Isolation and characterization of a beta-primeverosidase-like endo-manner beta-glycosidase from Aspergillus fumigatus AP-20.

Article abstract of DOI:10.1271/bbb.66.801

A novel beta-glycosidase-producing microorganism was isolated from soil and identified as Aspergillus fumigatus AP-20 based on its taxonomical characteristics. The enzyme was found to be an extracellular protein in the culture of the isolated fungus and w

Full text of DOI:10.1271/bbb.66.801

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Isolation and Characterization of A β-
Primeverosidase-like endo-manner β-Glycosidase
from Aspergillus fumigatus AP-20
Shigeru YAMAMOTOa, Masamichi OKADAa, Taichi USUIb & Kanzo SAKATAc
a Amano Enzyme Inc. Gifu R&D; CenterKakamigahara, Gifu 509-0108, Japan
b Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka
UniversityShizuoka 422-8529, Japan
c Institute for Chemical Research, Kyoto UniversityUji, Kyoto 611-0011, Japan
Published online: 22 May 2014.
To cite this article: Shigeru YAMAMOTO, Masamichi OKADA, Taichi USUI & Kanzo SAKATA (2002) Isolation and
Characterization of A β-Primeverosidase-like endo-manner β-Glycosidase from Aspergillus fumigatus AP-20, Bioscience,
Biotechnology, and Biochemistry, 66:4, 801-807, DOI: 10.1271/bbb.66.801
To link to this article: http://dx.doi.org/10.1271/bbb.66.801
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Biosci. Biotechnol. Biochem., 66 (4), 801–807, 2002
Isolation and Characterization of A b-Primeverosidase-like endo-manner
b
-Glycosidase from Aspergillus fumigatus AP-20
3,
†
Shigeru YAMAMOTO,1 Masamichi OKADA,1 Taichi USUI,2 and Kanzo SAKATA
1Amano Enzyme Inc. Gifu R&D Center, Kakamigahara, Gifu 509-0108, Japan
2Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University,
Shizuoka 422-8529, Japan
3Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
Received October 9, 2001; Accepted November 29, 2001
A novel b-glycosidase-producing microorganism was
kinds of enzymes will expand the possible applica-
tions in industry, for example, the control of aroma
formation or high recovery of aroma extraction. In
this study, we describe screening for microorganisms
isolated from soil and identiˆed as Aspergillus fumiga-
tus AP-20 based on its taxonomical characteristics. The
enzyme was found to be an extracellular protein in the
culture of the isolated fungus and was puriˆed 88-fold
by fractionation with ammonium sulfate followed by
successive column chromatographies on phenyl-
Sepharose HP and Mono P HR. The molecular mass
was estimated to be 47 kDa by SDS-PAGE and the isoe-
lectric point to be pH 6.0 by isoelectric focusing. The
producing b-primeveroside-hydrolyzing enzyme and
the puriˆcation and characterization of this unique b-
primeverosidase-like endo-manner b-glycosidase.
Materials and Methods
puriˆed enzyme was highly speciˆc for a substrate,
nitrophenyl -primeveroside (6- -xylopyranosyl-
p
-
Microorganisms. The microbial strain AP-20, iso-
lated from soil on a ˆeld in Aichi prefecture in Japan,
was used in this study. Strain AP-20 was identiˆed
taxonomically as Aspergillus fumigatus based on
Raper's Manual.12)
b
O
-
b
-
D
b-
D
-glucopyranoside), which was cleaved in an endo-man-
ner into primeverose and -nitrophenol.
p
Key words:
b
-glycosidase; Aspergillus fumigatus
;
-
puriˆcation;
primeverosidase
characterization;
b
Materials. Eugenyl
from young leaves of Camellia sasanqua.13)
Nitrophenyl ( NP) -primeveroside and primeverose
were enzymatically synthesized by our method.14)
NP gentiobioside (6- -glucopyranosyl-
glucopyranoside), NP monosaccharide glycosides
NP -gluco- and -xylopyranosides),
gentiobiose, sophorose (2-
-glucopyranose), and laminaribiose (3-
glucopyranosyl-
from Sigma Chemical Co. Cellobiose (4-
glucopyranosyl- -glucopyranose) was obtained
b-primeveroside was prepared
p
-
p
b
Aroma precursors of the ‰oral tea aroma like
linalool, 2-phenylethanol etc., have been isolated and
identiˆed as disaccharide glycosides. Most of them
p
O
-
b
-
D
b- -
D
p
were identiˆed as
xylopyranosyl-
-glucopyranoside).1–7) At the same
time -primeverosidases have been puriˆed from
b
-primeverosides (6-
O
-b
-
D
-
(
p
b
-
D
b
b
-
D
b
-D
O
-
-
D
-glucopyranosyl-
b
-
-
b
D
O
-
b
-
D
fresh leaves of cvs. Yabukita (Camellia sinensis var.
