Cloning, sequencing, and expression of the gene encoding the Clostridium stercorarium alpha-galactosidase Aga36A in Escherichia coli.

Article abstract of DOI:10.1271/bbb.67.2160

The alpha-galactosidase gene aga36A of Clostridium stercorarium F-9 was cloned, sequenced, and expressed in Escherichia coli. The aga36A gene consists of 2,208 nucleotides encoding a protein of 736 amino acids with a predicted molecular weight of 84,786.

Full text of DOI:10.1271/bbb.67.2160

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Cloning, Sequencing, and Expression of the
Gene Encoding the Clostridium stercorarium α-
Galactosidase Aga36A in Escherichia coli
a
a
a
a
SURYANI , Tetsuya KIMURA , Kazuo SAKKA & Kunio OHMIYA
a
Faculty of Bioresources, Mie UniversityTsu 514-8507, Japan
Published online: 22 May 2014.
To cite this article: SURYANI, Tetsuya KIMURA, Kazuo SAKKA & Kunio OHMIYA (2003) Cloning, Sequencing, and Expression
of the Gene Encoding the Clostridium stercorarium α-Galactosidase Aga36A in Escherichia coli , Bioscience, Biotechnology,
and Biochemistry, 67:10, 2160-2166, DOI: 10.1271/bbb.67.2160
To link to this article: http://dx.doi.org/10.1271/bbb.67.2160
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Biosci. Biotechnol. Biochem., 67 (10), 2160–2166, 2003
Cloning, Sequencing, and Expression of the Gene Encoding the Clostridium
stercorarium a-Galactosidase Aga36A in Escherichia coli
S
URYANI, Tetsuya KIMURA, Kazuo SAKKA,† and Kunio OHMIYA
Faculty of Bioresources, Mie University, Tsu 514-8507, Japan
Received April 14; 2003; Accepted June 30, 2003
The
a
-galactosidase gene aga36A of Clostridium ster-
some others are classiˆed in family 36. On the other
hand, most prokaryotic enzymes have been classiˆed
into family 4 or 36 with the exceptions of Clostridium
corarium F-9 was cloned, sequenced, and expressed in
Escherichia coli. The aga36A gene consists of 2,208
nucleotides encoding a protein of 736 amino acids with
a predicted molecular weight of 84,786. Aga36A is an
enzyme classiˆed in family 36 of the glycoside hydro-
lases and showed sequence similarity with some enzymes
of family 36 such as Geobacillus (formerly Bacillus
stearothermophilus GalA (57 ) and AgaN (52 ). The
enzyme puriˆed from a recombinant E. coli is optimally
active at 70 C and pH 6.0. The enzyme hydrolyzed
ra‹nose and guar gum with speciˆc activities of 3.0 U
mg and 0.46 U mg for the respective substrates.
7
)
josui Aga27A and Saccharopolyspora erythraea
MelA (DDBJ accession no. AF061331). Further-
more, a-Gals can be classiˆed into two groups based
8)
on their substrate speciˆcity, i.e., one group is
speciˆc for small saccharides such as -nitrophenyl
-galactopyranoside (PNP-Gal), melibiose, and
)
p
z
z
a
-
D
ra‹nose, and the other group can liberate galactose
from galactomannans such as guar (Cyamopsis
tetragonoloba) gum in addition to small substrates.
9
W
W
Clostridium stercorarium is a spore-forming
anaerobic thermophilic bacterium, which is known to
ferment various kinds of polysaccharides. Therefore,
this bacterium seems to be a good source of ther-
mophilic glycoside hydrolases and their genes, and
Key words: Clostridium stercorarium; a-galactosidase
a
-Galactosidase (
-galactosidic linkages at non-reducing ends in galac-
tose-containing oligosaccharides, galactolipids, and
a-Gal; EC 3.2.1.22) hydrolyzes
9
)
a
actually various genes encoding cellulases,
10) 11)
W
xylanase,
b
-xylosidase,
a- -arabinofuranosi-
L
1
)
12)
13)
or galactomannan.
usage in various ˆelds, e.g., in the sugar beet indus-
try, -Gal was used to increase the sucrose yield by
eliminating ra‹nose which prevents normal crystalli-
zation of beet sugar.
atulence caused by carbohydrates such as ra‹nose
and stachyose in beans, which can not be metabo-
a
-Gals have the potential for
dase, and
a- -rhamnosidase have been cloned
L
and characterized along with their gene products. In
a
this paper, we describe the C. stercorarium aga36A
gene encoding an
a-Gal classiˆed in family 36 of
2
)
a-Gals may be used to alleviate
glycoside hydrolases.
