Characterization of trehalose phosphorylase from Bacillus stearothermophilus SK-1 and nucleotide sequence of the corresponding gene

Article abstract of DOI:10.1271/bbb.66.1835

A bacterial trehalose phosphorylase (TPase; EC 2.4.1.64) was purified from the culture supernatant of Bacillus stearothermophilus SK-1 to apparent homogeneity, and some properties were investigated. Furthermore, a gene from SK-1 responsible for the TPase was cloned by Southern hybridization with a degenerate oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The M_r of the enzyme was estimated to be 150,000 by gel filtration and 83,000 by SDS-PAGE, so the enzyme is likely to be a homodimer. The enzyme had optimum activity at pH 7.0-8.0 or nearby and the optimum temperature was about 75°C. The deduced amino acid sequence of the SK-1 TPase encodes a theoretical protein with a M_r of 87,950. Alignment of amino acid sequences with a maltose phosphorylase from Lactobacillus brevis the crystal structure and active site of which had been analyzed suggested that these two phosphorylases evolved from a common ancestor. The Escherichia coli cells harboring the plasmid containing the cloned TPase gene had about 100 times the activity of SK-1.

Full text of DOI:10.1271/bbb.66.1835

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Characterization of Trehalose Phosphorylase from
Bacillus stearothermophilus SK-1 and Nucleotide
Sequence of the Corresponding Gene
Yasushi INOUEa, Keiko ISHIIa, Tetsuji TOMITAa, Tsuneya YATAKEa & Fumio FUKUIa
a Showa Sangyo Co., Ltd. 16, Sakura 1-chome, Tsukubashi, Ibaraki 305-0003, Japan
Published online: 22 May 2014.
To cite this article: Yasushi INOUE, Keiko ISHII, Tetsuji TOMITA, Tsuneya YATAKE & Fumio FUKUI (2002) Characterization
of Trehalose Phosphorylase from Bacillus stearothermophilus SK-1 and Nucleotide Sequence of the Corresponding Gene,
Bioscience, Biotechnology, and Biochemistry, 66:9, 1835-1843, DOI: 10.1271/bbb.66.1835
To link to this article: http://dx.doi.org/10.1271/bbb.66.1835
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Biosci. Biotechnol. Biochem., 66 (9), 1835–1843, 2002
Characterization of Trehalose Phosphorylase from Bacillus stearothermophilus
SK-1 and Nucleotide Sequence of the Corresponding Gene
Yasushi INOUE,† Keiko ISHII, Tetsuji TOMITA, Tsuneya YATAKE, and Fumio FUKUI
Showa Sangyo Co., Ltd., 16, Sakura 1-chome, Tsukubashi, Ibaraki 305-0003, Japan
Received February 18, 2002; Accepted May 13, 2002
A bacterial trehalose phosphorylase (TPase; EC
2.4.1.64) was puriˆed from the culture supernatant
of Bacillus stearothermophilus SK-1 to apparent
homogeneity, and some properties were investigated.
Furthermore, a gene from SK-1 responsible for the
TPase was cloned by Southern hybridization with a de-
generate oligonucleotide probe synthesized on the basis
of the N-terminal sequence of the puriˆed enzyme. The
Mr of the enzyme was estimated to be 150,000 by gel
ˆltration and 83,000 by SDS-PAGE, so the enzyme is
likely to be a homodimer. The enzyme had optimum ac-
tivity at pH 7.0–8.0 or nearby and the optimum temper-
We screened for microorganisms producing
TPase, and isolated Bacillus stearothermophilus