sinensis) for Japanese green tea, Shuixian (C. sinen-
sis var. sinensis) for oolong tea, and a cultivar (C.
sinensis var. assamica) for black tea. These enzymes
b- -glucopyranose) were purchased
D
O
-
b-D-
b
-
D
from Wako Pure Chemical Industries, Ltd. All other
chemicals were of analytical grade.
showed high substrate speciˆcity toward
b-
primeverosides to hydrolyze them into primeverose
and the corresponding aglycons.8–10) Quite recently,
Media. Solid medium for primary screening con-
Wang et al. have demonstrated that
b
-primeverosides
tained 0.5
0.05
primeveroside, and 2
Laboratories). The pH of the medium was adjusted
to 6.0. The separating solid media was Potato Dex-
trose Agar (Eiken Chemical Co. Ltd.). Liquid
medium base for secondary screening contained
z (w v) (NH4)2SO4, 0.1z (w v) KH2PO4,
W
W
・
z (w v) MgSO4 7H2O, 0.3z (w v) eugenyl b-
play an important role in tea aroma formation during
W
W
black tea manufacturing.11)
z
(w v) Bacto-Agar (Difco
W
However, no
b
-primeverosidase from a microor-
ganism has been reported. From such an aspect, we
tried to search for
from microbial sources, because the ˆnding of such
b-primeverosidase-like enzymes
†
To whom correspondence should be addressed. Kanzo SAKATA, Fax: +81-774-38-3229; E-mail: ksakata
＠
scl.kyoto-u.ac.jp
Abbreviations
:
p
NP, -nitrophenyl; FPLC, fast protein liquid chromatography; HPLC, high-pressure liquid chromatography
p

802
S. YAMAMOTO et al.
0.3
z
(w v) NaNO3, 0.1
z
z
(w v) KH2PO4, 0.05
z
z
The optimum temperature of the enzyme was
W
W
・
M
(w v) MgSO4 7H2O, 0.05
(w v) KCl, 0.001
measured with
dium citrate-HCl buŠer (pH 2.5) at various tempera-
tures (30–65 C). The thermal stability of the enzyme
was examined from the residual activities after
incubation in 20 m glycine–NaCl–NaOH buŠer (pH
8.0) at various temperatures (30–65 C) for 1 h. The
optimum pH of the enzyme was measured with NP
-primeveroside in 20 m disodium citrate-HCl
buŠer at diŠerent pHs (2.0–5.0) at 37 C. The pH sta-
pNP b-primeveroside in 20 m diso-
W
W
W
・
(w v) FeSO4 7H2O, and 2
z
(w v) glucose. The pH
W
of the medium was adjusted to 6.0. For medium P,
0.1 m NP -primeveroside was added to the liquid
medium of the secondary screening base and for
medium X, NP -xylopyranoside was added. For
9
M
p
b
M
p
b
-
D
9
the ˆrst cultivation for enzyme production, Dextrose
Peptone Broth (Eiken Chemical Co. Ltd.) was used.
The cultivation medium for enzyme production con-
p
b
M
9
tained 2
0.3 (w v) NaCl, 0.1
(w v) MgSO4 7H2O, 3
z
(w v) Soya‰our A (Nissin Oil Mills Ltd.),
bility of the enzyme was examined from the residual
activities after incubation in diŠerent pHs (3.0–12.0)
W
z
z (w v) K2HPO4, 0.05z
W
W
W
・
z
(w v) Soluble Starch, and
at 37
9
C for 1 h. The buŠers used were 20 m
M
citrate
W
#
z (w v) Gentose 80 (Nihon Syokuhin Kako Co.
M
1
buŠer (pH 3.0–6.0), 20 m Tris-malate-NaOH buŠer
W
Ltd.). The pH of the medium was adjusted to 5.6.