‰
Materials and Methods
3
)
lized by monogastric animals including humans.
-Gals can be used to improve the properties of guar
a
Bacterial strains and plasmids. C. stercorarium
gum, a type of galactomannan, as a gelation
promoter and cogelator, by enzymatic release of
strain F-9 used for isolation of the chromosomal
1
4)
DNA was described previously. Escherichia coli
4
)
galactose residues from the guar gum.
In this context, many genes encoding
DH5
a was used as the cloning host. Plasmids
a
-Gals have
pBluescript II KS ( ) and SK ( ) was used for clon-
＋ ＋
been isolated and characterized from various organ-
isms including ˆlamentous fungi, yeasts, bacteria,
plants, and humans along with the enzyme properties
of their translated products. On the basis of amino
ing experiments and analysis of the DNA sequence
of the aga36A gene.
Cloning and screening of
a-galactosidase gene
acid sequence homology, catalytic domains of
a
-Gals
(aga36A) from C. stercorarium F-9. The chro-
mosomal DNA from C. stercorarium F-9 was partial-
can be substantially classiˆed into three groups, fam-
5
,6)
ilies 4, 27 and 36 of glycoside hydrolases. Almost
ly digested with Sau3AI. The 3?-recessed ends of the
all of
a
-Gals from eukaryotic organisms including
DNA fragments were partially ˆlled with dG and dA
fungi, plants, and animals belong to family 27 and
by Klenow enzyme and ligated into the XhoI site of
†
To whom correspondence should be addressed. Tel:
＋
81-59-231-9621; Fax:
-galactopyranoside; ORF, open reading frame; PAGE, poly-
-nitrophenyl -galactopyranoside; TLC, thin-layer chro-
81-59-231-9684; E-mail: sakka bio.mie-u.ac.jp
＋ ＠
Abbreviations
:
a
-Gal, -galactosidase; 4MU-Gal, 4-methylumbelliferyl a-D
a
acrylamide gel electrophoresis; PCR, polymerase chain reaction; PNP-Gal,
matography
p
a-D

a
-Galactosidase of Clostridium stercorarium
2161
religating it or by subcloning speciˆc restriction frag-
ments into pBluescript II KS ( ). The nucleotide
＋
sequence data was analyzed with the GENETYX
computer software (Software Development Co.,
Ltd., Tokyo, Japan). Homology searches in Gen-
Bank were done with a BLAST program.
Puriˆcation of Aga36A. The EcoRI-PvuII frag-
ment of pANI1 was ligated between the EcoRI and
Fig. 1. Restriction Map of pANI1 Carrying the aga36A Gene.
Thin lines correspond to pBluescript II or pCR2.1, and bars
correspond to the inserted DNA fragments. The position and
EcoRV sites of pBluescript II SK (
pANI5. E. coli DH5 (pANI5) was grown on a rotary
shaker overnight at 37 C in 1000 ml of LB medium
containing ampicillin (50 ml) and 100
isopropyl -thiogalactopyranoside. The cells were
harvested by centrifugation and resupended in 50 ml
of 20 m Tris-HCl buŠer (pH 7.5). The cells were
＋
), yielding
direction of the ORF of aga36A are shown by an arrow. Plac
the lac promoter.
,
a
9
m
g
W
m
M
pBluescript II KS (
＋
), the 3
?
-recessed ends of which
b
-
D
had been also partially ˆlled with dC and dT. Recom-
binant plasmids were introduced into E. coli DH5
a
.