SK-1. This enzyme might be useful in some applica-
tions, such as the enzymatic synthesis of trehalose
from maltose coupled with maltose phosphorylase,
because it was produced and secreted by an aerobic
bacterium and had some advantageous characteris-
tics for commercial use. In this paper, we describe the
isolation and characterization of a TPase produced
by Bacillus stearothermophilus SK-1, and the cloning
and sequence of its gene.
ature was about 75
quence of the SK-1 TPase encodes a theoretical protein
with a r of 87,950. Alignment of amino acid sequences
with a maltose phosphorylase from Lactobacillus brevis
the crystal structure and active site of which had been
analyzed suggested that these two phosphorylases
evolved from a common ancestor. The Escherichia coli
cells harboring the plasmid containing the cloned TPase
gene had about 100 times the activity of SK-1.
9C. The deduced amino acid se-
Materials and Methods
M
Chemicals. Isomaltose was purchased from
Seikagaku Kogyo Co., Ltd., Japan. p-Nitrophenyl a-
D
-glucopyranoside,
p
-nitrophenyl
b- -glucopyrano-
D
side, trehalose, and
b
-D
-glucose 1-phosphate ( b-
G1P) were purchased from Sigma-Aldrich, St. Louis,
MO. DEAE-Toyopearl 650M and phenyl-Toyopearl
650M were products of Tosoh Corp., Tokyo, Japan.
Superdex 200 pg 16 60, MonoP HR 5 20, and Poly-
W
W
Key words: trehalose phosphorylase; trehalose phos-
buŠer 74 were products of Amersham Pharmacia
Biotech, Uppsala, Sweden. Pentax GH-0810M was a
product of Asahi Optical Co., Ltd., Tokyo. Other
chemicals were purchased from Wako Pure Chemical
Industries, Osaka, Japan.
phorylase gene; Bacillus stearothermophi-
lus
Trehalose phosphorylase (TPase;
a
,
a
-trehalose:
EC
orthophosphate -glucosyltransferase,
b
-
D
2.4.1.64) catalyzes the reversible phosphorolysis and
synthesis of trehalose as follows:
Medium. The test media used in screening were as
follows. The selection medium contained 0.2
z
trehalose, 0.05
0.1 sodium citrate, 0.02
KH2PO4, and 1.5 agar, pH 7.0. The standard
medium contained 0.5 peptone (Difco Laborato-
ries, Detroit, MI), 0.25 yeast extract, 0.1 glu-
cose, and 1.5 agar, pH 7.0. The medium YPT con-
tained 1 yeast extract, 2 Polypepton (Nihon
z z
yeast extract, 0.2 (NH4)2SO4,
Trehalose+orthophosphate
Ç
・
z MgSO4 7H2O, 0.35z
z
b
(a
)- -glucose 1-phosphate+ -glucose
D
D
z
z
z
This enzyme has been found in various organisms:
Thermoanaerobium brockii ATCC 35047,1)
a
z
3)
z
Plesiomonas sp.,2) Catellatospora ferruginea
,
4)
5)
z
z
Micrococcus varians
, Euglena gracilis, Flammuli-
7)
6)
Seiyaku Corp., Tokyo, Japan), and 2
pH 7.0.
z trehalose,
na velutipes
,
Schizophyllum commune
Grifola frondosa
Bradyrhizobium japonicum
,
Agaricus
10)
8)
9)
bisporus
,
,
Pichia fermentans
,
11)
,
and a cyanobacterial
Scytonema species.12) However, there are very few
reports about the genes and amino acid sequences of
this enzyme.
Screening method. Soils from various areas in
Japan were used. A small amount (about 2 g) of soil
was suspended in 5 ml of sterilized saline. Then the
†
To whom correspondence should be addressed. Yasushi INOUE, Fax: +81-298-50-6295; E-mail: brc
-G1P, -glucose 1-phosphate
＠
showa-sangyo.co.jp
Abbreviations: TPase, trehalose phosphorylase;
b
b-D

