Purification and characterization of a novel α-glucuronidase from Aspergillus niger specific for O-α-D-glucosyluronic acid α-D-glucosiduronic acid

Article abstract of DOI:10.1271/bbb.69.522

A new α-glucuronidase that specifically hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid (trehalose dicarboxylate, TreDC) was purified from a commercial enzyme preparation from Aspergillus niger, and its properties were examined. The enzyme did not degrade O-α-D-glucosyluronic acid α-D-glucoside, O-α-D-glucosyluronic acid β-D-glucosiduronic acid, O-α-D-glucosyluronic acid-(1 → 2)-β-D-fructosiduronic acid, p-nitrophenyl-O-α-D-glucosiduronic acid, methyl-O-α-D-glucosiduronic acid, or 6-O-α-(4-O-α-D- glucosyluronic acid)-D-glucosyl-β-cyclodextrine. Furthermore, it showed no activity on α-glucuronyl linkages of 4-O-methyl-D-glucosyluronic acid-α-(1 → 2)-xylooligosaccharides, derived from xylan, a supposed substrate of α-glucuronidases. The molecular mass of the enzyme was estimated to be 120 kDa by gel filtration and 58 kDa by SDS-PAGE suggesting, the enzyme is composed of two identical subunits. It was most active at pH 3.0-3.5 and at 40°C. It was stable in pH 2.0-4.5 and below 30°C. It hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid to produce α- and β-anomers of D-glucuronic acid in an equimolar ratio. This result suggests that inversion of the anomeric configuration of the substrate is involved in the hydrolysis mechanism.

Full text of DOI:10.1271/bbb.69.522

Biosci. Biotechnol. Biochem., 69 (3), 522–529, 2005
Purification and Characterization of a Novel ꢀ-Glucuronidase
from Aspergillus niger Specific for O-ꢀ-D-Glucosyluronic
Acid ꢀ-D-Glucosiduronic Acid
y
Takaaki KIRYU, Hirofumi NAKANO, Taro KISO, and Hiromi MURAKAMI
Department of Biochemistry and Materials for Human Life, Osaka Municipal Technical Research Institute,
6
-50, Morinomiya 1-chome, Joto-ku, Osaka 536-8553, Japan
Received September 24, 2004; Accepted December 15, 2004
A new ꢀ-glucuronidase that specifically hydrolyzed
O-ꢀ-D-glucosyluronic acid ꢀ-D-glucosiduronic acid (tre-
halose dicarboxylate, TreDC) was purified from a
commercial enzyme preparation from Aspergillus niger,
and its properties were examined. The enzyme did not
degrade O-ꢀ-D-glucosyluronic acid ꢀ-D-glucoside, O-ꢀ-
D-glucosyluronic acid ꢁ-D-glucosiduronic acid, O-ꢀ-D-
glucosyluronic acid-(1 ! 2)-ꢁ-D-fructosiduronic acid,
p-nitrophenyl-O-ꢀ-D-glucosiduronic acid, methyl-O-ꢀ-
D-glucosiduronic acid, or 6-O-ꢀ-(4-O-ꢀ-D-glucosyluronic
acid)-D-glucosyl-ꢁ-cyclodextrine. Furthermore, it show-
ed no activity on ꢀ-glucuronyl linkages of 4-O-methyl-D-
glucosyluronic acid-ꢀ-(1 ! 2)-xylooligosaccharides, de-
rived from xylan, a supposed substrate of ꢀ-glucuroni-
dases.
glucuronidases degrade ꢀ-1,2 glucuronyl linkages to
liberate 4-O-methyl D-glucuronic acids, which exist as
side chains of xylan, although the enzymes do not
hydrolyze p-nitrophenyl-O-ꢀ-D-glucosiduronic acid
1
–8)
(PNP ꢀ-glucuronide).
Among microbial ꢀ-glucuro-
nidases, ꢀ-glucuronidase from Thermotoga maritima is
the only enzyme that was known to hydrolyze PNP ꢀ-
9
)
glucuronide. Substrate specificities of the ꢀ-glucuroni-
dases, however, have not been examined in detail, due to
the unavailability of various ꢀ-glucuronyl oligosaccha-
rides.
A new method using Pseudogluconobactor saccha-
roketogenes has been developed to oxidize sugars.1
Since the bacterial cells are able to oxidize not only the
C-1 hydroxy group of glucopyranose residue but also the
C-6 hydroxymethyl group, the method makes it possible
to prepare various sugars containing glucuronyl resi-
dues. In our present study, O-ꢀ-D-glucosyluronic acid ꢀ-
D-glucosiduronic acid (trehalose dicarboxylate, TreDC)
was converted from trehalose by this microbial oxida-
tion method and used for the screening of ꢀ-glucuroni-
dases. In consequence, an enzyme obtained from
Aspergillus niger was found to hydrolyze TreDC.
It has been reported that ꢀ-glucuronidase from snail
0)
The molecular mass of the enzyme was estimated to
be 120 kDa by gel filtration and 58 kDa by SDS–PAGE
suggesting, the enzyme is composed of two identical
ꢀ
subunits. It was most active at pH 3.0–3.5 and at 40 C.