M
a
-Gal-producing clones were screened as follows.
disrupted by sonication and cell debris was removed
by centrifugation. The majority of the E. coli pro-
teins in the crude extract was inactivated and precipi-
Transformants were cultivated on LB agar (1
z
z
poly-
agar,
peptone, 0.5
pH 7.0) plates at 37
incubated at 60 C for 2 hr to inactivate the
activity of the host. Soft agar (0.5 agar in sodium
phosphate buŠer, pH 7.0) containing 10 m 4-
methylumbelliferyl -galactopyranoside (4MU-
Gal) was solidiˆed on the plates and then incubated
for 15 min at 60 C. Recombinant clones having
z yeast extract, 0.5z NaCl, 1.5
9
C overnight and the plates were
9
tated by heat treatment at 70 C for 15 min. This heat
9
a
-Gal
treatment did not decrease the total enzyme activity
in the crude extract. The precipitates were removed
by centrifugation to recover the supernatant. Pro-
z
M
a
-
D
teins were put onto a HiPrep 16
column (1.6 10 cm; Amersham Pharmacia Biotech,
Buckinghamshire, England) equilibrated with 20 m
Tris-HCl buŠer (pH 8.0). Adsorbed proteins were
eluted with a linear gradient of NaCl (0–0.35 ) in
W
10 DEAE FF
×
9
a
-
M
Gal activity were detected as colonies showing
uorescence under UV irradiation.
‰
M
the same buŠer. Active fractions were collected,
desalted, and put onto a RESOURCE PHE column
(1 ml; Amersham Pharmacia Biotech) equilibrated
Cloning of the upstream and downstream regions
of aga36A. Inverse polymerase chain reaction (PCR)
was used to amplify the upstream and downstream
regions of aga36A (Fig. 1). To clone the upstream
region, the C. stercorarium F-9 genomic DNA was
digested with NdeI, made circular by self-ligation,
and used for PCR as the template DNA with KOD
Dash DNA polymerase (Toyobo, Osaka, Japan) and
two primers complementary to the nucleotide se-
with 50 m
(NH SO
decreasing gradient of (NH
M
Tris-HCl buŠer (pH 8.0) containing 1
. Adsorbed proteins were eluted with a
SO (1–0 ). Fractions
M
4
)
2
4
4
)
2
4
M
with high activity were used for the characterization
of Aga36A.
Enzyme assays. Enzyme activity was routinely
quence upstream of aga36A, InvN1, 5
CGAACTGAAAGCG-3 , and InvN2, 5
ACCTTATGTGGCCTC-3 under the conditions
?
-CGGCCAT-
measured with 10 m
M
p
-nitrophenyl
D
a- -galac-
?
?
-TAAAG-
topyranoside (PNP-Gal) as a substrate. The reaction
mixture (ˆnal volume of 400 l), consisting of
appropriately diluted enzyme and PNP-Gal in
McIlvaine's buŠer (a mixture of 0.1 citric acid and
0.2 NaHPO , pH 7.0), was incubated at 60 C for
15 minutes. The amount of -nitrophenol released
?
m
recommended by Toyobo. The ampliˆed DNA frag-
ments were ligated into the TA cloning vector
pCR2.1 (Invitrogen, Carlsbad, USA) using a Takara
Ligation kit (Kyoto, Japan), yielding pANI3 (Fig. 1).
For cloning of the downstream region, HindIII was
used for digestion of the genomic DNA and primers
M
M
4
9
p
from the substrate was measured by the absorbance
at 410 nm. One unit (U) of enzyme for PNP-Gal
was deˆned as the amount of enzyme that liberates
InvC1, 5
?
-TCCAACCGAATACAGAACAC-3
?, and
InvC2,
5?
-TTTATCAGATACTGCTGAGG-3
?,
1.0
under the given assay conditions. Ra‹nose (ˆnal
concentration: 1 ) and guar gum (0.5 ) were also
mmol of p-nitrophenol per min from PNP-Gal
were used for PCR. The resultant plasmid was desig-
nated as pANI4 (Fig. 1).
z
z
used as natural substrates for the enzyme assay in the
same buŠer and temperature described above. The
amount of reducing sugars from each substrate was
measured with dinitrophthalic acid reagent. One
unit of enzyme for these substrates was deˆned as the
DNA sequencing. DNA sequencing of a plasmid
pANI1 was done on a Licor (Lincoln, Nebr.) model
1
5)
4
000L automated DNA sequencer, with appropriate
dye primers and a series of subclones. The subclones
were constructed by removing speciˆc restriction
fragments from the insert DNA of pANI1 and
amount of enzyme that produced 1
mmol of galactose
per min. The protein concentration was measured

2
162
S
URYANI et al
.
with a Micro BCA Protein Assay Kit (Pierce, Rock-
land, IL) with bovine serum albumin as a standard.