1836
Y. INOUE et al.
suspension (0.2 ml) was spread on agar plates of
selection medium. Colonies that appeared on the
plates were used to inoculate 3 ml of the YPT medi-
um in test tubes, which were shaken during cultiva-
Puriˆcation of TPase. The puriˆcation of TPase
was monitored by assays of TPase activity, and all
9
procedures were done at 10 C.
SK-1 was cultured aerobically for 3 d by shaking at
C in 3 l of YPT medium. Solid ammonium sulfate
was added to the culture supernatant after the cells
tion at 55
culture broth was homogenized with glass beads
0.3 mm). The homogenate was centrifuged at
9
C for 2 d. One milliliter portions of each
55
9
×
g for
(
q
had been removed by centrifugation at 10,000
10 min, and the precipitate that formed at between 40
and 60 saturation with ammonium sulfate was col-
lected by centrifugation at 15,000 for 20 min. The
precipitate was dissolved in a small volume of 5 m
×
10,000
precipitate that formed at 80
monium sulfate was collected by centrifugation at
g
for 15 min to remove cell debris, and the
z
saturation with am-
z
×
g
×
15,000
with 1 ml of 5 m
buŠer, pH 6.0, containing 80
g
for 10 min. The precipitate was washed
potassium phosphate-citrate
ammonium sulfate,
M
M
potassium phosphate-citrate buŠer, pH 6.0 (buŠer
A), and then dialyzed against the same buŠer. After
removal of the insoluble materials formed during the
dialysis by centrifugation, the dialyzed sample was
z
and dissolved in 0.2 ml of the same buŠer without
ammonium sulfate.
The trehalose phosphorolytic activity of this en-
zyme solution was tested under standard assay condi-
tions. The trehalose synthetic activity also was tested
under the following conditions. The enzyme solution
×
put on a column (2.5 20 cm) of DEAE-Toyopearl
650M equilibrated with buŠer A. After the column
was washed with buŠer A, the enzyme was eluted
with a linear gradient of NaCl from 0 to 0.5
M
in
was dialyzed against 5 m
M
acetate buŠer, pH 6.0.
buŠer A at the ‰ow rate of 4 ml min. The eluate was
W
The reaction mixture contained 0.03 ml of the en-
fractionated into 8-ml portions and the active frac-
tions were pooled. This step was repeated under the
same conditions except that the elution was done
zyme solution, 0.02 ml of 0.2
0.2 -G1P, 0.008 ml of 0.5
M
glucose, 0.02 ml of
acetate buŠer, pH
M
b
M
6.0, and water, in a total volume of 0.1 ml. The mix-
ture was incubated at 55 C for 1 d and then boiled
with a linear gradient of NaCl from 0 to 0.25
M
in
9
buŠer A. The active fractions were pooled and con-
centrated to a small volume by ultraˆltration with a
Centriprep-10 apparatus (Millipore Corp., Bedford,
MA). Solid ammonium sulfate was dissolved in this
for 10 min to stop the reaction. The detection of tre-
halose and phosphate was done by HPLC.
A strain capable of producing TPase was selected
as the enzyme producer. Identiˆcation of the isolated
bacterium was done by Japan Food Research
Laboratories (Tokyo) by the methods described in
``Bergey's Manual of Systematic Bacteriology''13)
and ``The Genus Bacillus''.14)
enzyme solution to a ˆnal concentration of 40
this solution was put on a column (2.5 20 cm) of
phenyl-Toyopearl 650M equilibrated with buŠer A
containing 40 ammonium sulfate. After the
column was washed with buŠer A containing 40
ammonium sulfate, elution was done with a linear
z, and
×
z
z
Assay of TPase activity. TPase activity was as-
sayed by the measurement of the glucose liberated
from trehalose in the presence of phosphate. The
z
gradient of ammonium sulfate from 40 to 0 in
buŠer A at the ‰ow rate of 4 ml min. The eluate was
W
fractionated into 8-ml portions and the active frac-
tions were pooled. The enzyme solution was concen-
trated to a small volume by ultraˆltration with a
standard assay system contained 0.6 ml of 2
z
treha-
lose, 0.06 ml of 0.5 potassium phosphate-citrate
M
buŠer, pH 6.0, an enzyme solution, and water in a
total volume of 1.2 ml. The mixture was incubated
Centriprep-10 and put on a Superdex 200 pg 16 60
W
column equilibrated with buŠer A containing 0.15
M
for 10 min at 60
9C and then boiled for 10 min to stop
NaCl. The enzyme was eluted with the same buŠer at
the ‰ow rate of 0.5 ml min and the active fractions
were pooled. The enzyme solution was put on a
hydroxyapatite column, Pentax GH-0810M equili-
the reaction. The amount of glucose liberated was
measured by the mutarotase-glucose oxidase method
with a Glucose CII-Test kit (Wako). One unit of
enzyme activity was deˆned as the amount of the
enzyme that liberates 1 micromole of glucose per
minute under these conditions. The speciˆc activity
was expressed by the enzyme activity per milligram of
protein.
W
brated with 1 m
M
potassium phosphate-citrate
CaCl2. After the
buŠer, pH 6.0, containing 0.3 m
M
column was washed with the same buŠer, elution was
done with a linear gradient of potassium phosphate-
citrate buŠer, pH 6.0, from 1 to 200 m
M
at the ‰ow
rate of 0.5 ml min. The eluate was fractionated into
1-ml portions and the active fractions were pooled as
the puriˆed TPase.
W
Assay of protein. In the puriˆcation by column
chromatography, the protein elution pattern was
monitored spectrophotometrically as the absorbance
at 280 nm. Protein concentrations were measured by
Amino-terminal amino acid sequence of TPase.
The amino-terminal amino acid sequence of puriˆed
TPase was analyzed by Edman degradation with a
gas-phase protein sequencer (ABI model 477A). The
the method of Lowry et al.
15) with bovine serum albu-
min as the standard.