ꢀ
It was stable in pH 2.0–4.5 and below 30 C. It hydro-
lyzed O-ꢀ-D-glucosyluronic acid ꢀ-D-glucosiduronic acid
to produce ꢀ- and ꢁ-anomers of D-glucuronic acid in an
equimolar ratio. This result suggests that inversion of
the anomeric configuration of the substrate is involved
in the hydrolysis mechanism.
1
1)
acetone powder hydrolyzed TreDC.
zyme, however, hydrolyzed other glucuronides such as
The snail en-
Key words: ꢀ-glucuronidase; O-ꢀ-D-glucosyluronic acid
PNP ꢀ-glucuronide and 4-O-methyl-D-glucosyluronic
ꢀ-D-glucosiduronic acid; Aspergillus niger;
purification; hydrolysis
acid-ꢀ-(1 ! 2)-xylooligosaccharide (Me-GA-X ) in ad-
n
dition. To our knowledge, there are no reports on ꢀ-
glucuronidase extremely specific for TreDC. In this
report, therefore, we tentatively call the TreDC specific
enzyme trehalose dicarboxylate hydrolase (TreDCase)
to distinguish it from other ꢀ-glucuronidases.
ꢀ
-Glucuronidases (EC 3.2.1.139) that catalyze the
hydrolysis of ꢀ-glucuronic linkages are distributed
1
–9)
widely in fungi and bacteria.
Most microbial ꢀ-
y
To whom correspondence should be addressed. Tel: +81-6-6963-8071; Fax: +81-6-6963-8079; E-mail: kiryu@omtri.city.osaka.jp
Abbreviations: TreDC, O-ꢀ-D-glucosyluronic acid ꢀ-D-glucosiduronic acid; PNP ꢀ-glucuronide, p-nitrophenyl-O-ꢀ-D-glucosiduronic acid; Me-
GA-Xn, 4-O-methyl-D-glucosyluronic acid-ꢀ-(1 ! 2)-xylooligosaccharide; TreDCase, trehalose dicarboxylate hydrolase; SucDC, O-ꢀ-D-glucosy-
luronic acid-(1 ! 2)-ꢁ-D-fructosiduronic acid; ꢀ,ꢁ-TreDC, O-ꢀ-D-glucosyluronic acid ꢁ-D-glucosiduronic acid; TreMC, O-ꢀ-D-glucosyluronic acid
ꢀ-D-glucoside; GUG-CD, 6-O-ꢀ-(4-O-ꢀ-D-glucosyluronic acid)-D-glucosyl-ꢁ-cyclodextrine; Me-GA-Xn-OH, 4-O-methyl-D-glucosyluronic acid-ꢀ-
(
1 ! 2)-xylooligosaccharyl-ꢀ-(1 ! 2)-xylitol; GlcUA, D-glucuronic acid; HPAEC-PAD, high-performance anion-exchange chromatography with
pulsed amperometric detection; TLC, thin layer chromatography; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel
electrophoresis; SDS, sodium dodecyl sulfate; DSS, 3-(trimehylsilyl)-1-propanesulfonic acid, sodium salt

A New ꢀ-Glucuronidase from Aspergillus niger
523
Materials and Methods
O-methyl-D-glucosyluronic acid-ꢀ-(1 ! 2)-D-xylosyl-
-(1 ! 4)-D-xylose, 4-O-methyl-D-glucosyluronic acid-
ꢁ
Materials. Oxidized saccharides, such as TreDC,
O-ꢀ-D-glucosyluronic acid-(1 ! 2)-ꢁ-D-fructosiduronic
acid (SucDC), O-ꢀ-D-glucosyluronic acid ꢁ-D-glucosi-
duronic acid (ꢀ,ꢁ-TreDC), PNP ꢀ-glucuronide, methyl-
O-ꢀ-D-glucosiduronic acid, and O-ꢀ-D-glucosyluronic
acid ꢀ-D-glucoside (TreMC), were prepared from treha-
lose, sucrose, ꢀ,ꢁ-trehalose (Sigma Chemicals, St.
Louis, MO), p-nitrophenyl-O-ꢀ-D-glucoside, and meth-
yl-O-ꢀ-D-glucoside respectively by microbial oxidation
as follows: Reaction mixtures consisting of 5 ml of 1%
trehalose, sucrose, ꢀ,ꢁ-trehalose, p-nitrophenyl-O-ꢀ-D-
glucoside, or methyl-O-ꢀ-D-glucoside were incubated
with 0.29 g (wet weight) of P. saccharoketogenes
IFO14483 cells prepared according to the method of
ꢀ-(1 ! 2)-D-xylosyl-ꢁ-(1 ! 4)-D-xylosyl-ꢁ-(1 ! 4)-
D-xylose, and 4-O-methyl-D-glucosyluronic acid-ꢀ-
(1 ! 2)-D-xylosyl-ꢁ-(1 ! 4)-D-xylosyl-ꢁ-(1 ! 4)-D-
xylosyl-ꢁ-(1 ! 4)-D-xylose (40:40:20, w/w). Me-GA-
Xn-OH was composed of 4-O-methyl-D-glucosyluronic
acid-ꢀ-(1 ! 2)-D-xylosyl-ꢁ-(1 ! 4)-xylitol, 4-O-meth-
yl-D-glucosyluronic acid-ꢀ-(1 ! 2)-D-xylosyl-ꢁ-(1 !
