolyzed nigerose, maltose, and kojibiose.
Glucans such as soluble starch and amylose also were
hydrolyzed to a considerable extent. Activities
toward a series of maltooligosaccharides were gener-
ally less than activities toward glucobioses. The
1
8)
glucobioses are 25, 95, 100, and 0
The Paecilomyces enzyme had much higher relative
activity forward nigerose (183 ). Such hydrolysis
speciˆcity may be related to the production of more
oligosaccharides containing
transglucosylation reaction as described below.
z
, respectively.
z
a-1,3-linkages in the
Transglucosylation activity of the enzyme
results suggested that the enzyme was a
dase. However, less -nitrophenyl -glucoside and
isomaltose were hydrolyzed less than the above
glucobioses. -1,1-Linked trehalose and -1,2-
linked sucrose were not degraded at all.
The pattern of substrate speciˆcities indicated that
a
-glucosi-
The puriˆed enzyme was allowed to react with
p
a
30z (w v) maltose and the products were analyzed
W
by ABEE conversion. Figure 3 shows a typical chro-
matogram of transglucosylation products by the
ABEE conversion. The retention time of each peak
was compared with the reaction time of authentic
a
,
a
a, b

3
4
I. KOBAYASHI et al.
Fig. 4. Changes in Time during Transglucosylation.
The transglucosylation reaction was started by the addition of
.5 ml of enzyme solution (12 units of enzyme activity) into
0
0
(
.5 ml of a maltose solution (0.6 g ml) in 40 m
M
acetate buŠer
C. The ˆnal concentrations of maltose and the
enzyme were 30 (w v) and 10 units g maltose, respectively.
Samples (10 l) were taken at various times and the reaction
W
pH 5.5) at 509
z
W
W
m
Fig. 3. Chromatogram of Transglucosylation Products from
Maltose by Paecilomyces lilacinus -Glucosidase.
A reaction mixture (1 ml) containing 30 (w v) maltose and
.23 unit of the enzyme in 20 m acetate buŠer (pH 5.5) was in-
cubated at 50 C for 24 h. Saccharides in the reaction mixture
were analyzed by the ABEE conversion described in Materials
and Methods. Peaks of 1, 2, 3, 4, 5, 6, and 7 were 4-
kojibiosyl- -glucose, kojibiose, nigerose, 4- -nigerosyl-
stopped by boiling of the mixture for 10 min. The sample was
derivatized with ABEE and analyzed by HPLC. The Y axis
shows the yield of oligosaccharides from 100 g of the substrate.
a
z
W
0
M
The composition of the saccharides is plotted.
maltose; , nigerose; , kojibiose; , maltotriose;
nigerosyl- -glucose; , 4- -kojibiosyl- -glucose.

, glucose;

,
-
9
#
%
$

, 4-O-a
D

O
-
a
D
O-a-
D-glu-
D
O
-
a
cose, maltotriose, maltose, and glucose respectively. The peaks
with an asterisk are unknown reducing saccharides. Note that
the peak area almost indicates molar concentrations of each sac-
charide. Non-reducing saccharides, if any, could not be detected
by this method.
substrate. In less than 10 h of the start of the reac-
tion, the synthesis of 4- -nigerosyl- -glucose and
maltotriose reached the maximum yield of about
and 12 , respectively, and afterward, these
-Kojibiosyl-
O
-
a
D
1
7
z
z
substances were degraded rapidly. 4-
-glucose was produced more slowly to give an
O
-
a
saccharides under two diŠerent conditions for HPLC
D
(
1
acetonitrile concentration in the mobile phase,
z
approximate yield of 8.6 at 22 h, and then it
0.5
products. Among the reaction products, at least three
trisaccharides, 4- -kojibiosyl- -glucose, 4-
nigerosyl- -glucose, and maltotriose, were synthe-
z
, or 12
z
) to identify the components of the
decreased slightly. Nigerose accumulated (the yield at
22 h, 16
trisaccharides. Kojibiose also was produced to reach
an yield of 8.4 at 46 h. During prolonged incuba-
tion (72 h), the yield of nigerose decreased slightly
and that of kojibiose increased further to 12 and
14 , respectively (not shown).
Nigerose and kojibiose seemed to be formed from
z) with the decrease of maltose and the
O
-
a
D
O-a-
D
z
sized with maltose as an acceptor, although panose
was not formed. As disaccharides, nigerose and
kojibiose also were formed, and isomaltose was scar-
z
z
cely detected (less than 1
z
of all disaccharides). Dis-
accharides were separated from the reaction mixture
by activated carbon column chromatography and the
mixture was analyzed by C NMR. Comparison of
the spectrum with the spectra of authentic nigerose
and kojibiose showed that the disaccharides mixture
contained these two disaccharides (not shown). There
three reactions. In the ˆrst one, the disaccharides
were produced by transglucosylation of glucose with
the maltose remaining in the reaction system as a
donor. The second one was also a transglucosylation,
although the enzyme transferred glucose residues
1
3
from trisaccharides such as 4-
O
-
a
-nigerosyl-
D
-
were no signals corresponding to
a
-
a
trehalose. There
glucose, 4- -kojibiosyl- -glucose, or maltotriose,
O
-
a
D
were several unidentiˆed peaks, presumed to be tri-
which also were transfer products, to glucose. The
third one was reverse hydrolysis reaction of glucose
to form disaccharides. The glucose concentration
may be not so high at an early reaction stage as
reverse reaction can occur, although it is possible to
form nigerose and kojibiose in reverse reaction, espe-
cially after long reaction time.
or tetrasaccharides. Trisaccharides such as nigero-
2
triose (3 -
O
-a
-
D
-glucosyl- -nigerose) and kojitriose
D
2
(
2 -
O
-
a
-
D
-glucosyl- -kojibiose) and tetrasaccharides
D
other than maltotetraose could not be identiˆed be-
cause of standard saccharides were not available.
Figure 4 shows the course of transglucosylation
3
)
catalyzed by the enzyme with 30
z
maltose as the
Chiba et al. reported that an a-glucosidase from