(pH 6.0–8.0), and 20 m
(pH 8.0–12.0).
M
glycine-NaCl-NaOH buŠer
Isolation of microorganisms and fermentation.
For the primary screening of microorganisms
Puriˆcation of a
b-glycosidase. All operations
producing
b
-primeveroside-hydrolyzing enzyme, soil
were done in 20 m potassium phosphate buŠer (pH
M
samples were suspended in sterilized saline and
spread on the solid media. After incubation at 30
6.0) at room temperature, unless otherwise stated.
The culture was ˆltered through ˆlter paper to re-
move mycelium, and the ˆltrate (8,600 ml) was con-
centrated to 710 ml by using ultraˆltration AIP-1010
9
C
for 5 days, the viable microorganisms were trans-
ferred to Potato Dextrose Agar slants. The secondary
screening was done as follows: the microorganisms
obtained by the primary screening were inoculated
into 100
30 h. Two hundreds
(Asahi Kasei Corporation) with 100
z recovery of
b
-primeverosidase activity. The enzyme solution
m
l of liquid media and incubated at 30
9
C for
(200 ml) was centrifuged to remove insolubles. To the
supernatant solution (184 ml), solid ammonium sul-
m
l of 0.5 Na2CO3 was added to
M
the medium. Microorganisms that were more deeply
colored in medium P than medium X by releasing
fate was added up to 50
and left overnight at 4 C. The resulting precipitates
were collected by centrifugation, dissolved in 10 ml
of the buŠer saturated with 20 ammonium sulfate.
The enzyme solution in the buŠer containing 20
z
saturation with stirring
9
p
-nitrophenol were selected. Each agar block (5
×
5 mm) taken from the Potato Dextrose Agar slant
was inoculated into 100 ml of ˆrst cultivation medi-
um in a 500 ml-Sakaguchi ‰ask and incubated at
z
z
ammonium sulfate was put onto a HiLoad 16 10
W
×
30
9
C for 24 h on a reciprocal shaker (140 strokes
phenyl-Sepharose HP column (16 mm i.d. 100 mm;
W
min). After this cultivation, 1 ml of the culture was
inoculated into the second cultivation medium in a
Pharmacia Biotech Co. Ltd.) using the FPLC sys-
tem, equilibrated with the same as shown in Fig. 2.
No b-primeverosidase was eluted when the column
was further washed with a high salt concentration.
After the column was washed with a linear gradient
500 ml-Sakaguchi ‰ask and incubated at 30
9C for 6
days on a reciprocal shaker (140 strokes min). The
W
culture ˆltrates were used as a crude enzyme solution.
of 20
z
(20 ml)
¿
0
z
(180 ml) ammonium sulfate,
55) were eluted with
Enzyme assay. During puriˆcation, the enzyme
fractions were measured for hydrolyzing activities
active enzyme fractions (Fr. 53
¿
a buŠer solution containing no ammonium sulfate.
Active fractions were combined and desalted using
by Econo-Pac 10DG columns (Bio-Rad Laborato-
toward
glucopyranoside and
NP -primeveroside. The incubation mixture
(245 l) consisted of 45 l of an enzyme sample solu-
tion and 200 l of 2 m substrate solution. The
buŠers of substrate solutions were 20 m acetate
buŠer (pH 5.5) for NP -monosaccharide glyco-
sides and 20 m acetate buŠer (pH 4.0) for NP
primeveroside. The reaction was started by adding an
enzyme sample to a substrate solution at 37 C and
stopped by addition of 250 l of 0.5 Na2CO3. The
liberated -nitrophenol was measured spec-
p
NP monosaccharide glycosides (
p
NP
b
-
D
-
p
NP
b
-
D
-xylopyranoside) and
p
b
ries) with a 25 m
M
triethanolamine-iminodiacetic
m
m
acid (pH 8.3) solution. The desalted solution (7.2 ml)
M
×
was put on a Mono P HR 5 20 column (5 mm i.d.
m
W
M
200 mm; Pharmacia Biotech Co. Ltd.) using the
FPLC system, equilibrated with 25 m triethanola-
mine-iminodiacetic acid (pH 8.3). When the column
was eluted with linear gradient from
(12 ml) 100 (72 ml) PolybuŠer (pH adjusted to
5.0 with iminodiacetic acid; 3 PolybuŠer 96, 7
PolybuŠer 74; Pharmacia Biotech Co. Ltd.), active
fractions (Fr. 31 32) were eluted with about 30
p
b
M
M
p
b-
a
0
z
9
¿
z
m
M
z
z
p
trophotometrically at 412 nm. One unit of the en-
zyme activity was deˆned as the amount of enzyme
liberating 1
these assay conditions.