4)-D-xylosyl-ꢁ-(1 ! 4)-xylitol, and 4-O-methyl-D-glu-
cosyluronic acid-ꢀ-(1 ! 2)-D-xylosyl-ꢁ-(1 ! 4)-D-xy-
loyl-ꢁ-(1 ! 4)-D-xylosyl-ꢁ-(1 ! 4)-xylitol (40:40:20,
w/w). Other chemicals were of the highest quality
commercially available.
Enzymes. The enzyme preparations, Cellurosine PE
60 and Orientase 20A, were donated by HBI Enzymes
(Hyogo, Japan). TreDCase was purified from Celluro-
sine PE 60 (HBI Enzymes). Porcine kidney trehalase
was purchased from Sigma Chemical. A. niger ꢀ-
1
0)
Ishiguro et al. The reactions were carried out in the
ꢀ
presence of 0.3% CaCO3 at 30 C for 24 h (TreDC,
SucDC, ꢀ,ꢁ-TreDC, PNP ꢀ-glucuronide, methyl-O-ꢀ-D-
glucosiduronic acid), or 6.5 h (TreMC). After oxidation,
the reaction mixtures were centrifuged at 5;000 g for
2
,12)
glucuronidase that hydrolyzed Me-GA-Xn
was par-
3
0 min to remove the cells, and put on an activated
tially purified from Orientase 20A (HBI Enzymes) by
DEAE-Toyopearl column chromatography, as follows:
Enzyme preparation was put on a DEAE-Toyopearl
column (3 ꢁ 20 cm, Tosoh, Tokyo, Japan). The enzyme
was eluted by linear gradient from zero to 0.5 M NaCl in
10 mM acetate buffer (pH 5.0). The active fractions were
carbon column (3:0 ꢁ 20 cm). The passed solutions were
evaporated, and applied onto a column (2:5 ꢁ 90 cm) of
Bio-Gel P2 (Bio-Rad Laboratories, Hercules, CA)
equilibrated with 10% ethanol. The eluates were
collected, and the fractions corresponding to the oxi-
dized products were combined, evaporated, and lyophi-
collected and dialyzed against 10 mM acetate buffer
13)
13
lized. Their structures were confirmed by C-NMR: (1)
0
(pH 5.0). Trehalase from A. niger
was partially
0
TreDC NMR ꢂc (D2O): 73.2 (C,C -4), 73.9 (C,C -2),
purified from Hemicellulase Amano 90 (Amano En-
zyme, Nagoya, Japan) by SP-Toyopearl column chro-
matography, as follows: Crude enzyme solution was
applied on a SP-Toyopearl column (3 ꢁ 20 cm, Tosoh).
Elution was carried out by linear gradient from zero to
1.0 M NaCl in 50 mM acetate buffer (pH 5.0). The active
fractions were combined and dialyzed against 10 mM
acetate buffer (pH 5.0).
0
0
0
0
7
6
7
3
1
4.2 (C,C -5), 74.8 (C,C -3), 97.1 (C,C -1), 175.9 (C,C -
0
). (2) SucDC NMR ꢂc (D2O): 64.2 (C -6), 73.8 (C-4),
0
0
4.8 (C-2), 75.3 (C-5), 75.5 (C-3), 78.5 (C -4), 78.8 (C -
0
0
0
), 82.4 (C -5), 94.5 (C -2), 107.2 (C-1), 179.4 (C -1),
79.5 (C-6), glucuronyl residue (C-1 to C-6), furucturo-
0
0
nyl residue (C -1 to C -6). (3) ꢀ,ꢁ-TreDC NMR ꢂc
D2O): 73.7 (C-4ꢀ), 73.8 (C-4ꢁ), 73.9 (C-2ꢀ), 74.0 (C-
(
5
ꢀ), 75.3 (C-3ꢀ), 75.3 (C-2ꢁ), 77.5 (C-5ꢁ), 77.6 (C-3ꢁ),
03.2 (C-1ꢀ), 105.8 (C-1ꢁ), 174.8 (C-6ꢁ), 175.6 (C-6ꢀ).
1
Enzyme activity assay.
(
4) PNP ꢀ-glucuronide NMR ꢂc (D2O): 73.5 (C-5), 74.5
(1) ꢀ-Glucuronidases. Standard assay method. A
reaction mixture consisting of 180 ml of 10 mM TreDC
in 100 mM sodium acetate–HCl buffer (pH 3.0) and
20 ml of enzyme solution was incubated at 40 C for
15 min. The reaction was stopped by adding an equal
0
(
C-2), 75.4 (C-4), 75.7 (C-3), 99.4 (C-1), 119.5 (C -2,6),
0 0 0
1
glucuronyl residue (C-1 to C-6), p-nitrophenyl residue
28.8 (C -3,5), 145.1 (C -4), 164.1 (C -1), 175.2 (C-6),
ꢀ
0
0
(
C -1 to C -6). (5) Methyl-O-ꢀ-D-glucosiduronic acid
NMR ꢂc (D2O): 57.9 (CH3–O–), 73.8 (C-4), 74.6 (C-2),
4.7 (C-5), 75.6 (C-3), 102.1 (C-1), 179.3 (C-6). (6)
volume (200 ml) of Somogyi solution. The reducing
sugars were measured by the Somogyi–Nelson method
7
0
0
14,15)
TreMC NMR ꢂc (D2O): 63.2 (C -6), 72.4 (C -4), 73.2
using D-glucuronic acid (GlcUA) as a standard.