Trehalose Phosphorylase from B. stearothermophilus SK-1 and Its Gene
1837
sample for sequence analysis was prepared by the
method of Matsudaira.16)
(Takara, Tokyo). Competent E. coli JM109 cells
were transformed with the ligation mixture and
spread on Luria-Bertani agar plates containing am-
Electrophoresis. SDS-PAGE of the puriˆed TPase
picillin (50 -galactoside
m
g ml), isopropyl-1-thio-
b
-
D
W
was done by the method of Laemmli17) with a Phast
(0.1 m ), and X-gal (40 mg ml). After screening of
M
W
System (Pharmacia, Uppsala, Sweden) and
a
the resulting 450 white colonies by Southern hybridi-
zation with the radioactive probes, two colonies car-
rying the recombinant plasmids containing inserts of
about 2 kb were obtained. The insert contained about
1.5 kb of the coding region of a putative trehalose
phosphorylase gene, but the terminal codon was
outside of the coding region. The DNA fragment cor-
responding to the missing C-terminal part was ampli-
PhastGel Gradient 10–15 (10–15
z polyacrylamide
gel; Pharmacia). An LMW electrophoresis calibra-
tion kit (Pharmacia) was used for standard proteins.
Isoelectric focusing was done with a Phast System
and PhastGel IEF 4–6.5 (Pharmacia). Protein bands
were stained with Coomassie Brilliant Blue R-250.
Molecular weight (Mr) measurement. A Superdex
ˆed by inverse PCR22) with two primers, TR (5
?-
200 pg 16 60 column was used to measure the Mr of
ATTTGCGCTGGGCAGCCAAAAGTGT-3
TL (5 -GTGCTTTGCCATCACGTTCGTGTAA-
), and a self-ligated MspI digest of the SK-1 chro-
?)
and
W
the puriˆed TPase. The analysis was done in 10 m
acetate buŠer, pH 6.0, containing 0.15 NaCl at
C at the ‰ow rate of 0.5 ml min. A Mr marker kit
M
?
M
3?
4
9
mosomal DNA as the template. The nucleotide se-
quence of the ampliˆed fragment was directly ana-
lyzed.
W
(Boehringer, Mannheim, Germany) was used for
standard proteins.
HPLC for identiˆcation of sugars and phosphate.
DNA sequence analysis. DNA was sequenced with
an ABI 373A sequencer (Applied Biosystems) and an
ABI Prism dye terminator cycle sequencing kit
(Perkin Elmer, Foster City, CA) or ABI Prism dye
primer cycle sequencing ready reaction kit (Perkin
Elmer). A homology search of protein databases was
done with the FASTA program.23) Amino acid se-
quences were aligned with the Genetyx Mac software
Version 10.1 (Software Development Co., Ltd.,
Tokyo, Japan).
HPLC conditions for measurement of trehalose and
×
＝
glucose were as follows: TSK gel Amido 80 (4.6
250 mm, Tosoh); mobile phase, acetonitrile:H2O
76:24 (w w); ‰ow rate, 0.8 ml min; column temp.,
W
W
80
HPLC conditions for measurement of
phosphate were as follows: TSK gel SAX (6.0
150 mm, Tosoh); mobile phase, 0.1 potassium
acetate (pH 5.0, adjusted with HCl); ‰ow rate,
1.0 ml min; column temp., 30 C, and detector, RI.
9
C and detector, refractive index indicator (RI).
b
-G1P and
×
M
9
W
Construction of the plasmid for expression of the
TPase gene in E. coli. On the basis of the entire
nucleotide sequence of the TPase from SK-1, two
Preparation of DNAs. B. stearothermophilus SK-1
chromosomal DNA was prepared by the method of
Saito and Miura18) except that achromopeptidase
from Achromobacter lyticus (Wako) was used as a
lytic enzyme. Plasmid DNA was prepared by the
method of Birnboim and Doly.19)
oligonucleotide primers, 5
AAACAAATCAGTTCAA-3
and 5 -AAACTGCAGTTAATCAACACGCCCGT-
TAT-3 (3 of the TPase gene), including PstI restric-
?
-AAACTGCAGTTGA-
?
(5 of the TPase gene)
?
?
?
?
tion sites (underlined), were synthesized (Espec Oligo
Service) and used together with total DNA from SK-1
to amplify the TPase gene. With Taq DNA poly-
merase (Takara), a 2448-bp fragment was ampliˆed.
The PCR fragment was cloned into the pCR2.1
Cloning of the TPase gene. On the basis of the par-
tial N-terminal sequence, QLNIEN, of TPase from
SK-1, a degenerate oligonucleotide probe, TN3
(5?-CARYTIAAYATHGARAA-3?), was synthesized
(Espec Oligo Service, Tsukuba, Japan) and used for
Southern hybridization with total DNA and a partial
genomic library of SK-1. Southern blotting was done
as described elsewhere.20) DNA manipulations and
vector and used to transform E. coli INVaF? with an
Original TA Cloning Kit (Invitrogen Corp.,
Carlsbad, CA) by the manufacturer's instructions.
Clones were analyzed for the correct insert by DNA
sequencing as described above. The resulting plasmid
was designated pSTP1.
transformation in Escherichia coli were done as de-
21)
scribed by Sambrook et al
with [
.
TN3 was end-labelled
g
-32P]ATP and hybridized to a Southern blot of
SK-1 chromosomal DNA cut with diŠerent restric-
tion enzymes. Speciˆc HindIII restriction fragments
of about 2 kb bound the radioactive probes. To clone
this fragment, a partial genomic library of SK-1 was
prepared as follows. Fragments (1.5–2.5 kb) from
SK-1 DNA cut with HindIII were isolated and ligated
with HindIII and phosphatase-treated pUC118
Results
Screening for TPase-producing bacteria
From among about 200 isolates that could grow at
9
55 C in a medium containing trehalose as the sole
carbon source, 11 isolates with trehalose degradation
activity were chosen and their trehalose phosphoro-

