Molecular cloning and characterization of an enzyme hydrolyzing p-nitrophenyl α-D-glucoside from Bacillus stearothermophilus SA0301

Article abstract of DOI:10.1271/bbb.70.495

Bacillus stearothermophilus SA0301 produces an extracellular oligo-1,6-glucosidase (bsO16G) that also hydrolyzes p-nitrophenyl α-D-glucoside (Tonozuka et al., J. Appl. Glycosci., 45, 397-400 (1998)). We cloned a gene for an enzyme hydrolyzing p-nitrophenyl α-D-glucoside, which was different from the one mentioned above, from B. stearothermophilus SA0301. The k₀/K_m values of bsO16G for isomaltotriose and isomaltose were 13.2 and 1.39 s^-1·mM^-1 respectively, while the newly cloned enzyme did not hydrolyze isomaltotriose, and the k₀/K_m value for isomaltose was 0.81 s ^-1·mM^-1. The primary structure of the cloned enzyme more closely resembled those of trehalose-6-phosphate hydrolases than those of oligo-1,6-glucosidases, and the cloned enzyme hydrolyzed trehalose 6-phosphate. An open reading frame encoding a protein homologous to the trehalose-specific IIBC component of the phopshotransferase system was also found upstream of the gene for this enzyme.

Full text of DOI:10.1271/bbb.70.495

Biosci. Biotechnol. Biochem., 70 (2), 495–499, 2006
Molecular Cloning and Characterization of an Enzyme Hydrolyzing
p-Nitrophenyl ꢀ-D-Glucoside from Bacillus stearothermophilus SA0301
y
*
Atsushi KOBAYASHI, Takashi TONOZUKA, Kimihiko SATO, Mikita SUYAMA,
**
Jun SASAKI, Batbold NYAMDAWAA, Masayoshi SAKAGUCHI, and Yoshiyuki SAKANO
Department of Applied Biological Science, Tokyo University of Agriculture and Technology,
3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
Received September 20, 2005; Accepted October 30, 2005
Bacillus stearothermophilus SA0301 produces an ex-
tracellular oligo-1,6-glucosidase (bsO16G) that also
hydrolyzes p-nitrophenyl ꢀ-D-glucoside (Tonozuka et
al., J. Appl. Glycosci., 45, 397–400 (1998)). We cloned a
gene for an enzyme hydrolyzing p-nitrophenyl ꢀ-D-
glucoside, which was different from the one mentioned
above, from B. stearothermophilus SA0301. The k0=Km
from Bacillus sp. SAM1606, also show broad substrate
specificity and hydrolyze ꢀ-1,1-, ꢀ-1,3-, ꢀ-1,4-, and
ꢀ-1,6-linked diglucoses as well as sucrose;7,8) (3)
trehalose-6-phosphate hydrolase (ꢀ,ꢀ0-trehalose-6-phos-
phate phosphoglucohydrolase, ꢀ,ꢀ0-phosphotrehalase,
EC 3.2.1.93), which hydrolyzes ꢀ,ꢀ0-trehalose-6-phos-
phate to produce glucose and glucose-6-phosphate;9,10)
and (4) dextran glucosidase (EC 3.2.1.70), which mainly
hydrolyzes ꢀ-1,6-glucosidic linkages of dextran.11)
Several researchers have reported that the primary
structures of these pNPG-hydrolyzing enzymes are
homologous.8,9,12)
Bacillus stearothermophilus SA0301 produces an
extracellular O16G (abbreviated bsO16G).1) We report-
ed previously that bsO16G hydrolyzes isomaltose and
isomaltotriose, but does not hydrolyze ꢀ,ꢀ0-trehalose.
This paper explains that B. stearothermophilus SA0301
has another gene encoding a pNPG-hydrolyzing en-
zyme, although molecular cloning of the bsO16G gene
was originally intended.
values of bsO16G for isomaltotriose and isomaltose
were 13.2 and 1.39 sÀ1ÁmM
À1
respectively, while the
newly cloned enzyme did not hydrolyze isomaltotrioÀse1,
and the k0=Km value for isomaltose was 0.81 sÀ1ÁmM
.