the substrate. As a result, two clones were selected as
a
-Gal-producing recombinants. Plasmids pAN1
(Fig. 1) and pAN2 were isolated from these two
EŠects of temperature and pH. The temperature
optimum was found under standard assay conditions
with PNP-Gal as the substrate by incubating the
recombinant clones and the restriction maps of the
inserted DNA fragments were constructed. Compari-
son of the restriction maps suggested that these plas-
mids contained the same inserted fragment but in the
opposite directions. We chose pAN1 for further
reaction mixtures at 50 9C to 80 9C . Thermal stability
was measured by incubating the enzyme for 10 min at
diŠerent temperatures. After the enzyme solution
was cooled on ice, its residual activity was measured
using the standard assay. For identifying the opti-
mum pH for enzyme activity, enzyme assays were
done in Britton and Robinson's universal buŠer
investigation. The
a-Gal gene carried by pAN1 was
referred to as aga36A
.
Nucleotide sequence of aga36A and its ‰anking
regions
(
80 m
acetic acid the pH was adjusted to 4 to 12 with 1
NaOH) by a 15-min incubation at 60
stability was measured by incubating 10
appropriately diluted enzyme in Britton and Robin-
son's universal buŠer at 4 C for 24 h. Enzyme ac-
M
phosphoric acid-80 m
M
boric acid-80 m
M
When we analyzed the nucleotide sequence of the
inserted fragment on the plasmid pANI1, we found
an incomplete open reading frame (ORF) upstream
of aga36A. Therefore, we ampliˆed the upstream
and downstream regions of aga36A by inverse PCR,
cloned the ampliˆed fragments (Fig. 1), and se-
quenced them. Figure 2 shows the complete nucleo-
tide sequence of the aga36A structural gene along
with its ‰anking regions. The ORF of aga36A, start-
ing at nucleotide position 1,046 and ending at posi-
tion 3,253, consists of 2,208 nucleotides encoding a
protein of 736 amino acids with a predicted molecu-
lar weight of 84,786. Upstream of aga36A, a com-
plete ORF (ORF1) encoding 276 amino acids and an
incomplete ORF (ORF2) encoding 54 amino acids
were identiˆed. Downstream of aga36A, on the other
hand, another incomplete ORF (ORF3) encoding 319
amino acids was found, but the direction of the ORF
is opposite to that of aga36A. The putative initiation
codon (ATG) of aga36A was preceded at a spacing of
N
9
C. The pH
m
l of the
9
tivity was then measured under the standard assay
conditions.
SDS-PAGE. SDS-polyacrylamide gel electropho-
resis (PAGE) was done using 10
z acrylamide gel as
1
6)
described by Laemmli.
Analysis of N-terminal amino acid sequence.
Aga36A puriˆed from the recombinant E. coli was
fractionated by SDS-PAGE and transferred onto a
polyvinylidene di‰uoride membrane by electroblot-
ting. The protein band of Aga36A was excised from
the Coomassie blue-stained membrane sheet and put
through N-terminal amino acid sequence analysis on
an Applied Biosystems model 476A protein sequen-
cer (Foster City, CA, USA).
6 bp by a potential ribosome-binding sequence (5
AGGGGG-3 ), which was homologous to the con-
sensus Shine-Dalgarno (SD) sequence (5 -AGGAGG-
) of E. coli. ORF1 was also preceded by a potential
ribosome-binding sequence (5 -AGGGGG-3 ). There
?-
?
?
Analysis of hydrolysis products. Thin-layer chro-
matography (TLC) of the hydrolysis products of
melibiose and ra‹nose was done on a silica gel 60
plastic sheet (Merck, Whitehouse Station, NJ, USA)
3?
?
?
are only 37-bp and 10-bp spacings between aga36A
and ORF1 and between ORF1 and ORF2, respec-
tively, suggesting that these genes are transcribed in a
polycistronic mRNA.
developed with a solvent of
n-butanol-acetic acid-
water (2:1:1), and mono- and oligosaccharides were
made visible by spraying the plate1 with
a
7)
diphenylamine-aniline-phosphate reagent.
Amino acid sequences of Aga36A and other ORFs
Comparison of the amino acid sequence of
Aga36A with entries in the DDBJ database indicated
that this enzyme is classiˆed in family 36 of glycoside
Nucleotide sequence accession number. The
nucleotide sequence reported in this paper will ap-
pear in the DDBJ, EMBL, and GenBank nucleotide
sequence databases with the accession number
AB089353.