¿
z
PolybuŠer. There was a symmetrical peak of the
enzyme activity associated with the protein peak,
mmol of p-nitrophenol per min under
which was almost devoid of other b-glycosidase ac-

b
-Glycosidase of Aspergillus fumigatus
803
tivities such as
b
-glucosidase or
b
-xylosidase.
(within 15
glucose (Determination kit for
agnostics K.K.) for -linked disaccharides or HPLC
for -glycosides. From these results, relative hydroly-
sis rates for various kinds of substrates were calculat-
ed, taking the reaction velocity for NP -primevero-
side as 100 ( ).
z
hydrolysis) was analyzed by using TC
D
-
D
-glucose, Roche Di-
Analytical methods. Protein was measured by the
method of Lowry et al
b
15)
.
using bovine serum
b
albumin as a standard. Sodium dodecyl sulfate-
polyacrylamide gel electorophoresis (SDS-PAGE)
was done with the PhastSystem (Pharmacia Biotech
Co. Ltd.) using a PhastGel Gradient 8–25 (Pharma-
cia Biotech Co. Ltd.). ``Perfect'' protein markers
15–150 kDa (Novagen) were used for the mass
calibration. Proteins were detected with a Pharmacia
silver stain. Isoelectric focusing was done on the
sucrose density-gradient isoelectric separation using
Ampholine pH 3.5–10.0 (Pharmacia Biotech Co.
Ltd.) as the carrier ampholyte.
p
b
z
Results and Discussion
Screening for microorganisms producing
primeveroside-hydrolyzing enzyme
b-
In the primary screening for
b-primeveroside-
hydrolyzing enzyme-producing strains, four positive
strains were obtained. The fermentation broth of
strain AP-20 showed the highest b-primeveroside-
hydrolyzing activity among these strains at the sec-
ondary screening. The crude enzyme solution hydro-
lyzed pNP b-primeveroside, which was cleaved in an
endo-manner into primeverose and p-nitrophenol.
HPLC was done a YMC-packed column ODS-AQ-
×
303 (ODS) (4.6 mm i.d. 250 mm) on a Shimadzu
liquid chromatograph equipped with a Shimadzu
SPD-10Avp UV detector. When
NP -primeveroside and
were used as substrates, elution was done with water
p
NP
b
-glycosides
(
p
b
p
NP
b
-gentiobioside)
W
MeOH (80 20, v v) at a ‰ow rate of 0.8 ml min at
Taxonomic characterization
W
W
W
40
9
C, and detected at 300 nm. With eugenyl
b
-glyco-
Culture tests were done by the procedures of
Raper11) by using Czapeck-Dox agar (CA) and Malt
extract (MEA) agar as test media. Morphological
examinations of the organism grown on CA and
sides (eugenyl
b
-primeveroside and eugenyl
b-gluco-
side), it was also done with water MeOH (55 45,
W
W
v v) at 280 nm.
W
Cleavage ratios and kinetic studies were done as
follows. The incubation mixture (0.5 ml) consists of
MEA at 259C for 7 days were done by slide-culture
methods with an optical microscope. The culture and
morphological characteristics are shown in Table 1.
This strain formed macroscopic colonies 73–75 mm
0.1–30 m
acetate buŠer (pH 4.0). Reactions were started by
adding the enzyme sample at 37 C. Samples (100 l)
were taken at 3-min intervals (3, 6, 9, 12 min) during
the incubation, and inactivated by adding 200 l of
0.1 trichroloacetic acid. The amount of
nitrophenol or eugenol formed from the initial sub-
strates at an early stage (15 hydrolysis) during incu-
M
p
NP or eugenyl
b
-glycoside in 50 m
M
9
m
in diameter at 37
9
C on CA after 3 days. Growth was
observed at 45 C on CA. Metulae and ascospores
9
m
were not formed. Conidial heads were strongly
columnar, not nodding, appearance, and its color
was dull green to grayish green. Conidia were globose
and their surfaces were echinulate. Conidiophores
M
p-
z
bation with the enzyme was analyzed by HPLC. The
amount of each product increased linearly as time
passed in the initial stage of the reaction. On the basis
of these data, the ratio of enzyme-catalyzed cleavage
of each glycosidic linkage was calculated. And the
initial velocity (
rectly from the initial slope of time vs product pro-
duced plots of the reaction. Six diŠerent substrate
was mostly 500 mm or less in length. The vesicles and
phialides were shown in Fig. 1. Based on these cultur-
al and morphological characteristics, this strain was
identiˆed as Aspergillus fumigatus
.