The primary structure of the cloned enzyme more
closely resembled those of trehalose-6-phosphate hydro-
lases than those of oligo-1,6-glucosidases, and the cloned
enzyme hydrolyzed trehalose 6-phosphate. An open
reading frame encoding a protein homologous to the
trehalose-specific IIBC component of the phopshotrans-
ferase system was also found upstream of the gene for
this enzyme.
Key words: oligo-1,6-glucosidase; p-nitrophenyl ꢀ-D-
glucoside; isomaltooligosaccharide; treha-
lose-6-phosphate hydrolase; Bacillus stear-
othermophilus SA0301
Materials and Methods
DNA manipulations, strains, and plasmids. The gene
manipulations were carried out based on those of
Sambrook et al.13) Escherichia coli MV1184 was used
as a host strain. A plasmid, pUC118, was obtained from
Takara Bio, Ohtsu, Japan. Bacillus stearothermophilus
SA0301 was prepared as described previously.1) Colo-
nies were screened for pNPG-hydrolyzing activity as
described.1) The DNA sequences of the constructed
plasmids were done using an Applied Biosystems 373A
DNA sequencer. The nucleotide sequence data have
been submitted to the DDBJ/EMBL/GenBank data-
bases (accession no. AB219424).
p-Nitrophenyl ꢀ-D-glucopyranoside (pNPG) is a
powerful tool for the study of glucosidases and related
enzymes. Enzymes hydrolyzing pNPG can be detected
easily, since the hydrolyzate, p-nitrophenol, is visible in
yellow. Several types of enzymes that hydrolyze pNPG
have been found. In the glycoside hydrolase family 13
(ꢀ-amylase family), the following enzymes have been
reported: (1) oligo-1,6-glucosidase (dextrin 6-ꢀ-gluca-
nohydrolase, EC 3.2.1.10, abbreviated O16G), which
hydrolyzes the ꢀ-1,6-glucosidic bonds of isomalto-
oligosaccharides;1–4) (2) ꢀ-glucosidase (EC 3.2.1.20),
which hydrolyzes ꢀ-1,4-glucosidic bonds and releases
D-glucose from the non-reducing end side of the
substrate5,6) and some of the enzymes, such as those
Preparation of the cloned pNPG-hydrolyzing enzyme.
Preparation of the crude recombinant enzyme was
carried out as follows: E. coli MV1184 harboring
y
To whom correspondence should be addressed. Fax: +81-42-367-5705; E-mail: tonozuka@cc.tuat.ac.jp
Present address: Graduate School of Engineering, Tohoku University, Aoba 6-6-11-513, Aoba-ku, Sendai, Miyagi 980-8579, Japan
Present address: Department of Applied Chemistry, Kogakuin University, 1-24-2 Nishi-shinjuku, Shinjuku-ku, Tokyo 163-8677, Japan
*
**

496
A. KOBAYASHI et al.
pIM04 was grown on 100 ml Luria-Bertani (LB)
medium containing ampicillin (50 mg/ml) to A600
Tris–HCl buffer (pH 7.0) was used instead of McIlvaine
buffer to eliminate the effect of phosphate on the
hydrolysis. The activities were calculated from the
amount of released glucose (the glucose-oxidase/per-
oxidase method, for trehalose 6-phosphate) or p-nitro-
phenol (for pNPG). Protein concentrations were deter-
mined by the method of Lowry et al., as described
previously.14)
¼
0:6{0:8, and then induced with isopropyl ꢁ-D-thio-
galactopyranoside (IPTG) to a final concentration of
0.5 mM and the incubation was continued overnight. The
cells were harvested by centrifugation at 10;000 ꢀ g for
5 min, resuspended in 40 ml of 10 mM Tris–HCl buffer
(pH 7.5), and disrupted by sonication. The supernatant
obtained by centrifugation at 10;000 ꢀ g for 15 min was
pooled as a crude bsPNPGH solution. The solution was
used for the protein purification and for the evaluation
of the ratio of the enzymatic activities for trehalose
6-phosphate and pNPG.
