Molecular cloning and expression in Pichia pastoris of a Irpex lacteus exo-/β-(1→3)-galactanase gene

Article abstract of DOI:10.1271/bbb.90433

A gene encoding exo-β-(1-3)-galactanase from Irpex lacteus was cloned by reverse transcriptase-PCR. The deduced amino acid sequence showed high similarity with exo-β-(1-3)-galactanases from other sources. The molecular mass of the mature form was calculat

Full text of DOI:10.1271/bbb.90433

Biosci. Biotechnol. Biochem., 73 (10), 2303–2309, 2009
Molecular Cloning and Expression in Pichia pastoris
of a Irpex lacteus Exo-ꢀ-(1 ! 3)-galactanase Gene
1;2
1
1
1
3
Toshihisa KOTAKE, Kiminari KITAZAWA, Ryohei TAKATA, Kohei OKABE, Hitomi ICHINOSE,
3
1;y
Satoshi KANEKO, and Yoichi TSUMURAYA
1
Division of Life Science, Graduate School of Science and Engineering, Saitama University,
2
55 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
Institute for Environmental Science and Technology, Saitama University,
2
2
55 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
Food Biotechnology Division, National Food Research Institute,
3
2
-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Received June 18, 2009; Accepted July 8, 2009; Online Publication, October 7, 2009
[doi:10.1271/bbb.90433]
A gene encoding exo-ꢀ-(1 ! 3)-galactanase from
from AGPs.8,9) Bacteria and fungi possess several
enzymes acting specifically on the carbohydrate moie-
ties of AGPs. A ꢀ-glucuronidase (EC 3.2.1.31) from
Aspergillus niger categorized in GHF 79 releases GlcA
Irpex lacteus was cloned by reverse transcriptase-PCR.
The deduced amino acid sequence showed high sim-
ilarity with exo-ꢀ-(1 ! 3)-galactanases from other
sources. The molecular mass of the mature form was
calculated to be 45,520 Da. The gene product expressed
in Pichia pastoris specifically hydrolyzed ꢀ-(1 ! 3)-
galactooligosaccharides, as did other exo-ꢀ-(1 ! 3)-
galactanases. The recombinant enzyme showed high
activity toward arabinogalactan-proteins (AGPs) from
radish as well as ꢀ-(1 ! 3)-galactan. Product analysis
revealed that the enzyme released ꢀ-(1 ! 6)-galacto-
biose, ꢀ-(1 ! 6)-galactotriose, and ꢁ-L-arabinofuranosyl-
1
0,11)
and 4-Me-GlcA from AGPs.
An endo-ꢀ-(1 ! 6)-
galactanase (EC 3.2.1.164) isolated from Trichoderma
viride is an enzyme that specifically hydrolyzes ꢀ-
(1 ! 6)-galactosyl side chains of AGPs, releasing Gal
1
2)
and ꢀ-(1 ! 6)-galactooligomers. This novel enzyme
has been categorized in GHF 5 based on the amino acid
1
3)
sequence. Exo-ꢀ-(1 ! 3)-galactanase (EC 3.2.1.145)
was first isolated from Driselase, a commercial enzyme
preparation from Irpex lacteus, and was found to act on
the ꢀ-(1 ! 3)-galactan backbones of the carbohydrate
moieties of AGPs. The enzyme, designated Il1,3Gal,
hydrolyzes ꢀ-(1 ! 3)-galactan bypassing side chains,
and releases at least 20 (1 ! 6)-linked ꢀ-galactosyl
groups and their acidic derivatives replaced with single
uronosyl residues at the nonreducing terminals from
(
1 ! 3)-ꢀ-galactosyl-(1 ! 6)-galactose together with Gal
from ꢀ-(1 ! 3)-galactans attached with and without ꢀ-
(
1 ! 6)-galactosyl branches prepared from acacia gum.
These results indicate that the exo-ꢀ-(1 ! 3)-galacta-
nase from I. lacteus efficiently hydrolyzes ꢀ-(1 ! 3)-
galactan main chains of AGPs by bypassing ꢀ-(1 ! 6)-
galactosyl side chains.