5
,6)
hydrolases. Aga36A shows overall sequence identi-
ty with some enzymes in this family, e.g., 58 se-
quence identity with Clostridium perfringens
z
1
8)
AgaN, 57
z z
and 52 identities with G. stearother-
Results and Discussion
mophilus GalA (DDBJ accession no. AF038547) and
1
9)
AgaN, respectively, 57
halodurans possible -Gal (BH2223), 43
with Lactobacillus plantarum MelA (AF189765),
42 and 35 identities with Pediococcus pen-
tosaceus AgaR and AgaS (L32093), respectively, and
37 identity with Streptococcus mutans Aga
z
identity with Bacillus
20)
Cloning of the aga36A gene from C. stercorarium
F-9
a
z identity
Approximately 5,000 transformants were obtained
from the C. stercorarium F-9 gene library and
z
z
screened for
a
-Gal productivity using 4MU-Gal as
z

a
-Galactosidase of Clostridium stercorarium
2163
Fig. 2. Nucleotide Sequence of the aga36A Gene and Deduced Amino Acid Sequence of the Gene Product.
The possible Shine-Dalgarno (SD) sequences are double-underlined. The amino acid sequence of the puriˆed enzyme experimentally
analyzed is boxed. Regions corresponding to PCR primers used for ampliˆcation of the upstream and downstream regions are under-
lined.
(
M77351). Furthermore, C. stercorarium Aga36A
had relatively high sequence similarities with some
-Gals such as Thermoanaerobacter ethanolicus
and ORF2p) were highly homologous with many
sugar transport proteins, e.g., ORF1p showed the
a
highest homology (74
z
identity) with a probable
2
1)
18)
MelA (Y08557), E. coli RafA, Biˆdobacterium
longum AglL (AF160969), and Biˆdobacterium
adolescentis Aga (AF124596), although their amino
acid sequences are not conserved in their N-terminal
membrane transport protein of C. perfringens and
the ORF2p (54 amino acids) showed 72
z identity
with a diŠerent probable membrane transport pro-
1
8)
tein of C. perfringens
.
and
showed lower sequence identities in restricted region
about 150 amino acids in the central region of
W
or C-terminal regions. By contrast, Aga36A
A deduced amino acid sequence (a region of about
270 amino acids) of the ORF3 polypeptide (ORF3p)
showed homology with many transcriptional regula-
(
Aga36A) with the other enzymes such as Thermus
thermophlus AgaT (AF135399), Thermotoga
tory proteins of the AraC
W
XylS family, e.g., 37
z
identity with a probable transcriptional regulator of
2
2)
18)
neapolitana AglA,
AgaT.
Deduced amino acid sequences of the overall
region of the ORF1 and ORF2 polypeptides (ORF1p
and Thermus brockianus
C. perfringens
regulatory protein of Streptococcus pneumoniae
(AE007480), and 33 identity with a probable tran-
scriptional regulator protein of B. halodurans
,
32z identity with the msm operon
2
3)
z
20)
.

2
164
S
URYANI et al
.
Fig. 3. Comparison of
a-Gal Gene Clusters of C. stercorarium
,
C. perfringens, B. halodurans, and G. stearothermophilus.
Related gene products are shown in the same patterns. Tra, probable transporter protein; Reg, probable transcription regulator; Ccp,
catabolite control protein, Chp, conserved hypothetical protein, MsmE, multiple sugar-binding protein. Sequence identities ( ) of
z
hypothetical gene products to Aga36A, ORF1p, ORF2p, and ORF3p of C. stercorarium are shown above related gene products. ND
means no sequence identity detected by homology searches with the BLAST program.
Comparison of the a-Gal gene clusters
As seen in the E. coli lac operon, gene(s) responsi-
ble for the transport of substrates or hydrolysis
products are often found in the vicinity of a glycoside
hydrolase. In case of the C. stercorarium aga36A
gene, two ORFs encoding probable transport pro-
teins and a gene encoding a possible transcriptional
regulator were found in its vicinity (Fig. 3). There-
fore, we compared the organization of the
clusters that encoded -Gals highly homologous with
Aga36A, i.e., the gene clusters of C. perfringens aga
G. stearothermophilus galA, and B. halodurans gal
BH2223). As shown in Fig. 3, two genes encoding
a-Gal gene
a
,
(
probable transport proteins are found upstream of
C. perfringens aga and B. halodurans gal but no gene
responsible for sugar transport is identiˆed in the
vicinity of G. stearothermophilus galA. Amino acid
sequences of ORF1p and ORF2p showed the highest
homology with those of the corresponding probable
Fig. 4. SDS-PAGE of the Puriˆed Recombinant Aga36A.