One
0
0
(C -5), 73.6 (C -2), 73.8 (C-4), 74.9 (C-2), 75.0 (C-5),
unit of activity was defined as the amount of enzyme
liberating 2 mmol of GlcUA per min.
(2) Assay of hydrolysis activity against Me-GA-Xn-
0
0
7
5.2 (C -3), 75.7 (C-3), 96.0 (C-1), 96.0 (C -1), 175.6
C-6), glucuronyl residue (C-1 to C-6), glucosyl residue
(
(
0
0
C -1 to C -6). 6-O-ꢀ-(4-O-ꢀ-D-glucosyluronic acid)-D-
OH. The hydrolysis activity against Me-GA-X -OH was
n
ꢀ
glucosyl-ꢁ-cyclodextrine (GUG-CD) was supplied by
the Bio Research Corporation of Yokohama (Yoko-
measured at 40 C in a 15 min incubated reaction
mixture consisting of 180 ml of 5.8 mg/ml Me-GA-Xn-
OH in 100 mM sodium acetate–HCl buffer (pH 4.0) and
20 ml of enzyme solution. The reaction was terminated
by boiling for 5 min. The amount of 4-O-methyl-D-
glucuronic acid was measured by high-performance
anion-exchange chromatography with pulsed ampero-
1
0)
hama, Japan). Me-GA-Xn (Aldouronic Acid Mixture)
and 4-O-methyl-D-glucosyluronic acid-ꢀ-(1 ! 2)-xylo-
oligosaccharyl-ꢀ-(1 ! 2)-xylitol (Reduced Aldo-uronic
Acids, Me-GA-X -OH) were purchased from Mega-
n
zyme (Dublin, Ireland). Me-GA-X was composed of 4-
n

524
T. KIRYU et al.
metric detection (HPAEC-PAD), as described below,
using GlcUA as a standard. One unit of enzyme activity
was defined as the amount of enzyme liberating 1 mmol
of 4-O-methyl-D-glucuronic acid per min.
PAGE were done by the methods of Davis and Laemmli
1
6,17)
respectively.
The molecular mass standards were
phospholylase b (97.0 kDa), albumin (66.0 kDa), oval-
bumin (45.0 kDa), carbonic anhydrase (30.0 kDa), and
trypsin inhibitor (20.1 kDa) (Amasham Biosciences UK,
Buckinghamshire, England). The gel was stained for
protein with a Protein Silver Staining Kit (Bexel
Biotechnology, Union City, CA).
(
3) Trehalases. A substrate solution consisting of
1
80 ml of 10 mM trehalose in 100 mM acetate buffer
(
pH 5.0) and 20 ml of enzyme solution was mixed and
ꢀ
incubated at 40 C for 15 min. The reaction mixture was
boiled for 5 min to stop the reaction, and the amount of
D-glucose was measured by glucose oxidase regent
Estimation of molecular mass by gel filtration. The
molecular mass of TreDCase was estimated by gel
filtration under the following conditions: system, FPLC
system; pump, P-500; controller, LCC-500; detection,
absorbance at 280 nm; column, Superdex 200 (Pharma-
cia). The molecular mass standards used were alcohol
dehydrogenase (150.0 kDa), albumin (66.0 kDa), and
carbonic anhydrase (29.0 kDa) (Sigma Chemical).
(Diacolor GC, Toyobo, Osaka, Japan). One unit of
enzyme activity was defined as the amount of enzyme
liberating 2 mmol of D-glucose per min.
Purification procedure for TreDCase.
Step 1. Sephadex G-25 column chromatography.
Forty g of Cellurosine PE60 was dissolved in 20 ml of
1
0 mM acetate buffer (pH 4.0) (buffer A). After centri-
NMR. H- (300 MHz) and 13C-NMR (75 MHz) spec-
tra were recorded on a solution of about 1% in D2O
using a JEOL AL-FTNMR spectrometer. 3-(Trimehyl-
silyl)-1-propanesulfonic acid, sodium salt (DSS) was
used as an internal standard.
1
fugation, the supernatant was applied onto a Sephadex
G-25 column (4:0 ꢁ 40 cm, Tosoh) equilibrated with
buffer A for desalting. The enzyme was eluted by the
same buffer at a flow rate of 1.0 ml/min. The protein
fractions were combined.