1
4)
AGPs. Recently, we isolated a gene encoding exo-ꢀ-
(
1 ! 3)-galactanase, Pc1,3Gal, from Phanerochaete
Key words: arabinogalactan-protein;
exo-ꢀ-(1 ! 3)-
chrysosporium based on the amino acid sequences of
native Il1,3Gal. Pc1,3Gal appeared to belong to GHF 43,
and had a functional domain with similarity to carbohy-
drate-binding module (CBM) 6. We also found that
functional exo-ꢀ-(1 ! 3)-galactanase is distributed in
bacteria such as Clostridium thermocellum and Strepto-
galactanase; Irpex lacteus; Pichia pastoris;
recombinant enzyme
1
5)
The carbohydrate moieties of arabinogalactan-pro-
teins (AGPs) consisting of ꢀ-(1 ! 3)(1 ! 6)-galactan
backbones and other auxiliary sugars such as glucuronic
acid (GlcA), 4-O-methyl-GlcA (4-Me-GlcA), L-rham-
nose, and L-fucose are degraded by various glycoside
1
6,17)
myces avermitilis,
but the gene encoding the native
exo-ꢀ-(1 ! 3)-galactanase of I. lacteus remains to be
isolated.
1
–3)
hydrolases in nature.
(
A type of plant ꢀ-galactosidase
EC 3.2.1.23) belonging to glycoside hydrolase family
Here we report the isolation of a cDNA clone
encoding exo-ꢀ-(1 ! 3)-galactanase from I. lacteus
and the properties of the recombinant enzyme expressed
in Pichia pastoris. The results indicate that Il1,3Gal
shares high sequence similarity with Pc1,3Gal and
efficiently hydrolyzes ꢀ-(1 ! 3)-galactan main chains
of AGPs by bypassing ꢀ-(1 ! 6)-galactosyl side chains.
(
ꢀ
GHF) 35 specifically hydrolyzes the ꢀ-(1 ! 3)- and
4
–7)
-(1 ! 6)-galactosyl residues of AGPs.
In addition,
an enzyme of GHF 3 having both ꢁ-L-arabinofuranosi-
dase (EC 3.2.1.55) and ꢀ-xylosidase (EC 3.2.1.37)
activities from radish has been found to release L-Ara
y
To whom correspondence should be addressed. Fax: +81-48-858-3384; E-mail: tsumura@mail.saitama-u.ac.jp
Abbreviations: ABEE, p-aminobenzoic acid ethyl ester; AGP, arabinogalactan-protein; An1,3Gal, Aspergillus niger exo-ꢀ-(1 ! 3)-galactanase;
CBM, carbohydrate-binding module; CM, carboxymethyl; Endo H, endo-glycosidase H; GHF, glycoside hydrolase family; GlcA, glucuronic acid;
HPLC, high performance liquid chromatography; Il1,3Gal, Irpex lacteus exo-ꢀ-(1 ! 3)-galactanase; 4-Me-GlcA, 4-O-methyl-glucuronic acid;
Nc1,3Gal, Neurospora crassa exo-ꢀ-(1 ! 3)-galactanase; PAGE, polyacrylamide gel electrophoresis; Pc1,3Gal, Phanerochaete chrysosporium exo-
ꢀ
-(1 ! 3)-galactanase; PNP, p-nitrophenyl; RACE, rapid amplification of cDNA ends; RT-PCR, reverse-transcriptase-PCR; TLC, thin layer
chromatography