v
) of each reaction was obtained di-
Puriˆcation and characterization of the
dase
An enzyme hydrolyzing NP b-primeveroside
b-glycosi-
p
concentrations (0.1–30 m
periment. The Km and Vmax for
primeverosides were calculated from plots of
M
) were used for each ex-
from A. fumigatus AP-20 was puriˆed by ammoni-
um sulfate preciptation followed by successive
column chromatographies using phenyl-Sepharose
HP and Mono P HR. An elution proˆle showed a
p
NP and eugenyl b-
v
against substrate concentrations using the Michaelis-
Menten equation and the least squares method.16)
separation of b-primeverosidase activity from the b-
Relative hydrolysis rates of various kinds of
linked disaccharides and -glycosides by the
glycosidase were measured by incubating 1 m
substrates in 1.0 ml of 25 m acetate buŠer (pH 4.0)
with the enzyme. Reactions were started by adding
each enzyme sample at 37 C. Samples (200 l) were
b
-
glycosidase one on phenyl-Sepharose HP (Fig. 2).
The active fraction was further puriˆed by Mono P
HR. A summary of the puriˆcation is presented in
Table 2. The ˆnal recovery of the enzyme activity was
b
b
-
M
of
M
40
z and the speciˆc activity increased 88-fold after
9
m
the ˆrst ˆltration. The puriˆed enzyme gave a single
band corresponding to a molecular mass of about
47 kDa on SDS-PAGE (Fig. 3). The isoelectric point
was pH 6.0 by isoelectric focusing. The enzyme had a
taken at intervals (3, 6, 9, 12 min) during the incuba-
tion, and inactivated by boiling for 5 min. The
amount of hydrolysates formed at an early stage

804
S. YAMAMOTO et al.
Table 1. Characterization of the Strain AP-20 Identiˆed as Aspergillus fumigatus
A. Culture characteristics
Growth medium
(after 7 days at 279C)
Czapek-Dox agar
Malt extract agar
Growth rate (diameter, mm)
Surface texture
Conidiogenesis
Soluble pigment
Reverse
48–50
Velutinous to powdery
Dull green to grayish green, light to moderate
None
Light yellowish brown to brown
78–80
Velutinous to powdery
Dull green to grayish green, abundant
None
Colorless to yellowish white
B. Morphological characteristics
Conidial heads
Conidiophores
Strongly columnar, 48–128
Smooth, 125–800 m (mostly less than 500
from submerged hyphae
m
m in length by 16–52
m
m in diameter, dull green to grayish green
m in diameter, arising directly
m
m
m) in length. Based in length by 5–10 m
Vesicles
Flask shaped, 10–25
None
5.6–12 by 2.4–3.2 mm
Green in the mass, echinulate, globose to subglobose, 2.6–3.6
None
mm in diameter
Metulae
Phialides
Conidia
mm in diameter
Ascospores
Table 2. Puriˆcation of the b-Glycosidase from Aspergillus fumigatus AP-20
Total
Total
Speciˆc
Puriˆcation
fold
Puriˆcation step
Ultraˆltration
Recovery (z)
activity (u)
protein (mg)
activity (u mg)
W
45.2
41.2
23.3
18.2
828
199.8
8.0
0.05
0.20
2.9
1
4
58
88
100
91
52
50
z (NH4)2SO4 ppt
Phenyl Sepharose
Mono P
4.1
4.4
40
The enzyme activities were measured with
pNP b-primeveroside.
z 9
(À90 ) from 50 to 60 C.
Relative hydrolysis rates of
and -glycosides by the -glycosidase
The relative hydrolysis rate of the puriˆed enzyme
b-linked disaccharides
b
b
on
p
NP
b
-gentiobioside compared to
p
NP
b
-
-
primeveroside at 100 was 41, while that of eugenyl
b
primeveroside was 1.8, a 55-fold diŠerence (Table 3).