1838
Y. INOUE et al.
Table 1. Puriˆcation of TPase from Bacillus stearothermophilus SK-1
Total protein
(mg)
Total activity
(units)
Speciˆc activity
Yield
Puriˆcation
(fold)
Step
(units mg)
(z)
W
Culture supernatanta)
Ammonium sulfate (40–60
DEAE-Toyopearl 650M (ˆrst)
DEAE-Toyopearl 650M (second)
Phenyl-Toyopearl 650M
Superdex 200 pg
2845
1845
253
73.8
15.2
3.7
945
864
389
279
117
81.6
52.3
0.33
0.47
1.54
3.78
7.70
22.1
23.8
100
1
1.4
4.7
11.5
23.3
67.0
72.1
z)
91.4
41.2
29.5
12.4
8.63
5.53
Pentax GH-0810M
2.2
a)
Corresponding to 5 liters of the culture broth.
Table 2. Substrate Speciˆcity of Phosphorolysis of TPase
Substrate
Relative activity (z)
Trehalose
Isomaltose
Maltose
100
3
1
Sucrose
1
Neotrehalose
Cellobiose
1
2
2
2
p
p
-Nitrophenyl
-Nitrophenyl
a
b
-
-
D
-glucopyranoside
-glucopyranoside
D
Fig. 1. EŠects of pH (A) and Temperature (B) on the TPase
Activity.
The enzyme activity was assayed as described in the text in the presence of
substrate, except that incubation was for 20 min. The activity with tre-
halose was taken to be 100
1
z
(A) To examine the optimum pH, we measured TPase activi-
z.
ties in 25 m
used were:
(+25 m
zyme solution was kept at 60
tions at various pHs and the activity remaining was measured.
M
buŠer solutions at various pHs. The buŠer systems