Primary sequence analysis. Similarity searches were
performed of the DDBJ database (http://www.ddbj.nig.
ac.jp/) using the BLAST program. Alignment of the
sequences and their phylogenetic distances were calcu-
lated at the DDBJ database using the ClustalW program.
The phylogenetic tree was illustrated with the TreeView
program.15)
Purification of the cloned pNPG-hydrolyzing enzyme.
The crude enzyme was dialyzed against 10 mM Tris–HCl
buffer (pH 7.5), and applied onto a DEAE-Toyopearl
650S column (1:8 ꢀ 16 cm, Tosoh, Tokyo, Japan)
equilibrated with the same buffer. The enzyme was
eluted with a linear gradient of 0–0.2 M sodium chloride
in the same buffer at a flow rate of 3 ml/min. The active
fractions were collected and dialyzed against 10 mM
Tris–HCl buffer (pH 7.5), and applied onto a Mono Q
HR 5/5 column (0:5 ꢀ 5 cm, Amersham). The proce-
dures of equilibration and elution were the same as for
the DEAE-Toyopearl 650S column.
Results and Discussion
Molecular cloning of a gene for a pNPG-hydrolyzing
enzyme
A genomic DNA of B. stearothermophilus SA0301
was prepared and partially digested with Sau3AI. After
separation using electrophoresis on a 1% agarose gel,
fragments of 4 to 10 kb in length were recovered and
inserted at the BamHI site of the plasmid pUC118.
E. coli MV1184 cells were transformed with the
recombinant plasmids. Colonies were screened for
pNPG-hydrolyzing activity and one clone was obtained.
The plasmid was designated pIM01 and the physical
map was determined (Fig. 1). The 2.2-kb KpnI-AccI
fragment of pIM01 was subcloned into pUC118, result-
ing in plasmid pIM04 (Fig. 1). pNPG-hydrolyzing
activity was detected in the E. coli MV1184 cells
harboring pIM04 when 0.5 mM IPTG was present in
the medium. It was not clear that the gene encodes the
enzyme we initially intended to clone (bsO16G), and
hence the newly cloned enzyme was tentatively des-
ignated bsPNPGH.
Preparation of bsO16G. Preparation and purification
of the extracellular enzyme (bsO16G) from B. stearo-
thermophilus SA0301 were carried out as described
previously.1)
Substrates for enzyme assays. Isomaltose (Glcp-ꢀ-
(1!6)-Glc), isomaltotriose (Glcp-ꢀ-(1!6)-Glcp-ꢀ-
(1!6)-Glc) and palatinose (Glcp-ꢀ-(1!6)-Fruf) were
purchased from Wako Pure Chemicals, Osaka, Japan.
ꢀ,ꢀ0-Trehalose (Glcp-ꢀ-(1!1)-Glcp) was obtained
from Hayashibara, Okayama, Japan. pNPG and treha-
lose 6-phosphate was purchased from Sigma, St. Louis,
MO, USA.
Purification and effect of pH and temperature of
recombinant bsPNPGH
Enzyme and protein assays. The pNPG-hydrolyzing
activity (for 5 mM pNPG in 50 mM McIlvaine buffer,
pH 6.5, at 60 ꢁC) was evaluated as described pervious-
ly.1) One unit of enzymatic activity was defined as the
amount of 1 mmol of released p-nitrophenol per min.