The gel was stained with Coomassie brilliant blue: lane M,
protein molecular mass standard (molecular masses shown at
the left); lane 1, crude extract; lane 2, proteins after heat trea-
tent; lane 3, proteins after a HiPrep 16 10 DEAE FF column;
W
transport proteins of C. perfringens
identities, respectively. ORF1p was less
homologous with the corresponding protein of B.
halodurans i.e., ORF1p showed only 25 identity
, i.e., 74z and
lane 4, Aga36A after a RESOURCE PHE column.
72
z
,
z
with a probable transport protein and ORF2p
showed no sequence identity with another probable
transport protein of B. halodurans. Although a
probable transcriptional regulator gene (ORF3) is
found downstream of aga36A in C. stercorarium, a
Puriˆcation and characterization of the recom-
binant Aga36A
The recombinant Aga36A was puriˆed 40-fold
from the cell-free extract of E. coli DH5a(pANI5) by
HiPrep 16
PHE column chromatographies with the yield of
18 . Speciˆc activity of the ˆnally puriˆed enzyme
was 450 U mg. The puriˆed preparation gave a sin-
W
10 DEAE FF column and RESOURCE
homologous gene (37
z
sequence identity) was
conserved upstream of the C. perfringens aga cluster
and the direction of the probable transcriptional
regulator gene was opposite to that of the C. perfrin-
gens aga gene; a BLAST search indicated that
ORF3p is the most homologous with the transcrip-
tional regulator of C. perfringens among the entries
in DDBJ database. These results suggest that the
C. stercorarium aga36A gene cluster is closely related
to the C. perfringens aga gene cluster, i.e., these
genes are derived from a common ancestor, and rear-
rangement of the gene organization occurred during
their evolution.
z
W
gle band on SDS-PAGE, and the molecular weight of
the puriˆed enzyme was estimated to be around
70,000 (Fig. 4). However, the molecular weight
estimated by SDS-PAGE appeared to be smaller than
that deduced from the amino acid sequence of
Aga36A (84,786). The N-terminal amino acid
sequence of the puriˆed enzyme was found to be
Phe-His-Glu-Glu-Ser-Gly-Glu, which is in complete
agreement with the amino acid sequence (amino acid
positions from 5 to 11) deduced for the DNA se-
quence. Therefore, it is likely that Aga36A suŠered
from partial proteolysis by host protease(s) at its
C-terminal region. Although it is possible that the

a
-Galactosidase of Clostridium stercorarium
2165
Lineweaver-Burk plot, the
calculated to be 4.3 m
respectively. This
G. stearothermophilus AgaN for the same substrate
0.38 m ) although the max value was comparable
with that of G. stearothermophilus AgaN (0.52 mol
K
m
and
V
max values were
M
and 0.43
W W
mmol min mg,
K value was higher that that of
m
(
M
V
m
1
9)
W
min
As described above, C. stercorarium Aga36A has
overall sequence identity with some -Gals of family
6 from C. perfringens G. stearothermophilus B.
halodurans L. plantarum, and P. pentosaceus
Among them, G. stearothermophilus AgaN was
W
mg).
a
3
,
,
,
.
1
9)
examined for its enzyme properties. AgaN puriˆed
from E. coli was maximally active at 75 C and highly
thermostable, i.e., its half-life of inactivation at 70
9
9
C
was 19 h. AgaN showed a high a‹nity for oligomeric
substrates such as melibiose and ra‹nose. Properties
of C. stercorarium Aga36A seem to be similar to
those of G. stearothermophilus AgaN.
Fig. 5. Thin-layer Chromatography of Hydrolysis Products
from Ra‹nose and Melibiose.
Ra‹nose (20
bated with the puriˆed enzyme (0.5 unit) in 10
m
g) and melibiose (20
m
g) were separately incu-
l of McIlvaine
m
buŠer (pH 7.0) for 6 hr and the hydrolysates were analyzed by
TLC. Gal, galactose; Glu, glucose; Suc, sucrose; Raf, ra‹nose;
Acknowledgments
Mel, melibiose; Raf
＋
E, ra‹nose digested with the enzyme; Mel
This work was supported in part by a Grant-in-Aid
for University and Society collaboration, the
Ministry of Education, Culture, Sports, Science and
Technology of Japan. We thank Dr. S. Karita of this
university for the operation of the protein sequencer.