2304
T. KOTAKE et al.
ꢀ
Materials and Methods
1% (w/v) glycerol at 30 C with shaking at 100 rpm for 2 d. The cells
were harvested by centrifugation at 3,000 rpm for 5 min, washed with
ice-cold distilled water, and then suspended in 50 ml of YPM medium
containing 1% (w/v) yeast extract, 2% (w/v) peptone, and 1% (v/v)
Culture of I. lacteus. I. lacteus (NBRC5367) was purchased from
NITE Biological Resource Center (Tokyo). Mycelia of I. lacteus were
cultured in liquid PS medium containing 20% (w/v) potato extract and
ꢀ
methanol. The yeast cells were cultured for a further 14 d at 30 C,
ꢀ
during which time 0.5 ml of methanol was added each day, to induce
the recombinant enzyme. For purification of the recombinant enzyme,
the culture medium of Pichia cells was centrifuged at 8,000 rpm for
2% (w/v) sucrose (pH 6.0) at 25 C for 7 d. The submerged culture
was then inoculated to PS medium containing 1.5% (w/v) agar and
ꢀ
cultured at 25 C for 3 d.
1
5 min and the supernatant was collected. After dialysis against 10 mM
sodium acetate buffer (pH 4.6), the sample was first applied to a
:2 ꢁ 23-cm Toyopearl HW40 (Tosoh, Tokyo) column to remove
pigments, and then adsorbed onto a 1:4 ꢁ 5:5-cm SP-Toyopearl 650M
Tosoh) column that had been equilibrated with the buffer. The column
was eluted with a linear gradient of 0–500 mM NaCl in the buffer
total 100 ml). The active fractions were collected and applied to a
cDNA isolation. The cDNA encoding exo-ꢀ-(1 ! 3)-galactanase
was cloned by reverse-transcriptase-PCR (RT-PCR). Total RNA was
extracted from mycelia of I. lacteus. The mycelia were frozen in liquid
nitrogen, homogenized with mortar and pestle, and extracted with an
Isogen kit (Nippon Gene, Tokyo) according to the manufacturer’s
instructions. Single-strand cDNA was synthesized from approximately
2
(
(
2
ꢁ 84-cm Sephacryl S-200 (GE Healthcare Bio-Sciences) column
1
ReverTra Ace-ꢁ-(Toyobo, Osaka, Japan), and oligo(dT)-adaptor
mg of total RNA from the mycelia using a reverse-transcriptase,
equilibrated with the same buffer. The purity of the recombinant
enzyme was determined on SDS-polyacrylamide gel electrophoresis
0
0
primer (5 -GCGACATCATCGAATTCCGATGTTTTTTTTTTTTT-3 ).
0
(
PAGE).18) The enzyme in the gel was stained with Coomassie
For cloning of the cDNA, a set of degenerate primers, F-1 (5 -GCN-
0
Brilliant Blue R-250. The concentration of protein was determined by
the method of Bradford using bovine serum albumin as the standard.1
0
GCNTGGACNGAYAC-3 ) and R-1 (5 -TGNGCNGARTCRTCRT-
0
9)
CYTG-3 ), was designed based on the amino acid sequences,
AAWTDT and QDDDSAQ, respectively, determined for the native
Digestion of exo-ꢀ-(1 ! 3)-galactanase with endo-glycosidase.
Native exo-ꢀ-(1 ! 3)-galactanase was purified by conventional
chromatographic techniques from Driselase, a commercial enzyme
preparation from I. lacteus, as described previously.14) The native
enzyme. The determined amino acid sequences were reported in a
previous paper.15) PCR was performed with ExTaq (Takara Bio, Ohtsu,
ꢀ
Japan) under the following conditions: denaturing at 94 C for 0.5 min,
ꢀ
ꢀ
annealing at 55 C for 0.5 min, and amplification at 72 C for 1.0 min,
5 cycles. The amplified cDNA fragment (0.4 kb) was subcloned into a
pGEM T-Easy vector (Promega, Madison, WI), and the nucleotide
(
3 mg) and recombinant (3 mg) exo-ꢀ-(1 ! 3)-galactanases were
3
ꢀ
denatured in the denaturing buffer at 100 C from 5 min and then
digested with 50 units of endo-glycosidase H (Endo H, New England
sequence was determined with an ABI PRISM 310 genetic analyzer
0
ꢀ
Biolabs, Ipswich, UK) at 37 C for 14 h. The enzymes were separated
on SDS–PAGE.