Puriˆcation of Paecilomyces lilacinus
a-Glucosidase
35
buckwheat synthesizes nigerose and kojibiose from
starch. In addition, the plant enzyme accumulates
isomaltose. The Paecilomyces enzyme was diŠerent
in causing no synthesis of 1,6-glucosidic linkages.
The enzyme from the Acremonium sp. was the only
6) Murosaki, S., Muroyama, K., Yamamoto, Y.,
Kusaka, H., Liu, T., and Yoshikai, Y., Im-
munopotentiating activity of nigerooligosaccharides
for the T helper 1-like immune response in mice.
Biosci. Biotechnol. Biochem., 63, 373–378 (1999).
7
)
Murosaki, S., Muroyama, K., Yamamoto, Y., Liu,
T., and Yoshikai, Y., Nigerooligosaccharides aug-
ments natural killer activity of hepatic mononuclear
cells in mice. Int. Immunopharmacol., 2, 151–159
enzyme that produces oligosaccharides containing
a-
1
,3-glucosidic linkages from maltose or maltoo-
4
)
ligosaccharides. The Paecilomyces enzyme reported
here was similar to the Acremonium enzyme in its
(
2002).
production of oligosaccharides containing
a-1,3-
8
)
Chaen, H., Yamamoto, T., Nishimoto, T., Nakada,
T., Fukuda, S., Sugimoto, T., Kurimoto, M., and
Tsujisaka, Y., Puriˆcation and characterization of a
novel phosphorylase, kojibiose phosphorylase, from
Thermoanaerobium brockii. J. Appl. Glycosci., 46,
glucosidic linkages. Our enzyme can be clearly distin-
guished from the Acremonium enzyme by the
production of oligosaccharides containing
a-1,2-
glucosidic linkages in addition to ones containing
a
-
4
23–429 (1999).
Shiota, M., and Yatake, T., Japan Tokkyo Koho,
022958 (Jan. 31, 1991).
1
,3-glucosidic linkages. To our knowledge, there are
9
)
no reports describing microbial
a-glucosidases that
3
synthesize considerable amounts of oligosaccharides
1
1
0) Chaen, H., Nishimoto, T., Nakada, T., Fukuda, S.,
Kurimoto, M., and Tsujisaka, Y., Enzymatic synthe-
sis of kojioligosaccharides using kojibiose phos-
phorylase. J. Biosci. Bioeng., 92, 177–182 (2001).
1) Kobayashi, I., Yasuno, S., Hashimoto, H., Ikura,
K., Nakano, H., and Kitahata, S., Analysis of posi-
containing both a-1,2- and a-1,3-glucosidic linkages
in transglucosylation reactions.
Kojibiose and nigerose are contained in sake,
honey, and beer, but at low concentrations. The a-
glucosidase of Paecilomyces lilacinus may be useful
as a new sweetener containing ``rare'' disaccharides
from a maltose or starch hydrolyte.
tional isomers of
a-linked glucooligosaccharides by
p
-aminobenzoic ethyl ester-conversion method. J.
Appl. Glycosci., 49, 461–468 (2002).
1
2) Yasuno, S., Murata, T., Kokubo, K., Yamaguchi,
T., and Kamei, M., Two-mode analysis by high-per-
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