,
potassium phosphate-citrate;
#
, Tris-HCl
M
K2HPO4). For examination of pH stability, the en-
C for 24 h in 25 m buŠer solu-
9
M
lytic activity was tested. Only one isolate was ob-
tained and conˆrmed to have trehalose synthetic
activity. The isolate was identiˆed as Bacillus
stearothermophilus and named SK-1.
The buŠer systems used were:
, Tris-HCl.

, potassium phosphate-citrate;
$
(B) To examine the optimum temperature, we measured
TPase activities at various temperatures in 25 m potassium
phosphate-citrate buŠer (pH 6.0) ( ). For examination of ther-
mal stability, the enzyme solution was kept at various tempera-
tures for 15 min in 10 m acetate buŠer (pH 6.0) and the activity
).
M

Puriˆcation and amino-terminal amino acid se-
quence analysis of TPase
M
SK-1 produced TPase almost completely extracel-
lularly. Most of the TPase activity was detected in the
culture supernatant after cultivation for 1 to 3 d with
remaining was measured (

shaking at 55
ˆed TPase from the culture supernatant of SK-1. The
puriˆcation procedure resulted in 5.53 recovery of
9
C in YPT medium. Therefore, we puri-
Substrate speciˆcity
The speciˆcity of phosphorolysis of various disac-
charides and related compounds by the puriˆed en-
zyme was examined (Table 2). All substrates except
trehalose were nearly non-reactive with TPase. These
results suggested that this enzyme had high substrate
z
the enzyme activity and a 72-fold enrichment of
TPase compared with the culture supernatant of a 5-
liter culture (Table 1). SDS-PAGE of this puriˆed
TPase gave a single protein band (data not shown).
The ˆrst 19 residues of the amino-terminal sequence
of the puriˆed TPase, identiˆed chemically, were
SWSISSNQLNIENLLNEES.
speciˆcity for the
a
, 1-1 glycosidic linkage between -
D
glucose and -glucose.
D
EŠects of pH and temperature
The activities were measured in the range of pH 4.0
to 9.0 with 25 m potassium phosphate-citrate buŠer
(pH 4.0–7.5) and 25 m Tris-HCl buŠer (pH
7.5–9.0). When Tris-HCl buŠer was used, 25 m
Mr and pI
M
The apparent Mr of the puriˆed enzyme was esti-
mated to be 150,000 by gel ˆltration on a Superdex
200 pg column. SDS-PAGE of the puriˆed enzyme
gave a single protein band with an apparent Mr of
83,000 (data not shown). These results suggested that
the enzyme consisted of two identical subunits. The
M
M
K2HPO4 was added to the reaction mixture, and the
pH after mixing in the latter compound is indicated.
The enzyme had an optimum pH range from 7.0 to
8.0 (Fig. 1(A)). The pH stability was assessed by
p
I
of the puriˆed enzyme was estimated to be 5.0 by
treatment at 60
25 m potassium phosphate-citrate buŠer (pH
4.0–8.0) and 25 m Tris-HCl buŠer (pH 7.5–9.0).
9
C for 24 h from pH 4.0 to 9.0 with
isoelectric focusing (data not shown).
M
M