Kinetic parameters (including parameters for pNPG)
were determined as follows: A reaction mixture (1 ml)
containing the substrate, 50 mM McIlvaine buffer
(pH 6.5), and bsPNPGH or bsO16G was incubated at
60 ꢁC, and the portions (125 ml) were taken at 5-min
intervals to confirm the linearity of the reaction. The
reaction was stopped by adding equal amounts of 40 mM
sodium hydroxide, and the amount of liberated glucose
was measured using glucose-oxidase and peroxidase as
described1,14) for all the substrates listed in Table 2. For
evaluation of the ratio of the enzymatic activities for
trehalose 6-phosphate and pNPG (5 mM each), 10 mM
E. coli MV1184 harboring pIM04 was cultivated, and
recombinant bsPNPGH was purified in two chromatog-
raphy steps using a DEAE-Toyopearl 650S column and
a Mono Q HR 5/5 column (Table 1). SDS–PAGE with
Coomassie Brilliant Blue R staining gave a single band,
and the molecular mass was found to be approximately
65 kDa (data not shown). The N-terminal amino acid
Table 1. Summary of Purification of Recombinant bsPNPGH
Total
activity
(unit)
Specific
activity
(unit/mg)
Yield
(%)
Purification
(fold)
Crude enzyme
DEAE-Toyopearl 650S
Mono Q HR 5/5
348
178
112
100
51
12.8
38.8
65.9
1
3.0
5.1
32

Bacillus p-Nitrophenyl ꢀ-D-Glucoside Hydrolyzing Enzyme
497
0
1
2
3
4
5
(kb)
ORF –1
bsPNPGH
pIM01
(5.1 kb)
pIM04
(2.2 kb)
BamHI
HincII
KpnI HincII BamHI AccI BamHI
Fig. 1. Physical Maps of Plasmids Carrying the bsPNPGH Gene.
Open reading frames (ORF ꢂ1 and bsPNPGH) are shown and the directions are indicated by arrows. The sequenced region in this study
(3.3 kb) of pIM01 is printed in black.
Table 2. Kinetic Parameters for Hydrolysis of Various Substrates with bsO16G and bsPNPGH
bsO16G
bsPNPGH
k0
(sꢂ1
Km
k0=Km
k0
(sꢂ1
Km
k0=Km
(sꢂ1ꢃmM
11.9
0.81
Substrate
pNPG
Isomaltose
ꢂ1
ꢂ1
)
(mM)
(sꢂ1ꢃmM
101
)
)
(mM)
)
164 ꢄ 0:23
5:60 ꢄ 0:07
12:6 ꢄ 0:41
1:62 ꢄ 0:09
4:02 ꢄ 0:15
0:95 ꢄ 0:06
ND
52:0 ꢄ 0:21
4:97 ꢄ 0:11
4:37 ꢄ 0:18
6:15 ꢄ 0:30
ND
1.39
13.2
Isomaltotriose
ꢀ,ꢀ0-Trehalose
Palatinose
0:30 ꢄ 0:03
3:90 ꢄ 0:07
0:23 ꢄ 0:01
4:40 ꢄ 0:18
4:14 ꢄ 0:28
7:29 ꢄ 0:41
0.068
0.94
0.032
0:60 ꢄ 0:05
0:20 ꢄ 0:01
1:34 ꢄ 0:03
5:98 ꢄ 0:53
0.45
0.033
Methyl ꢀ-D-glucoside
ND, Activity not detected.
sequence of the recombinant bsPNPGH was analyzed
using a Beckman L3000 protein sequencer and deter-
mined to be MNKQPWWKK-. We reported previously
that the N-terminal amino acid sequence of bsO16G was
determined to be MERKWWEA-,1) which is different
from that of recombinant bsPNPGH.
isomaltooligosaccharides. In contrast, bsPNPGH did not
hydrolyze isomaltotriose but did hydrolyze ꢀ,ꢀ0-treha-
lose. Other than with an artificial substrate, pNPG, the
k0=Km values of bsPNPGH were low, and thus none of
the natural sugars listed in Table 2 were found to be the
substrate for bsPNPGH.
The effect of pH and temperature on bsPNPGH
activity was examined using pNPG as a substrate. The
optimal pH and temperature were pH 5.5–6.5 and 50–
60 ꢁC respectively. The enzyme was stable (residual
activity > 90%) in the range from 5.5 to 9.5 (at 50 ꢁC
for 10 min), and also stable up to 65 ꢁC (at pH 6.5 for
10 min). These values were similar to those for
bsO16G.1)
Comparison of the primary structures of bsPNPGH
and related enzymes
The nucleotide sequence of a portion of 3.3 kb of
pIM01 (printed in black in Fig. 1) was determined. The
open reading frame of bsPNPGH consisted of 1,689
nucleotides starting with an ATG codon, which corre-
sponds to a protein of 563 amino acid residues. The
calculated molecular mass was 66 kDa. The deduced N-
terminal amino acid sequence of bsPNPGH was iden-
tified as MNKQPWWKK-, identical to the N-terminal
amino acid sequence determined with the protein
sequencer. No sequence for the N-terminal amino acid
sequence of bsO16G, MERKWWEA-,1) was found in
the deduced primary structure of bsPNPGH, indicating
that the bsPNPGH gene is different from the bsO16G
gene we originally intended to clone.