(Applied Biosystems, Foster City, CA). The 3 -region was amplified
with proofreading polymerase (KOD-Plus, Toyobo) using an internal
0
0
0
specific primer, 3 RACE-1 (5 -ACAACATCGTTGAGCGTCCC-3 ),
0
0
Substrate specificity. The enzymatic activity of the purified
recombinant enzyme was measured using 0.25% (w/v) polysaccharide
or 2.5 mM galactooligosaccharide as the substrate. The reaction mixture
and an adaptor primer (5 -GCGACATCATCGAATTCCGATG-3 )
ꢀ
under the following conditions: denaturing at 94 C for 0.5 min,
ꢀ
ꢀ
annealing at 55 C for 0.5 min, and amplification at 68 C for 2.0 min,
0
35 cycles. The 5 -region of the cDNA was amplified with a kit for rapid
amplification of 5 cDNA ends (5 -RACE kit, Invitrogen, Carlsbad)
(total volume, 0.1 ml) consisting of the recombinant enzyme, substrate,
and 50 mM sodium acetate buffer (pH 4.6) was incubated at 37 ꢀC, and
0
0
0
0
this condition was defined as the standard assay condition. The activity
of the recombinant enzyme toward polysaccharides was determined as
the increase in reducing terminals of liberated sugars by the Somogyi-
Nelson method.20,21) One unit of enzyme activity was defined as the
amount that liberates 1 mmol of reducing sugars per min. Mono- and
oligosaccharides in enzymatic hydrolysates were separated by thin
layer chromatography (TLC) on Silica gel 60F254 (Merck, Darmstadt,
Germany) using 7:1:2 (v/v/v) 1-propanol/ethanol/water as the
solvent, and were detected by charring after spraying TLC plates with
using internal specific primers, 5 RACE-0 (5 -GTTTTCGCAGTTGC-
0
0
0
0
GACACC-3 ), 5 RACE-1 (5 -ACAAGGCACTGTTCTCCGTC-3 ),
0
0
0
and 5 RACE-2 (5 -AGTAAAACGTGCTTCCGACC-3 ). The coding
region was amplified with proofreading polymerase (KOD-Plus), and
the nucleotide sequence was determined.
Oligo- and polysaccharides. Carboxymethyl (CM)-cellulose 4M, ꢀ-
(
1 ! 4)-galactan from lupin, and ꢀ-glucan from barley were pur-
chased from Megazyme (Wicklow, Ireland). Acacia gum from acacia
tree, laminarin from Laminaria digitata, 4-O-methyl-glucuronoxylan,
xylan from birch wood, p-nitrophenyl (PNP)-ꢁ-L-arabinofuranoside,
PNP-ꢀ-galactopyranoside, PNP-ꢀ-glucopyranoside, PNP-ꢀ-glucuro-
nide, and PNP-ꢀ-xylopyranoside were from Sigma (St. Louis, MO).
CM-curdlan was from Wako (Osaka, Japan). ꢀ-(1 ! 3)–Galactan and
related ꢀ-galactans were prepared from acacia gum by once, twice, and
2
0% (v/v) H2SO4-methanol.
Mode of action. The mode of action of the recombinant enzyme was
analyzed using ꢀ-(1 ! 3)-galactan, polymers I and II, and native AGP
from radish roots. The reaction mixtures (total volume, 50 ml)
containing 0.1% (w/v) polysaccharide, the recombinant enzyme, and
ꢀ
thrice Smith degradation, and were designated polymers I, II, and III
respectively as described in a previous paper.14) In the present study,
50 mM sodium acetate buffer (pH 4.6) were incubated at 37 C for
0–24 h. At appropriate time intervals, a portion was withdrawn and
inactivated by heating. The reducing sugars liberated were coupled at
their reducing terminals with p-aminobenzoic acid ethyl ester (ABEE)
by the method of Matsuura and Imaoka.22) The ABEE-derivatized
sugars were analyzed on an high performance liquid chromatography
polymer III was used as ꢀ-(1 ! 3)–galactan, although it contains a
few ꢀ-(1 ! 6)-galactosyl side chains (Supplemental Fig. 1; see Biosci.