Trehalose Phosphorylase from B. stearothermophilus SK-1 and Its Gene
1839
The TPase was stable at pHs between 6.0 and 8.0 un-
der these conditions.
producing TPase, and puriˆed and characterized the
enzyme. We have cloned its gene and expressed it in
E. coli. This is the ˆrst report of the production of
TPase, not to mention its gene, by Bacillus bacteria.
TPase has been found in various organisms and
some properties are compared in Table 3. Generally
speaking, bacterial TPases are similar to each other
in many points, to judge from known properties.
One diŠerence is the diversity of the number of subu-
nits, although the Mr of the subunits is not very
diŠerent. TPase from SK-1 was one of the most ther-
mostable ones. From the viewpoint of large-scale fer-
mentation, together with the extracellular production
described below, high thermostability is advan-
tageous when the enzyme is to be prepared for com-
mercial use.
The eŠects of temperature on activity were exam-
ined. The enzyme activities were measured at various
temperatures under the standard assay conditions.
The maximum activity was at 75
thermal stabilities of the enzyme were assessed in
25 m potassium phosphate-citrate buŠer, pH 6.0,
9C (Fig. 1(B)). The
M
after incubation at various temperatures for 15 min.
The puriˆed TPase was completely stable up to 60 C.
9
Kinetics
When the reciprocals of the initial velocities were
plotted against those of the initial concentrations of
trehalose at several ˆxed concentrations of Pi, the
lines intersected in the left quadrant, so the kinetic
mechanism was sequential (data not shown). The Km
The metabolism of trehalose has been studied in
many organisms, and TPase may be located in the
cell to degrade trehalose transported into the cell
from the culture medium. Also, with TPase from
SK-1, low Km for trehalose and Pi suggest that the
TPase from SK-1 can proceed in the direction of
phosphorolysis under physiological conditions. In
this study, most of the TPase activity was detected in
the culture supernatant of SK-1, but the DNA se-
quence of the TPase gene showed that there was no
secretion signal peptide, and analysis of the amino-
terminal residues of the TPase produced in SK-1 sug-
gested that it is processed by a methionine aminopep-
tidase. One possibility is that this observation arose
because of autolysis of the cells, but the reason why
SK-1 produces TPase extracellularly remains
unknown.
for trehalose and Pi were 4.1 and 1.1 m
tively.
M
, respec-
DNA sequence of the TPase gene
The DNA sequence of the TPase gene, consisting
of 2796 bp, was identiˆed (Fig. 2). The open reading
frame of 765 codons, from positions 389 to 2683, en-
coded a theoretical protein with a
Mr of 87,950. This
agreed with the results of SDS-PAGE of puriˆed
TPase from SK-1 (data not shown). The amino acid
sequence deduced from the DNA sequence contained
the amino-terminal amino acid sequence of the puri-
ˆed TPase identiˆed chemically (amino acid residues
2 through 20 [Fig. 2]). This conˆrmed that the open
reading frame was the coding sequence for TPase.
That the amino-terminal amino acid sequence con-
tained no signal sequence is indicated that it was syn-
thesized in the cytoplasm. It is not known why TPase
is found in the culture supernatant of SK-1. Analysis
of the DNA sequences upstream of the TPase gene
showed that there was a potential „35 sequence,
„10 sequence, and a ribosomal binding site at nt 257
to 262, 280 to 285, and 376 to 380, respectively.
The deduced amino acid sequence of the TPase
gene had 46
z
similarity to the TPase from Ther-
moanaerobium brockii ATCC 35047 (Table 4). Some
highly conserved regions were observed, suggesting
them to be crucial for the expression of its activity.
On the other hand, two TPase genes of basidiomy-
cetes, Grifola frondosa (AB010104) and Pleurotus
z
sajor-caju (AF149777) were less than 20 similar to
Expression of the protein encoded by TPase gene
in E. coli
The expression of the recombinant TPase encoded
by pSTP1 was checked by an assay of the TPase ac-
tivity. E. coli cells carrying pSTP1, designated STP1,
were cultured aerobically for 1 d with shaking at
those of SK-1 and T. brockii ATCC 35047, and there
were no conserved regions. These observations may
suggest that TPase evolved independently in eukary-
otes and prokaryotes. We could not ˆnd any other
similar genes in the databases of the DDBJ including
phosphorylases, glucosyltransferases, and trehalases.
TPases have been classiˆed in family 65 of the
glycoside hydrolases, which includes sequences cod-
ing for maltose phosphorylases, trehalases, and un-
characterized proteins. During this work, the crystal
structure of the maltose phosphorylase from Lac-
tobacillus brevis (Lb-MP) was published and the
catalytic residue of Glu487 was identiˆed.24) The
37
(50
glass beads (
9
m
C in Luria-Bertani medium containing ampicillin
g ml). The culture broth was homogenized with
W
f
0.3 mm), followed by centrifugation at
×
10,000
g for 15 min to remove cell debris, and the
supernatant was assayed for TPase activity. STP1
produced about 20,000 units per liter culture of
TPase, about 100 times that produced by SK-1.
TPase from SK-1 had only 32
z similarity to Lb-MP
as a whole, but residues that form the putative active-
site pocket of Lb-MP agreed well with those of the
TPase. Especially, the hypothetic catalytic acid and
Discussion
We isolated Bacillus stearothermophilus SK-1