A homology search of the deduced primary structure
of bsPNPGH was carried out, and the phylogenetic tree
of bsPNPGH and related enzymes is shown in Fig. 2A.
bsPNPGH most resembled an ꢀ-glucosidase from
Bacillus sp. DG0303 (TrEMBL no. Q9L872, 88%
identity), and also resembled trehalose-6-phosphate
hydrolases from several Bacilli, such as Bacillus sp.
Comparison of the kinetic parameters of bsPNPGH
and bsO16G
The kinetic parameters of bsPNPGH and bsO16G for
several saccharides were determined. Both enzymes
were unable to hydrolyze maltose. Although both
enzymes hydrolyzed pNPG, isomaltose, palatinose,
and methyl ꢀ-D-glucoside, their kinetic parameters were
different (Table 2). The k0=Km values of bsO16G for
ꢂ1
pNPG and isomaltose (101 and 1.39 sꢂ1ꢃmM respec-
tively) wereꢂh1igher than those of bsPNPGH (11.9 and
0.81 sꢂ1ꢃmM
respectively). The k0=Km value of
bsO16G for isomaltotriose was higher than that for
ꢂ1
isomaltose (13.2 and 1.39 sꢂ1ꢃmM respectively), and
bsO16G did not hydrolyze ꢀ,ꢀ0-trehalose. These results
indicate that the principal substrates of bsO16G are

498
A. KOBAYASHI et al.
bsPNPGH
BSP-DG0303-AGLU
BSP-GP16-PTRE
A
B
BSU-168-PTRE
BTG-O16G
BCE-O16G
BSP-SAM1606-AGLU
SEQ-DGLU
SMU-DGLU
*
*
*
332
191
205 252
263 323
bsPNPGH
FQKGIDGFRLDVINL
MTVGEMSSTTID
NALFWCNHDQ
NALFWCNHDQ
NALFWCNHDQ
NALFWCNHDQ
NSLYLNNHDQ
NSLYWNNHDQ
NSLYWTNHDQ
NSLFWNNHDL
NSLFWNNHDL
BSP–DG0303–AGLU
BSP–GP16–PTRE
BSU–168–PTRE
BTG–O16G
FQKGVDGFRLDVVNL
LKKGVDGFRLDVINL
FEKGIDGFRLDVINL
LDKGVDGFRMDVINM
LEKGIDGFRMDVINF
LDKGIDGFRMDVINA
IAKGIGGFRMDVIDL
IDKGIGGFRMDVIDM
MTVGEMSSTTID
MTVGEMSSTTID
MTVGEMSSTTVD
MTVGETPGVTPK
MTVGEMPGVTTE
MTVGETGGVTTK
MTVGETWGATPE
LTVGETWGATPE
BCE–O16G
BSP–SAM1606–AGLU
SEQ–DGLU
SMU–DGLU
Fig. 2. Comparison of the Primary Structures of bsPNPGH and Related Enzymes.