Biotechnol. Biochem. Web site). Native and ꢁ-L-arabinofuranosidase-
treated AGPs from radish roots and leaves, ꢀ-(1 ! 3)(1 ! 6)-galactan
from Prototheca zopfii, ꢀ-(1 ! 3)-, ꢀ-(1 ! 4)-, and ꢀ-(1 ! 6)-
(HPLC) system equipped with a TSKgel Amide-80 (4:6 mm ꢁ
250 mm; Tosoh) column. The column was eluted with a linear gradient
galactooligosaccharides, and sugar beet arabinan were prepared as
described previously.6,12)
of CH CN/water, from 74:26 to 58:42 (v/v), for 40 min at a flow rate
3
ꢀ
of 1 ml/min at 40 C. The ABEE-sugars were monitored with
fluorescence detector model RF-10AXL at 305 nm (excitation) and
Heterologous expression of exo-ꢀ-(1 ! 3)-galactanase in Pichia
pastoris. Partial cDNA corresponding to the region from Glu21 to
Tyr448 was amplified with specific primers and subcloned into a
pGEM5zfþ vector (Promega). After confirmation of the nucleotide
sequence, the cDNA fragment was inserted between the EcoRI and
XbaI sites, preceded by yeast ꢁ-factor of pPICZꢁC (Invitrogen).
Methylotrophic yeast P. pastoris strain KM71 was transformed with
the linearized plasmid construct with a multicopy Pichia expression kit
3
60 nm (emission).
Results and Discussion
Isolation of cDNA for exo-ꢀ-(1 ! 3)-galactanase and
heterologous expression in P. pastoris
In our previous study, partial amino acid sequences
of native exo-ꢀ-(1 ! 3)-galactanase, Il1,3Gal, purified
from Driselase were determined, and this led to the
(
Invitrogen). Transformants resistant to zeocin were screened accord-
ing to the manufacturer’s instructions. The zeocin-resistent colony was
cultured in 800 ml of 1% (w/v) yeast extract, 2% (w/v) peptone and

Exo-ꢀ-(1 ! 3)-galactanase from Irpex lacteus
2305
*
*
*
Fig. 1. Amino Acid Sequence of Il1,3Gal.
The amino acid sequence of Il1,3Gal was aligned with those of Pc1,3Gal from P. chrysosporium, Nc1,3Gal from N. crassa, and An1,3Gal
from A. niger by the pairwise method using the ClustalW program. Solid lines indicate the amino acid sequences corresponding to those
determined for native Il1,3Gal purified from Driselase. Asterisks indicate putative N-glycosylation sites of Il1,3Gal. The conserved residues are
highlighted in black. A region corresponding to CBM6 is boxed. Accession numbers for the clones are as follows: An1,3Gal, XP 001393182;
Il1,3Gal, AB461394; Nc1,3Gal, XP 958027; Pc1,3Gal43A, BAD98241.
isolation of several genes encoding exo-ꢀ-(1 ! 3)-
for this discrepancy is not clear, but it might arise
because the strain used in the preparation of the native
enzyme and that used in cDNA cloning were different.
The amino acid sequence of Il1,3Gal showed highest
similarity to exo-ꢀ-(1 ! 3)-galactanase from P. chrys-
osporium (Pc1,3Gal, identity, 85%), and shared signifi-
cant similarity with other exo-ꢀ-(1 ! 3)-galactanases
from other sources, including Neurospora crassa
(Nc1,3Gal, identity 41%) and Aspergillus niger
(An1,3Gal, identity 38%) (Fig. 1). It is probable that
an exo-ꢀ-(1 ! 3)-galactanase identified previously in
1
5–17)
galactanases from various microorganisms.
In the
present study, we isolated the cDNA encoding Il1,3Gal
based on the amino acid sequences and expressed the
recombinant protein in P. pastoris.