1840
Y. INOUE et al.
Fig. 2. Nucleotide and Deduced Amino Acid Sequence of the TPase Gene of SK-1.
Numbers on the right indicate nucleotide positions. The putative „35 sequence, „10 sequence, and ribosomal binding site are
shown. The restriction enzyme sites of HindIII and MspI are shown. The stop codon is indicated by an asterisk. The deduced amino acid
sequences are shown below the nucleotide sequence. The amino-terminal amino acid sequence of TPase, identiˆed chemically, is under-
lined. The sequences corresponding to those of primers used in the text are also underlined (TR and TL).
residues contacting the phosphate coincided com-
pletely (Fig. 3). TPase resembles maltose phosphory-
lase in its reaction mechanism, that is, both enzymes
catalyze reversible phosphorolysis of a disaccharide
a common ancestor.
An overexpression system for the e‹cient produc-
tion of TPase is under investigation. The DNA se-
quence in this report will appear in the DDBJ,
EMBL, and GenBank databases with the accession
number of AB079610.
with b-G1P as a glucosyl donor and with inversion of
the anomeric conˆguration. These observations sug-
gest that TPase and MPase could have evolved from

Trehalose Phosphorylase from B. stearothermophilus SK-1 and Its Gene
1841

1842
Y. INOUE et al.
Fig. 3. Alignment of the Amino Acid Sequences of the TPase from SK-1 and Maltose Phosphorylases.
The alignment includes sequences of the TPase of Bacillus stearothermophilus SK-1 (Bs-TP), the maltose phosphorylases of Lac-
tobacillus brevis (Lb-MP), Lactobacillus sanfranciscensis (Ls-MP), and Neisseria meningitidis (Nm-MP); of the putative maltose phos-
phorylases of Mycobacterium tuberculosis (Mt-MP), Mycobacterium leprae (Ml-MP), and Bacillus subtilis (Bs-MP). The residues that
form the active-site pocket of Lb-MP are marked ( ). The hypothetic catalytic acid and residues contacting the phosphate are shaded.
&
Table 4. Similarities of Amino Acid Sequences of Various
TPases
Acknowledgments
We thank to Professor K. Yamane and Dr. K.
Nakamura (Tsukuba University, Japan) for their
valuable discussions and advice.
1
2
3
4
1 Bs SK-1
100.0
46.0
17.6
18.1
46.0
100.0
18.6
17.5
17.6
18.6
100.0
73.7
18.1
17.5
73.7
100.0
2 Tb (ATCC 35047)
3 Gf
4 Ps
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Trehalose Phosphorylase from B. stearothermophilus SK-1 and Its Gene
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