Replacement of the eugenyl group by NP greatly
decreased the hydrolysis rate. -Glucobioses and
p
b
primeverose acted slightly as substrates. These results
show that primeverose and aglycon units are essential
for the hydrolytic action of the enzyme.
Kinetic studies and cleavage ratio
Kinetic parameters for the hydrolysis of three
kinds of disaccharide
4. NP -primeveroside had a
max of 2.8 and was the best substrate. The Km Vmax
b
-glycosides are listed in Table
p
b
K
m of 2.88 m and Km
M
V
W
W
of
pNP b-gentiobioside is 68z of that of pNP b-
primeveroside and it still acts as a fairly good sub-
strate. On the contrary, eugenyl
b
-primeveroside act-
max: 1.5 of that
pNP b-primeveroside). This suggests that the
ed only slightly as a substrate (Km
V
z
W
Fig. 1. Light Microphotograph of Aspergillus fumigatus AP-20,
Grown on MEA Medium for 7 Days at 25 C.
of
9
chemical property of the aglycon moiety at the termi-
nal position is strictly required for the hydrolytic
action.
broad pH optimality in the range of pH 2.5–3.0 and
was stable from pH 7.0–8.0. It had its optimum
The action modes of the enzyme on three kinds of
activity at 55
9
C and showed near-optimum activities
disaccharide b-glycosides were further examined.

b
-Glycosidase of Aspergillus fumigatus
805
Fig. 2. FPLC Elution Proˆle of the
b
-Glycosidase Fraction on a Phenyl-Sepharose Column from Aspergillus fumigatus AP-20.
potassium phosphate buŠer (pH 6.0); ammonium sulfate gradient elu-
-glucosidase (Glc) activities were measured using NP -primeveroside and
Column, HiLoad 16 10 phenyl-Sepharose HP; elution, 20 m
M
W
tion; ‰ow rate, 2 ml min. The
p
b
-primeverosidase (Pri) and
b
p
b
W
NP b-glucoside, respectively.
Table 3. Relative Hydrolysis Rates on Various Kinds of
b-
Linked Disaccharides and
b-Glycosides by the b-Glycosidase
Substrate
Relative rate of hydrolysis (
z)
p
p
-Nitrophenyl
-Nitrophenyl
b
b
-primeveroside
-gentiobioside
100
41
Eugenyl
Sophorose (Glc
Laminaribiose (Glc
Cellobiose (Glc 1-4Glc)
Gentiobiose (Glc 1-6Glc)
Primeverose (Xyl 1-6Glc)
b
-primeveroside
1-2Glc)
1-3Glc)
1.8
0.5
1.6
0.6
5.1
0.2
b
b
b
b
b
eugenyl group aŠected the position of glycosides to
be hydrolyzed against the action of the enzyme.
From these results, we suppose a subsite structure
Fig. 3. SDS-PAGE of the
lus fumigatus AP-20.
b-Glycosidase Puriˆed from Aspergil-
so that pNP b-primeveroside has a matching shape,
which can accommodate a chain of three residues to
ˆt into the active site. As a matter of convenience, it
is assumed that the active site of the enzyme consists
of four subsites (S1, S2, S3, and S4), and that the
glycosodic bonds of the substrates are split between
S2 and S3 (Fig. 4). Reducing disaccharides,
The gel was stained with silver. Lane 1, molecular mass mar-
ker; Lane 2, puriˆed enzyme protein.
From the HPLC chromatograms of the digest of
each substrate, the amounts of the products were cal-
culated from their peak areas. The amounts of each
product increased linearly with time in the initial
stage of the reaction (data not shown). Based on the
product analysis, the ratio of the enzyme-catalyzed
cleavage of glycosidic linkages in the three substrates
were diŠerent from each other. The number of sus-
ceptible bonds varied with the structual diŠerences of
primeverose and other b-linked glucobioses, act only
slightly as substrates. It shows that disaccharide
glycoside structure is at least needed for the binding
to the active site. A comparison of the catalytic
e‹ciency for
pNP b-primeveroside with that for eu-
genyl -primeveroside shows that the eugenyl group
b
did prohibit reaction of the enzyme. Subsite 3 was
strict for binding the aglycon group. Replacement of
the substrates.
predominantly in an endo-manner into primeverose
and -nitrophenol. The action pattern on NP -gen-
NP
pNP b-primeveroside was hydrolyzed
p
NP b-gentiobioside by pNP b-primeveroside does
p
p
b
appreciabily in‰uence the enzyme action, although
the former substrate acts as a fairly good substrate.