＋
E, melibiose digested with the enzyme.
properties of the puriˆed enzyme are not necessarily
identical to those of the non-truncated enzyme, we
could not purify the latter molecular species. Since
the puriˆed enzyme showed high speciˆc activity, we
used this preparation for further characterization.
The puriˆed enzyme had relatively high speciˆc activ-
References
1
)
Dey, P. M., and Campillo, E. D.,
Adv. Enzymol. Relat. Areas Mol. Biol., 56, 141–249
1984).
Yamane, T., Decomposition of ra‹nose by
a-Galactosidase.
ity toward ra‹nose (3.0 U
toward to guar gum (0.46 U
W
mg) and low activity
mg). The action of the
(
W
2
)
a
-galac-
enzyme on ra‹nose and melibiose was qualitatively
analyzed by TLC. As shown in Fig. 5, the recom-
binant Aga36A hydrolyzed ra‹nose to yield galac-
tose and sucrose, and hydrolyzed melibose to yield
galactose and glucose. The enzyme was optimally
active at pH 6.0 and showed high activity, i.e., more
tosidase. An enzymatic reaction applied in the
factory-process in Japanese beet sugar factories.
W
Sucr. Belge Sugar Ind. Abstr., 90, 345–348 (1971).
3
)
Lindley, M. G., Cellobiase, milibiase and other disac-
charidases. In ``Development in Food Carbohy-
drates'' vol. 3, eds. Lee, C. K., and Lindley, M. G.,
Elsevier Applied Science Publishers, London, pp.
141–165 (1982).
than 90
of 5 to 8 and about 95
pH 7.0 when the enzyme activity was assayed with
PNP-Gal by 15-min incubation at 60 C in Britton
z
of the maximum activity, in the pH range
z
of the maximum activity at
4
5
)
)
McCleary, B. V., and Neukom, J., EŠect of enzyme
modiˆcation on the solution and interaction proper-
ties of galactomannan. Prog. Food Nutr. Sci., 6,
9
and Robinson's universal buŠer solutions at various
pHs. The enzyme was stable in the pH range of 6 to
0 when incubated at 4 C for 24 h in the same buŠer
9
1
09–118 (1982).
Henrissat, B., and Bairoch, A., Updating the sequen-
ce-based classiˆcation of glycosyl hydrolases.
Biochem. J., 316, 695–696 (1996).
1
solutions without the substrate. The eŠects of tem-
perature on the activity and stability of the enzyme
were examined. The optimum temperature of the
6) Henrissat, B., and Coutinho, P., [Online] Glycosyl
hydrolase families. Architecture et Fonction des
enzyme was found at 70
up to 70 C for 10 minutes in the absence of sub-
strate. Enzyme assays in the presence of various
metal ions (ˆnal concentration of 1 m ) such as
9C. The enzyme was stable
Macromol eá ules Biologiques, CNRS, Marseille,
France, http: // afmb.cnrs-mrs.fr
index.html.
W W W
¿cazy CAZY
9
7
)
Jindou, S., Karita, S., Fujino, E., Fujino, T.,
Hayashi, H., Kimura, T., Sakka, K., and Ohmiya,
M
2
＋
2
＋
2
＋
2
＋
2
＋
2
＋
2
＋
2
＋
Ba , Ca , Cu , Fe , Mg , Mn , Ni , Zn
K.,
a-Galactosidase Aga27A, an enzymatic compo-
suggested that Aga36A does not have any metal ion
requirement. The initial rates of the reaction were
measured with various concentrations of PNP-Gal at
nent of the Clostridium josui cellulosome. J. Bac-
teriol., 184, 600–604 (2002).
8) Dey, P. M., Patel, S., and Brouwnleader, M. D.,
6
0
9
C in McIlvaine's buŠer (pH 7.0). From the
Induction of
a-galactosidase in Penicellium

2
166
S .
URYANI et al
ochrochloron by guar (Cyamopsis tetragonoloba
)
during the assembly of the head of bacteriophage T4.
gum. Biotecnol. Appl. Biochem., 17, 361–371 (1993).