Step 2. Q-Sepharose column chromatography. The
protein fraction was put on a column of Q-Sepharose
HPAEC-PAD. HPAEC-PAD was done under the
following conditions: system, Dionex DX-500; detector,
Model PAD II pulsed amperometric detector; column,
Dionex Carbopac PA-1 (4:0 ꢁ 250 mm); elution, linear
gradient from 150 to 300 mM sodium acetate in 100 mM
(3:0 ꢁ 20 cm, Pharmacia, Uppsala, Sweden) equilibrated
with buffer A. The enzyme was eluted by linear gradient
from zero to 0.5 M NaCl in buffer A at 0.5 ml/min. The
enzyme fractions were combined and dialyzed against
buffer A.
ꢀ
NaOH; flow rate, 1.0 ml/min; temperature, 35 C.
Step 3. SP-Toyopearl column chromatography. The
dialyzed solution was applied onto a column of SP-
Toyopearl (1:5 ꢁ 20 cm, Tosoh) equilibrated with buf-
fer A. The elution was done by linear gradient from zero
to 1.0 M NaCl in the same buffer at 0.5 ml/min. The
active fractions were collected.
Step 4. !-Aminododecyl-agarose column chromatog-
raphy. The active fraction was dialyzed against buffer A
and put on a column of !-aminododecyl-agarose
Thin layer chromatography. Thin layer chromatog-
raphy (TLC) was done with TLC plates (Kaisel Gel 60,
Merck, Darmstadt, Germany) and a solvent system of
ethyl acetate–acetic acid–water (3:1:1, v/v). Spots of
sugars were detected by heating the plate to 150 C after
spraying with 50% (w/w) sulfonic acid in methanol.
ꢀ
Effect of chemicals and metal ions. Reaction mixtures
(160 ml) consisting of TreDCase (0.01 U/ml) in 100 mM
sodium acetate–HCl buffer (pH 3.0) were incubated
with 20 ml of 20 mM CaCl , CuCl , MnCl , FeCl ,
(
1:5 ꢁ 10 cm, Sigma Chemical) equilibrated with buf-
fer A. The enzyme was eluted by linear gradient from
zero to 0.1 M NaCl in the buffer A at 0.5 ml/min. The
active fractions were collected and dialyzed against
buffer A.
2
2
2
3
MgSO , KCl, NaCl, AgNO , CoCl , NiCl , FeSO ,
4
3
2
2
4
ꢀ
HgCl2; SnCl2, SDS, or dithiothreitol at 40 C for 30 min.
Step 5. ProtEX-DEAE column chromatography.
ProtEX-DEAE was done under the following condi-
tions: system, fast protein liquid chromatography
After incubation, 20 ml of 100 mM TreDC was added and
incubated at 40 C for 15 min. The reaction was
terminated by boiling for5 min. The amount of GlcUA
was measured by HPAEC-PAD, and the remaining
activity was estimated.
ꢀ
(
FPLC, Pharmacia) system; pump, P-500; controller,
LCC-500; detection, absorbance at 280 nm; column,
ProtEX-DEAE (0:8 ꢁ 10 cm, Mitsubishi Chemical,
Tokyo); elution, linear gradient from zero to 0.1 M NaCl
in buffer A; flow rate, 0.5 ml/min. The active fractions
were combined and dialyzed against buffer A. The
dialyzed solution was used as a purified enzyme
preparation.
Kinetic parameters toward TreDC. The Km and Vmax
values toward TreDC were measured as follows: The
enzyme (0.042 U/ml) in 100 mM sodium acetate-HCl
buffer (pH 3.0) was incubated with TreDC in various
ꢀ
concentrations (2.5, 5.0, 10, 20 mM) at 40 C for 15 min.
After incubation, the amount of GlcUA was measured
Electrophoresis. Native polyacrylamide gel electro-
phoresis (PAGE) and sodium dodecyl sulfate (SDS)–
by the standard assay method. The Km and Vmax values
1
8)
were calculated from a Hanes–Woolf plot.
The

A New ꢀ-Glucuronidase from Aspergillus niger
Table 1. Summary of Purification of TreDCase
525
Total protein
(
Total activity
(Unit)
Specific activity
(Unit/A280)
Purification
(-fold)
Yield
(%)
Procedure
Crude enzyme
A280)
16,200
2,900
848
120
0.007
0.027
0.083
0.54
1.6
1
4
100
66
Sephadex G-25
Q-Sepharose
SP-Toyopearl
78.5
70.2
21.1
5.70
0.91
12
77
59
18
39.0
3.50
0.22
!-Aminododesyl-agarose
ProtEX-DEAE
229
586
4.8
0.76
4.1
amount of protein was estimated by the absorbance at
80 nm (light path, 1 cm), assuming that E1% was 10.0.
2
Substrate specificity. Enzyme solution (0.1 U/ml,
80 ml) in 100 mM sodium acetate–HCl buffer (pH 3.0)
1
was incubated with 20 ml of 100 mM TreDC, PNP ꢀ-
glucuronide, ꢀ,ꢁ-TreDC, TreMC, methyl-O-ꢀ-D-gluco-
siduronic acid, SucDC, GUG-CD, trehalose, or 58 mg/
ꢀ
ml Me-GA-X at 40 C. After 24 h of incubation, the
n
amounts of GlcUA and 4-O-methyl-D-glucuronic acid
were measured by HPAEC-PAD.