This may explain the ‰exibility of subsite 1 for bind-
ing the glucosyl group at the nonreducing end.
In conclusion, in the screening of microorganisms
tiobioside was somewhat diŠerent from that of
p
b
-primeveroside and the cleavage occurred at both
the ˆrst and second bonds from the
ratio of 0.62:0.38. Eugenyl
p
NP group in the
b-primeveroside was
hydrolyzed at the bond between Xyl and Glu with the
enzyme, although it was hydrolyzed slowly. Taking
the kinetic data into account, this indicates that the
producing any
we found a ˆlamentous fungus A. fumigatus produc-
ing a novel -glycosidase which hydrolyzes not only
b-primeveroside-hydrolyzing enzyme,
b

806
S. YAMAMOTO et al.
Table 4. Kinetic Parameters of the
b
-Glycosidase
Vmax
Substrate
Km (m
M
)
Vmax Km
W
(
m
mol min mg protein)
W
W
p
p
-Nitrophenyl
-Nitrophenyl
b
b
-primeveroside
-gentiobioside
2.9
6.3
6.3
8.1
12
0.26
2.8
1.9
0.041
Eugenyl
b
-primeveroside
D
-gluco-pyranosides as aroma precursors from tea
enyl
b-
leaves for oolong tea., Biosci. Biotechnol. Biochem., 60,
1815–1819 (1996).
5) Nishikitani, M., Kubota, K., Kobayashi, A., and Sugawara,
F., Geranyl 6-O-a-
L-arabinopranosyl-
b-D-glucopyrano-
side isolated as an aroma precursor from leaves of a
green tea cultivar. Biosci. Biotechnol. Biochem., 60,
929–931 (1996).
Fig. 4. Schematic Representation of Substrate Binding to Sub-
sites and the Action of the -Glycosidase on the Substrates.
6) Nishikitani, M., Wang, D., Kubota, K., Kobayashi,
b
A., and Sugawara, F., (Z)-3-Hexenyl and trans
-
X, xylosyl residue; G, glucosyl residue; P,
p-nitrophenyl
linalool 3,7-oxide b-primeverosides as aroma precur-
$
residue; E, eugenyl residue; , the catalytic site.
sors from leaves of a green tea cultivar. Biosci.
Biotechnol. Biochem., 60, 1631–1633 (1996).
7) Wang, D., Yoshimura, T., Kubota, K., and
Kobayashi, A. Analysis of glycosidically bound aro-
ma precursors in tea leaves. 1. Qualitative and quan-
titative analyses of glycosides with aglycons as aroma
compounds. J. Agric. Food Chem., 48, 5411–5418
(2000).
b
-primeverosides but also
endo-manner into disaccharide and aglycon. As a
result, NP -primeveroside was very suitable as a
synthetic substrate for analytical use of
primeverosidase-like endo-manner -glycosidase.
b-gentiobiosides in an
p
b
b
-
b
8) Guo, W., Ogawa, K., Yamauchi, K., Watanabe, N.,
Usui, T., Luo, S., and Sakata, K., Isolation and
Further investigations on the substrate speciˆcities
are now in progress.
characterization of a
b-primeverosidase concerned
with the alcoholic aroma formation in tea leaves.
Biosci. Biotechnol. Biochem., 60, 1810–1814 (1996).
9) Ogawa, K., Iijima, Y., Guo, W., Watanabe, N.,
Usui, T., Dong, S., Tong, S., and Sakata, K., Puriˆ-
Acknowledgments
We thank Dr. M. Yamamoto, Nihon Syokuhin
Kako Co. Ltd., and M. Okada, Pola Co., for valua-
ble discussions. We also thank M. Yokoe, Amano
Enzyme Inc., for his useful advice in the identiˆca-
tion of the microorganism.
cation of a
b-primeverosidase concerned with alco-
holic aroma formation in tea leaves (cv. Shuixian) to
be processed to oolong tea. J. Agric. Food Chem.
,
45, 877–882 (1997).
10) Iijima, Y., Ogawa, K., Watanabe, N., Usui, T.,
Kameyama, M.-O., Nagata, T., and Sakata, K.,
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