A, Phylogenetic tree of the enzymes. The tree was created with the programs Clustal W (DDBJ) and TreeView. Abbreviations: BSP-DG0303-
AGLU, Bacillus sp. DG0303 ꢀ-glucosidase (TrEMBL Q9L872); BSP-GP16-PTRE, Bacillus sp. GP16 trehalose-6-phosphate hydrolase (PRF
2922284A); BSU-168-PTRE, Bacillus subtilis trehalose-6-phosphate hydrolase (Swiss-Prot P39795); BTG-O16G, Bacillus thermoglucosidasius
oligo-1,6-glucosidase (Swiss-Prot P29094); BCE-O16G Bacillus cereus oligo-1,6-glucosidase (Swiss-Prot P21332); BSP-SAM1606-AGLU,
Bacillus sp. SAM1606 ꢀ-glucosidase (TrEMBL Q45517); SEQ-DGLU, Streptococcus equisimilis dextran glucosidase (Swiss-Prot Q59905);
SMU-DGLU, Streptococcus mutans dextran glucosidase (Swiss-Prot Q99040). B, Comparison of the amino acid sequences in the vicinity of the
catalytic residues. The numbering of the amino acid sequence of bsPNPGH is given. Three catalytic residues of ꢀ-amylase family enzymes are
indicated by asterisks. Arrows above the sequences indicated the residues that are important for the substrate specificities of the Bacillus sp.
SAM1606 ꢀ-glucosidase proposed by Noguchi et al.8)
GP16 (PRF no. 2922284A, 82% identity) and Bacillus
subtilis 1689) (Swiss-Prot no. P39795, 71% identity).
bsPNPGH showed homology with O16Gs and dextran
glucosidases, although the scores (about 50–60% iden-
tity) were lower than those for trehalose-6-phosphate
hydrolases (Fig. 2A).
(Fig. 1). The ORF ꢂ1 gene was transcribed in the same
direction as the bsPNPGH gene. The ORF ꢂ1 gene
consisted of 1,413 nucleotides, starting with an ATG
codon, which corresponds to a protein of 471 amino acid
residues. The deduced primary structure of ORF ꢂ1 was
highly homologous to bacillary trehalose-specific IIBC
components (about 70–90% identity) of the phospho-
transferase system (PTS), the major sugar transport
system in many Gram-positive and Gram-negative
bacterial species.18,19)
Enzymes belonging to the ꢀ-amylase family have
three acidic residues, which are identified as catalytic
residues.16,17) Based on the alignment of the primary
structures of bsPNPGH, trehalose-6-phosphate hydro-
lases, O16Gs, and dextran glucosidases, Asp201,
Glu256, and Asp331 of bsPNPGH, appear to function
as catalytic residues (Fig. 2B). Sequences in the vicinity
of the catalytic residues of these enzymes were found to
be highly conserved (Fig. 2B). Noguchi et al. reported
that the replacement of the two amino acid residues
indicated by arrows in Fig. 2B of Bacillus sp. SAM1606
ꢀ-glucosidase significantly altered the substrate specific-
ity.8) These two residues (Ser258 and Cys328) of
bsPNPGH were identical to those of Bacillus sp.
DG0303 ꢀ-glucosidase and trehalose-6-phosphate hy-
drolases (Fig. 2B).
bsPNPGH hydrolyzed trehalose 6-phosphate
The observations described above strongly suggest
that bsPNPGH exhibits the activity of trehalose-6-
phosphate hydrolase. To confirm this, a crude bsPNPGH
solution was prepared and the ratio of the enzymatic
activities for trehalose 6-phosphate and pNPG (5 mM
each) was determined. The activity for trehalose 6-
phosphate (101 mmolꢃminꢂ1ꢃmlꢂ1) was 45 times as high
as that for pNPG (2.26 mmolꢃminꢂ1ꢃmlꢂ1). The crude
extract from E. coli MV1184 harboring pUC118 was
also prepared by the same procedures. The extract
hydrolyzed neither trehalose 6-phosphate nor pNPG
under the same conditions. The results suggest that
bsPNPGH is a trehalose-6-phosphate hydrolase, and it is
reasonable to redesignate it as bsPTRE for future
studies.
Nucleotide sequence of an open reading frame
located upstream of the bsPNPGH gene
An open reading frame was located 80 bp upstream of
the bsPNPGH gene, and was designated ORF ꢂ1

Bacillus p-Nitrophenyl ꢀ-D-Glucoside Hydrolyzing Enzyme
499
properties of an oligo-1,6-D-glucosidase from an alkalo-
philic Bacillus species. Carbohydr. Res., 197, 227–235
(1990).