Anomeric types of hydrolysis products from TreDC.
The anomeric configurations of the hydrolysis products
were determined by the following method: A solution
containing 2.0 U of the purified enzyme was frozen by
liquid N2 and lyophilized. After lyophilization, the
substrate solution consisting 0.5 ml of 50 mM TreDC in
D2O at pH 3.0 was added (the pH was adjusted by
mixing 50 mM sodium TreDC and 50 mM TreDC). The
Fig. 1. SDS–PAGE of Purified Enzyme.
SDS–PAGE was done by 8% polyacrylamide slab gel. The gel
was stained for protein with silver staining regent. Lane 1, the
purified TreDCase. Ten mg of purified enzyme was applied. Lane 2,
SDS molecular mass standards including phospholylase b (97.0
kDa), albumin (66.0 kDa), ovalbumin (45.0 kDa), carbonic anhy-
drase (30.0 kDa), and trypsin inhibitor (20.1 kDa).
ꢀ
reaction mixture was incubated at 25 C for 8–50 min,
1
and H-NMR spectra were recorded.
58 kDa (Fig. 1). These results indicate that the enzyme
is composed of two identical subunits. Fungal ꢀ-
Results
glucuronidases are monomers, and no dimeric enzymes
1
,2,6–8)
Screening and purification of TreDCase
Commercial enzyme preparations were incubated
with 1.0% TreDC at 40 C for 18 h and the product,
from fungal origin have been reported. It is known
that ꢀ-glucuronidases from bacteria and snail are
3–5,9,11)
composed of two subunits (Table 2).
ꢀ
GlcUA, was detected by TLC. Out of 120 different
preparations, 6 exhibited TreDC hydrolysis activity. We
purified the enzyme, which was involved in the
hydrolysis of TreDC (TreDCase), from a commercial
product from A. niger, Cellurosine PE60 (HBI En-
zymes), which showed the strongest hydrolysis activity.
The results of purification are summarized in Table 1.
TreDCase was purified 586-fold. The yield was 0.76%.
At all steps, a single active peak was observed.
SP-Toyopearl, !-Aminododecyl-agarose, and ProtEX-
DEAE column chromatography were effective for
increasing specific activities, although the recoveries in
these steps were relatively low. Only one protein band
was observed, when 10 mg of the purified TreDCase was
analyzed with native PAGE followed by staining with a
silver staining regent. The molecular mass of TreDCase
was 120 kDa as estimated by gel filtration. On the other
hand, one protein band was also observed by SDS–
PAGE, and its molecular mass was estimated to be
Effects of pH, temperature, and chemicals
As shown in Fig. 2, the enzyme was stable in a pH
range of 2.0–4.0 at 30 C, and the optimum pH was
pH 3.0–3.5. TreDCase had the most acidic optimum pH
ꢀ
among the fungal and bacterial ꢀ-glucuronidases pre-
2
–9)
viously reported (Table 2).
ꢀ
The enzyme was stable
up to 30 C after incubation for 24 h at pH 4.0, and
ꢀ
activity was highest at 40 C. When the enzyme was
treated with 2.0 mM of various chemicals and metal ions,
2
þ
Hg (residual activity, 42%) and SDS (52%) inhibited
þ
þ
2þ
2þ
2þ
activity (data not shown). K , Na , Ca , Cu , Mg ,
þ
2
Ag , and dithiothreitol slightly activated TreDCase
2
þ
3þ
2þ
(110–118%), and Mn , Fe , and Fe marginally
2
þ
2þ
2þ
inhibited it (80–87%). Co , Ni , and Sn showed no
effect (101–103%).
Substrate specificity
The kinetic parameters of TreDCase toward TreDC

526
T. KIRYU et al.
Table 2. Comparison of Properties of ꢀ-Glucuronidases
A. niger
Intracellular)
Properties
TreDCase
Snail acetone powder
(
Molecular weight
by SDS–PAGE (kDa)
by gel filtration (kDa)
Optimum pH
58 (dimer)
120
130 (monomer)
97 (dimer)
180
3.0
150
4.8
3.0–3.5
2.0–4.0
40
pH stability
Optimum temperature ( C)
4.5–7.0
60
<50
3.0–7.0
50
<50
ꢀ
ꢀ
Thermostability ( C)
<30
Reference
This study
1
11, 20
Fig. 2. Effect of pH (A) and Temperature (B) on Activity and Stability of TreDCase.