First we amplified a partial cDNA fragment of
Il1,3Gal by RT-PCR using a set of degenerate primers
designed based on the amino acid sequences determined
for native Il1,3Gal. The sequence of full-length mRNA
0
0
was determined by 5 RACE and 3 RACE procedures.
The cloned cDNA appeared to encode a polypeptide of
2
3)
4
48 amino acids, including sequences that corresponded
A. niger is encoded by the An1,3Gal gene.
to the amino acid sequences of native Il1,3Gal (Fig. 1).
The sequence contained a putative signal sequence (20
amino acid residues) preceding the N-terminal sequence
of the mature enzyme. Il1,3Gal appeared to have a
CBM6 in the C-terminal region (Glu329-Val447),
sharing high similarity with Pc1,3Gal. Three putative
N-glycosylation sites were found in the deduced protein
sequence (Fig. 1, indicated by asterisks). The amino
acid sequence deduced from Il1,3Gal cDNA did not
completely match the peptide sequences determined for
the native enzymes: among the N-terminal 24 residues,
three residues differed from those determined for the
native enzyme, and two residues out of 30 also
mismatched in the internal region (Fig. 1). The reason
Expression of recombinant Il1,3Gal
Partial cDNA corresponding to mature exo-ꢀ-
(1 ! 3)-galactanase (Glu21-Tyr448) was fused to a
yeast secretion signal sequence (ꢁ-factor) and expressed
in the methylotrophic yeast P. pastoris. Recombinant
Il1,3Gal (rIl1,3Gal) was induced under the control of the
alcohol oxidase promotor and purified from the culture
medium by conventional chromatography (Table 1).
Since the recombinant enzyme was produced at high
concentration in the culture medium by prolonged
induction (14 d), the specific activity did not increase
during the purification procedures. The specific activity
(47.6 units/mg protein) of rIl1,3Gal was comparable to
1
5)

2306
T. KOTAKE et al.
1
1
20
00
S
1
2
3
4
S
Sodium phosphate
Sodium acetate
MES-NaOH
kDa
A
9
4
8
0
0
Tris-HCl
6
7
6
4
3
3
0
40
2
0
0
2
3
4
5
pH
6
7
8
1
20
2
0
100
B
8
6
4
0
0
0
Fig. 2. SDS–PAGE of Native and Recombinant Il1,3Gals.
The native and recombinant exo-ꢀ-(1 ! 3)-galactanases (approx-
imately 1 mg) were analyzed by SDS–PAGE. The enzymes were
digested with Endo H to remove N-linked glycans. Protein in the gel
was stained with Coomassie Brilliant Blue R-250. Lanes S,
molecular mass markers; lane 1, native Il1,3Gal purified from
Driselase; lane 2, native Il1,3Gal digested with Endo H; lane 3,
rIl1,3Gal expressed in P. pastoris; lane 4, rIl1,3Gal digested with
Endo H.
20
0
1
0
20
30
40
50
60
Temperature (ºC)
Fig. 3. Effects of pH and Temperature on the Activity of rIl1,3Gal.
A, Activity-pH curves resulting from experiments with reaction
mixtures with the following buffers (50 mM) of differing pH:
diamond, sodium phosphate; square, sodium acetate; triangle,
Table 1. Purification of rIl1,3Gal Expressed in P. pastoris
Total
Total
Specific
Purifi
protein activity activity -cation
Yield
2
The enzyme was incubated with 0.25% (w/v) ꢀ-1,3-galactan at
-morpholinoethanesulfonic acid (MES)-NaOH; circle, Tris–HCl.
ꢀ
mg
units
units/mg
protein
65.4
-fold
%
37 C. B, Activity-temperature curve of exo-ꢀ-(1 ! 3)-galactanase
activity resulting from incubation at different temperatures. A
reaction mixture containing 50 mM acetate buffer (pH 4.6) and
0.25% (w/v) ꢀ-1,3-galactan as substrate was used. Activity is
expressed as percent of that determined under standard assay
condition (see ‘‘Materials and Methods’’).