ꢀ-Amylase family enzymes are composed of three
common domains, A, B, and C.17) Janecek et al. analyzed
ˇ
the amino acid sequences of domain B of ꢀ-amylase
family enzymes.12) Domain B from Bacillus cereus
O16G and Streptococcus mutans dextran glucosidase
resemble each other very closely, and the report refers to
‘‘domain B of the oligo-1,6-glucosidase type.’’ Interest-
ingly, S. mutans dextran glucosidase not only attacks
dextran, but also splits pNPG, a molecule much smaller
than dextran.11) In this study, a BLAST search for
bsPNPGH retrieved O16Gs, ꢀ-glucosidases, trehalose-6-
phosphate hydrolases, and dextran glucosidases, all of
which are known to be pNPG-hydrolyzing enzymes.
These findings suggest that the primary structures of the
enzymes hydrolyzing pNPG are homologous, while their
enzymatic and physiological properties are quite diverse.
We have reported that intracellular and extracellular
pNPG-hydrolyzing activities were observed in different
periods during the cultivation of B. stearothermophilus
SA0301.1) The intracellular and the extracelluar activ-
ities were detected mainly during the logarithmic and
stationary phases respectively. On the basis of the results
from kinetic studies and primary structure analysis, it is
likely that the intracellular enzyme is to be identified as
bsPNPGH, which participates in PTS-mediated metab-
olism, while the extracelluar enzyme is to be identified as
bsO16G, which engages in hydrolyzing isomaltooligo-
saccharides on the outside of the cell. Although the
primary structures of the pNPG-hydrolyzing enzymes
are homologous to each other, their substrate specificities
for natural saccharides are diverse. Our observations
suggest that B. stearothermophilus produces two pNPG-
hydrolyzing enzymes whose physiological roles are
different. Further studies of these two enzymes might
help to elucidate the mechanism of the substrate
recognition of the pNPG-hydrolyzing enzymes. Oligo-
nucleotides encoding the N-terminal amino acid se-
quence of bsO16G and also amino acid sequences
conserved among O16Gs were prepared, and PCR
amplifications were carried out using several combina-
tions of these nucleotides. No clone for bsO16G has been
obtained so far, and molecular cloning of bsO16G is now
in progress.
5) Chiba, S., Molecular mechanism in ꢀ-glucosidase and
glucoamylase. Biosci. Biotechnol. Biochem., 61, 1233–
1239 (1997).
6) Nishio, T., Hakamata, W., Kimura, A., Chiba, S.,
Takatsuki, A., Kawachi, R., and Oku, T., Glycon
specificity profiling of ꢀ-glucosidases using monodeoxy
and mono-O-methyl derivatives of p-nitrophenyl ꢀ-D-
glucopyranoside. Carbohydr. Res., 337, 629–634 (2002).
7) Inohara-Ochiai, M., Nakayama, T., Goto, R., Nakao, M.,
Ueda, T., and Shibano, Y., Altering substrate specificity
of Bacillus sp. SAM1606 ꢀ-glucosidase by comparative
site-specific mutagenesis. J. Biol. Chem., 272, 1601–
1607 (1997).
8) Noguchi, A., Yano, M., Ohshima, Y., Hemmi, H.,
Inohara-Ochiai, M., Okada, M., Min, K.-S., Nakayama,
T., and Nishino, T., Deciphering the molecular basis of
the broad substrate specificity of ꢀ-glucosidase from
Bacillus sp. SAM1606. J. Biochem., 134, 543–550
(2003).
9) Gotsche, S., and Dahl, M. K., Purification and character-
ization of the phospho-ꢀ(1,1)glucosidase (TreA) of
Bacillus subtilis 168. J. Bacteriol., 177, 2721–2726
(1995).
10) Rimmele, M., and Boos, W., Trehalose-6-phosphate
hydrolase of Escherichia coli. J. Bacteriol., 176, 5654–
5664 (1994).
11) Russell, R. R. B., and Ferretti, J. J., Nucleotide sequence
of the dextran glucosidase (dexB) gene of Streptococcus
mutans. J. Gen. Microbiol., 136, 803–810 (1990).
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ˇ
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