(
A) Effects on activity (—): Reaction mixtures (180 ml) consisting of TreDCase (0.02 U/ml) in 100 mM sodium acetate–HCl buffer (pH 1.5–
ꢀ
4
.0, ) or acetate buffer (pH 4.0–5.5, ) were incubated with 20 ml of 100 mM TreDC at 40 C for 15 min. Effect on stability (- - -): TreDCase
(
0.2 U/ml, 200 ml) in 50 mM sodium acetate–HCl buffer (pH 1.5–4.0, ), acetate buffer (pH 4.0–5.5, ), or MacIlvain buffer (pH 5.0–7.0,
)
were incubated at 30 C for 24 h. Then these solutions were diluted with 1.8 ml of 10 mM TreDC in 100 mM sodium acetate–HCl buffer (pH 3.0),
ꢀ
and the residual activities were assayed by the standard assay method. (B) Effect on activity (—): Reaction mixtures (180 ml) consisting
TreDCase (0.02 U/ml) and TreDC (10 mM) in 100 mM sodium acetate–HCl buffer (pH 3.0) were incubated at various temperatures (20–60 C)
ꢀ
for 15 min. Effect on stability (- - -): The enzyme solutions (180 ml) consisting of TreDCase (0.02 U/ml) in 100 mM sodium acetate–HCl buffer
ꢀ
(
pH 3.0) were incubated at various temperatures (5–50 C) for 24 h. Twenty ml of 100 mM TreDC was added and the remaining activities were
measured by the standard method.
were measured as described in Materials and Methods.
The Km and Vmax values were calculated to be 4.4 mM
and 50 mmol of TreDC hydrolyzed/min/mg protein
respectively. Specificities of TreDCase on other
substrates are shown in Table 3. TreDCase did not
hydrolyze TreMC in which one of two glucosyl residues
of trehalose was changed to a glucuronyl residue, ꢀ,ꢁ-
TreDC, which had the ꢀ,ꢁ-1,1 glucuronyl linkage, and
SucDC. Furthermore, other ꢀ-glucuronides, such as PNP
or Me-GA-Xn-OH.
Figure 3B shows a comparison of the substrate
specificity of TreDCase with those of trehalases.
TreDCase did not hydrolyze trehalose. Trehalases from
both porcine kidney and A. niger did not degrade
TreDC. These results indicate that trehalases and
TreDCase are entirely different enzymes.
Time course of TreDC hydrolysis by TreDCase under
high substrate concentration conditions
ꢀ
-glucuronide and methyl-O-ꢀ-D-glucosiduronic acid,
and GUG-CD, which had an ꢀ-1,4 glucuronyl linkage,
were not degraded at all.
The hydrolysis activities of TreDCase for Me-GA-Xn
In view of possible industrial applications of
TreDCase, in, for instance, the production of GlcUA
from TreDC, the effects of high concentrations of
substrate on reaction rate were studied by incubating the
enzyme with 100–300 mM of TreDC (Fig. 4). TreDCase
completely hydrolyzed TreDC below 200 mM of TreDC
and produced a stoichiometric amount of GlcUA. Even
at a higher concentration of TreDC (300 mM), about
80% of TreDC was degraded.
and Me-GA-X -OH, which were known as substrates of
n
usual ꢀ-glucuronidases (Table 3 and Fig. 3A) was
examined. The Me-GA-Xn hydrolyzing ꢀ-glucuronidase
2
,12)
from A. niger was used as a control enzyme.
The
previously reported ꢀ-glucuronidase from A. niger
degraded Me-GA-Xn-OH and hydrolyzed TreDC slight-
ly. In contrast, TreDCase did not hydrolyze Me-GA-Xn

A New ꢀ-Glucuronidase from Aspergillus niger
527
Table 3. Substrate Specificities of Various ꢀ-Glucuronidases
Hydrolysis activity
Substrate
TreDCase
A. niger (Intracellular)
Snail acetone powder
T. maritima
TreDC
Me-GA-Xn
PNP-ꢀ-GA
þ
—
þ
þ
N.D.
—
—
—
—
—
—
—
—
þ
—
þ
þ
ꢀ,ꢁ-TreDC
TreDC
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Me-ꢀ-GA
SucDC
GUG-CD
Reference
This study
1, 27
11, 23
9
Me-GA-Xn, 4-O-methyl glucosyluronic acid xylooligosaccharide; PNP-ꢀ-GA, PNP ꢀ-glucuronide; Me-ꢀ-GA, methyl ꢀ-D-glucosiduronic acid; N.D., not determined.
Fig. 3. Courses of Hydrolysis of TreDC, Me-GA-Xn-OH, and Trehalose by TreDCase, ꢀ-Glucuronidase, and Trehalases.
A) Courses of hydrolysis of TreDC and Me-GA-Xn-OH by TreDCase and ꢀ-glucuronidase. Reaction mixtures (1.0 ml) containing 0.01 U of
(
enzyme (TreDCase, ; partially purified A. niger ꢀ-glucuronidase, ), and substrate (0.01 mmol of TreDC, —; 5.8 mg of Me-GA-Xn-OH, - - -) in
ꢀ
1
00 mM sodium acetate–HCl buffer (pH 3.0, TreDCase) or acetate buffer (pH 4.0, partially purified ꢀ-glucuronidase) were incubated at 40 C.
After incubation, the amounts of GlcUA (—) and 4-O-methyl-D-glcuronic acid (- - -) were measured by HPAEC-PAD. Note that no 4-O-methyl-D-
glcuronic acid was liberated by TreDCase from Me-GA-Xn-OH. (B) Courses of hydrolysis of TreDC and trehalose by trehalases. Reaction
mixtures (0.9 ml) consisting of 10 mM substrate (TreDC, —; trehalose, - - -) in 100 mM sodium acetate buffer (pH 3.0, TreDCase) or acetate buffer
(pH 5.0, trehalases) were incubated with 0.1 ml of 0.01 U/ml enzyme (TreDCase, ; porcine kidney trehalase, ; partially purified trehalase of
A. niger, ) at 40 C. The amounts of GlcUA (—) and glucose (- - -) were measured by HPAEC-PAD and glucose oxidase regent respectively.