Culture medium
SP-Toyopearl
Sephacryl S-200
23.7
17.8
9.8
1550
1032
467
1.0
0.8
0.7
100.0
66.6
30.1
58.0
47.6
Activity was measured using ꢀ-1,3-galactan as the substrate under standard
assay conditions (see ‘‘Materials and Methods’’).
Table 2. Substrate Specificity of rIl1,3Gal toward Polysaccharides
Gal
β-(1→3)-Gal2
β-(1→3)-Gal3
Relative
activity
(
%)a
Polysaccharides
ꢀ-(1 ! 3)-Galactan
100
74
Polymer II
Polymer I
51
Acacia gum from acacia tree
Native AGP from radish roots
0.4
70
74
26
7
ꢁ-L-Arabinofuranosidase-treated AGP from radish
Native AGP from radish leaves
ꢀ
-(1 ! 3)(1 ! 6)-Galactan from P. zopfii
-(1 ! 4)-Galactan from lupin
ꢀ
Arabinan from sugar beat
0
0
CM-cellulose [ꢀ-(1 ! 4)-glucan]
CM-curdlan [ꢀ-(1 ! 3)-glucan]
0
1
2
3
4
5
6
7
8
9 10
0
ꢀ
-Glucan from barley [ꢀ-(1 ! 3)(1 ! 4)-glucan]
0
Fig. 4. Substrate Specificity of rIl1,3Gal.
Laminarin from L. digitata [ꢀ-(1 ! 3)(1 ! 6)-glucan]
-O-Methyl-glucuronoxylan
0
Hydrolysis products of various oligosaccharides by rIl1,3Gal were
analyzed on TLC. Lanes 1 and 10, standard Gal, ꢀ-(1 ! 3)-
galactobiose, and ꢀ-(1 ! 3)-galactotriose; lanes 2–8, products from
ꢀ-(1 ! 3)-galactobiose, ꢀ-(1 ! 3)-galactotriose, ꢀ-(1 ! 4)-galac-
tobiose, ꢀ-(1 ! 4)-galactotriose, ꢀ-(1 ! 6)-galactobiose, ꢀ-
(1 ! 6)-galactotriose, and laminaribiose respectively; lane 9, stand-
ard Glc. Localizations of standard Gal and ꢀ-(1 ! 3)-galactobiose
and of ꢀ-(1 ! 3)-galactotriose are indicated on the left.
4
0
Xylan from birchwood
PNP-sugarsb
0
PNP-ꢁ-L-arabinofurnoside
PNP-ꢀ-D-galactopyranoside
PNP-ꢀ-D-glucopyranoside
PNP-ꢀ-D-glucuronide
PNP-ꢀ-D-xylopyranoside
0
0
0
0
0
a
Activity is expressed as % of that toward ꢀ-(1 ! 3)-galactan, taken as 100.
b
The enzyme was incubated with PNP-substrate at a final concentration of
2.5 mM: 2.5 mg/ml was employed for polymers.

Exo-ꢀ-(1 ! 3)-galactanase from Irpex lacteus
2307
A [β-(1→3)-Galactan]
B (Polymer II)
Gal
Gal
β-(1→6)-Gal2
ABEE
β-(1→6)-Gal2
ABEE
2
1
0
4 h
24 h
10 min
0 min
min
0 min
0
5
10 15 20 25 30
0
5
10 15 20 25 30
Retention time (min)
C (Polymer I)
D (Native AGP)
ABEE
β-(1→6)-Gal2
α-L-Ara-(1→3)-β-Gal-(1→6)-Gal
Gal
Gal
ABEE
β-(1→6)-Gal2
β-(1→6)-Gal3
2
1
0
4 h
24 h
0 min
min
10 min
0 min
0
5
10 15 20 25 30
0
5
10 15 20 25 30
Retention time (min)
Fig. 5. Mode of Action of rIl1,3Gal.