Bronnenmeier, K., Kundt, K., Riedel, K., Schwarz,
W. H., and Staudenbauer, W. L., Structure of the
Clostridium stercorarium gene celY encoding the
Nature, 227, 680–685 (1970).
9
)
17) Gasparic, J., and Churacek, J., Laboratory han-
dbook of paper and thin-layer chromatography. Ellis
Horwood Ltd., Chichester, England, pp. 153–154
(1978).
18) Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima,
K., Yamashita, A., Shiba, T., Ogasawara, N.,
Hattori, M., Kuhara, S., and Hayashi, H., Complete
genome sequence of Clostridium perfringens, an
exo-
b-1,4-glucanase Avicelase II. Microbiology, 143,
8
91–898 (1997).
1
1
0) Ali, M. K., Kimura, T., Sakka, K., and Ohmiya, K.,
The multidomain xylanase Xyn10B as a cellulose-
binding protein in Clostridium stercorarium
Microbiol. Lett., 198, 79–83 (2001).
. FEMS
anaerobic ‰esh-eater. Proc. Natl. Acad. Sci. U.S.A.
99, 996–1001 (2002).
,
1) Sakka,K., Yoshikawa, K., Kojima, Y., Karita, S.,
Ohmiya, K., and Shimada, K., Sequence of the
Clostridium stercorarium xylA gene encoding a bi-
19) Fridjonsson, O., Watzlawick, H., Gehweiler, A., and
Mattes, R., Thermostable -galactosidase from Bacil-
lus stearothermophilus NUB3621: cloning, sequenc-
a
functional protein with
b- -xylosidase and a- -
D L
arabinofuranosidase activities and properties of the
product. Biotechnol. Biochem., 57, 268–272 (1993).
2) Schwarz, W. H., Bronnenmeier, K., Krause, B.,
Lottspeich, F., and Staudenbauer, W. L., Debranch-
ing of arabinoxylan: properties of the thermoactive
ing, and characterization. FEMS Microbiol. Lett.
,
176, 147–153 (1999).
1
1
20) Takami, H., Nakasone, K., Takaki, Y., Maeno, G.,
Sasaki, R., Masui, N., Fuji, F., Hirama, C.,
Nakamura, Y., Ogasawara, N., Kuhara, S., and
Horikoshi, K., Complete genome sequence of the
alkaliphilic bacterium Bacillus halodurans and
recombinant
a
-
L
-arabinofuranosidase from Clostridi-
(ArfB). Appl. Microbiol.
um stercorarium
Biotechnol., 43, 856–860 (1995).
genomic sequence comparison with Bacillus subtilis
Nucleic Acids Res., 28, 4317–4331 (2000).
.
3) Zverlov, V. V., Hertel, C., Bronnenmeier, K.,
Hroch, A., Kellermann, J., and Schwarz, W. H., The
21) Aslanidis, C., Schmid, K., and Schmitt, R., Nucleo-
tide sequences and operon structure of plasmid-borne
genes mediating uptake and utilization of ra‹nose in
thermostable
stercorarium
primary structure of a bacterial
a
-
L
-ramnosidase RamA of Clostridium
biochemical characterization and
-rhamnoside
:
a
-
L
Escherichia coli
22) King, M. R., Yernool, D. A., Eveleigh, D. E., and
Chassy, B. M., Thermostable -galactosidase from
. J. Bacteriol., 171, 6753–6763 (1989).
hydrolase, a new type of inverting glycoside hydro-
lase. Mol. Microbiol., 35, 173–179 (2000).
a
1
4) Sakka, K., Kojima, Y., Yoshikawa, K., and
Shimada, K., Cloning and expression in Escherichia
coli of Clostridium stercorarium strain F-9 genes
related to xylan hydrolysis. Agric. Biol. Chem., 54,
Thermotoga neapolitana: cloning, sequencing and ex-
pression. FEMS Microbiol. Lett., 163, 37–42 (1998).
23) Fridjonsson, O., Watzlawick, H., Gehweiler, A.,
Rohrhirsch, T., and Mattes, R., Cloning of the gene
3
37–342 (1990).
encoding a novel thermostable a-galactosidase from
1
1
5) Momose, T., ``Yukiteiseibunseki'' (in Japanese),
Hirokawa Shoten, Tokyo, Japan, p. 226 (1972).
6) Laemmli, U. K., Cleavage of structural proteins
Thermus brockianus ITI360. Appl. Environ.
Microbiol., 65, 3955–3963 (1999).