ꢀ
Anomeric type of hydrolysis products from TreDC
The anomeric configurations of the hydrolysis prod-
ucts of TreDC were determined by measuring the ratio
1
of ꢀ-GlcUA and ꢁ-GlcUA with H-NMR (Fig. 5). A
3
doublet at 5.19 ppm ( J 3.9 Hz) was assigned to H-1ꢀ
0
3
and H -1ꢀ of TreDC, and doublets at 5.25 ppm ( J
3
3.9 Hz) and 4.48 ppm ( J 7.9 Hz) were assigned to H-1ꢀ
and H-1ꢁ of GlcUA respectively. The intensity of a
doublet from TreDC decreased and the intensity of
doublets from ꢀ- and ꢁ-GlcUA increased with the
progress of hydrolysis. The intensity ratios of the
products showed equimolar production of the two
anomars during the early stage of reaction (8–50 min).
Since the rate of mutarotation from ꢀ-GlcUA to ꢁ-
GlcUA was slow enough not to affect these results, ꢁ-
GlcUA was probably formed by the inversion of
anomeric configuration.
Fig. 4. Time Courses of TreDC Hydrolysis by TreDCase at High
Substrate Concentration.
Reaction mixtures (1.0 ml) consisting TreDC (100 mM,
00 mM, ; 300 mM, ) and TreDCase (1.2 U/ml) in 100 mM
sodium acetate–HCl buffer (pH 3.0) were incubated at 40 C for
–72 h. The amounts of TreDC (ꢂ ꢂ ꢂ) and GlcUA (—) were measured
by HPAEC-PAD.
;
2
ꢀ
0

528
T. KIRYU et al.
9
)
ing that TreDC is a poor substrate for the enzyme. The
broad substrate specificity of the snail enzyme was
antipodal against the strict substrate specificity of
TreDCase.
TreDCase inverted the anomeric configuration of the
substrate (Fig. 5). Biely et al. reported that extracellular
A. tubingensis ꢀ-glucuronidase produced products with
2
1)
an inverted anomeric configuration. Since most ꢀ-
glucuronidases have been classified into glycosyl hydro-
lase family 67 based on amino-acid sequence similarity,
they proposed that the ꢀ-glucuronidases that belong to
2
2,23)
the family are inverting enzymes.
inversion and retention, have been proposed for the
reaction of glycosylases.
Two mechanisms,
2
4)
In general, inverting en-
zymes catalyze hydrolysis by the single displacement
mechanism, whereas retaining enzymes hydrolyze sub-
strates by the double displacement mechanism. Some
glycosidases, such as glucoamylase and trehalase, are
2
5–27)
known to be inverting enzymes.
properties of TreDCase, high substrate specificity for
,ꢀ-1,1 glycosidic linkage and inversion of the anomeric
The catalytic
ꢀ
Fig. 5. 1H-NMR Spectra of TreDC and Hydrolysis Products by
TreDCase.
A reaction mixture (0.5 ml) consisting of TreDCase (1.0 U/ml)
configuration of substrates, are similar to those of
trehalases.
and TreDC (25 mM) was incubated for 8–50 min. The 1H-NMR
spectra of the reaction mixture were measured. The assignments of
Since TreDC is composed of two glucuronyl residues,
TreDCase is probably available for producing GlcUA.
GlcUA has been used as a medicine, because it helps in
detoxification, improvement of liver function, and
recovery from fatigue. GlcUA is manufactured by
chemical oxidation of starch followed by acid-cata-
lyzed-hydrolysis. The yield of GlcUA chemical produc-
tion, however, is still low (less than 20%), and many
byproducts are produced. TreDCase completely hydro-
lyzed 200 mM of TreDC in 72 h to produce GlcUA in
100% yield (Fig. 4). TreDCase, therefore, might be
applicable in the production of GlcUA.
3
crucial signals are indicated. A doublet 5.19 ppm ( J 3.9 Hz) was
0
3
assigned to H-1ꢀ and H -1ꢀ of TreDC. Doublets at 5.24 ppm ( J
3
3.9 Hz) and 4.64 ppm ( J 7.9 Hz) were assigned to H-1ꢀ and H-1ꢁ of
GlcUA respectively. The numbers on the doublets indicate rates of
H-1ꢀ and H-1ꢁ of GlcUA signals.
Discussion
We obtained a new type of ꢀ-glucuronidase which
was highly specific for TreDC. The enzyme did not
hydrolyze Me-GA-Xn or its derivatives, the common
substrates for the reported ꢀ-glucuronidases. The en-
zyme described here hydrolyzed neither TreMC, ꢀ,ꢁ-
TreDC, SucDC, GUG-CD, nor Me-GA-X (Table 3).
n
This suggests that the enzyme is highly specific for
TreDC, and that it recognized and required the two ꢀ,ꢀ-
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