The mode of action of rIl1,3Gal on various polymers was analyzed. Enzymatic hydrolysates, at incubation times 0 min, 10 min, and 24 h,
released from ꢀ-(1 ! 3)-galactan (A), polymers II (B) and I (C) prepared from acacia gum by Smith degradation, and native AGP from radish
roots (D) were derivatized with ABEE and analyzed by HPLC equipped with an Amide-80 column. Arrows indicate the elution positions of
standard ABEE and ABEE-derivatized Gal and ꢀ-(1 ! 6)-galactobiose [ꢀ-(1 ! 6)-Gal2] and galactotriose [ꢀ-(1 ! 6)-Gal3], and ꢁ-L-Ara-
(
1 ! 3)-ꢀ-Gal-(1 ! 6)-Gal.
that of native Il1,3Gal (87.8 units/mg protein) and
remarkably higher than those of recombinant exo-ꢀ-
rIl1,3Gal showed maximum activity at pH 4.5, and
hardly acted on ꢀ-(1 ! 3)-galactan below pH 3.0 or
above pH 6.5. The optimum temperature for enzyme
(
cellum (21 units/mg protein), and S. avermitilis
1 ! 3)-galactanases from bacteria such as C. thermo-
ꢀ
action was 40 C (Fig. 3). The activity of rIl1,3Gal was
1
4,16,17)
(
3.7 units/mg protein).
The purified rIl1,3Gal
not diminished by incubation at pH 2–9 for 10 min,
preceding the enzyme reaction. On the other hand,
approximately 90% of the activity of rIl1,3Gal was lost
appeared as two bands with relative molecular masses
of 54 and 49 kDa on SDS–PAGE (Fig. 2), larger than
the apparent molecular mass predicted from the se-
quence deduced from the cDNA (45,520 Da). To
examine whether the higher molecular masses of
rIl1,3Gal were derived from N-glycans attached to the
enzymes, the enzyme proteins were digested with Endo
H. The apparent molecular masses of rIl1,3Gal shifted to
ꢀ
under treatment at 60 C for 10 min (Supplemental
Fig. 2; see Biosci. Biotechnol. Biochem. Web site).
Substrate specificity of rIl1,3Gal
Glycoside hydrolases are categorized into more than
110 families based on hydrophobic cluster analysis.
Il1,3Gal belongs to GHF 43, as do other exo-ꢀ-(1 ! 3)-
galactanases identified to date. This family includes other
glucoside hydrolases such as ꢀ-xylosidase (EC 3.2.1.37),
ꢁ-L-arabinofuranosidase (EC 3.2.1.55), arabinanase (EC
3.2.1.99), and xylanase (EC 3.2.1.8) (Carbohydrate
Active Enzymes database, http://www.cazy.org/). Using
4
5 kDa. Additionally, the apparent molecular mass of
the native enzyme also decreased to 44 kDa. The results
clearly indicated that in P. pastoris, rIl1,3Gal undergoes
N-glycosylation different from that for the native
enzyme. It has been found that yeast attaches large
2
4)
high-mannose type glycans to secreted proteins.

2308
T. KOTAKE et al.
various polysaccharides and PNP-substrates, the activ-
ities of rIl1,3Gal were examined. rIl1,3Gal failed to
hydrolyze polysaccharides lacking ꢀ-(1 ! 3)-galactosyl
residues (Table 2). Furthermore, rIl1,3Gal hydrolyzed
ꢀ-(1 ! 3)-galactobiose and -triose into Gal, whereas the
other galactooligosaccharides and laminaribiose [ꢀ-Glc-
Acknowledgments
This research was supported in part by a Grant-in-Aid
for Scientific Research (to T.K, no. 17770028) from
Ministry of Education, Culture, Sports, Science, and
Technology of Japan.
(
1 ! 3)-Glc] did not serve as substrates (Fig. 4). These
results indicate that the cloned cDNA encodes bona fide
exo-ꢀ-(1 ! 3)-galactanase. They also revealed that
rIl1,3Gal has relatively high activity toward AGPs: the
specific activity of rIl1,3Gal toward native AGP was
estimated to be 33.3 units/mg protein, apparently higher
than those of other recombinant exo-ꢀ-(1 ! 3)-galacta-
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1
0,11)
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