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Applied and Environmental Microbiology, May 1999, p. 1991-1997, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Purification and Characterization of an Extremely
Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated
Hyperthermophilic Archaeon, a Thermococcus sp.
Yoshihisa
Tachibana,1,*
Akiko
Kuramura,1
Naoki
Shirasaka,1
Yuji
Suzuki,2
Tomoko
Yamamoto,3
Shinsuke
Fujiwara,3
Masahiro
Takagi,3 and
Tadayuki
Imanaka4
Research and Development Center, Nagase Co., Ltd., 2-2-3 Murotani, Nishi-ku, Kobe 651-2241,1
Nagase Biochemicals, Ltd., 1-52 Osadano-cho, Fukuchiyama,
Kyoto 620-0853,2 Department of
Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871,3 and
Department of Synthetic Chemistry and Biological Chemistry,
Graduate School of Engineering, Kyoto University, Kyoto
606-8501,4 Japan
Received 4 December 1998/Accepted 25 February 1999
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ABSTRACT |
The extremely thermophilic anaerobic archaeon strain B1001 was
isolated from a hot-spring environment in Japan. The cells were
irregular cocci, 0.5 to 1.0 µm in diameter. The new isolate grew at
temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to
9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The
G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene
sequencing of strain B1001 indicated that it belongs to the genus
Thermococcus. During growth on starch, the strain produced
a thermostable cyclomaltodextrin glucanotransferase (CGTase). The
enzyme was purified 1,750-fold, and the molecular mass was determined
to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for
starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was
pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at
110°C. The enzyme formed mainly 
-cyclodextrin with small amounts
of 
- and 
-cyclodextrins from starch. This is the first report on
the presence of the extremely thermostable CGTase from
hyperthermophilic archaea.
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INTRODUCTION |
Cyclomaltodextrin
glucanotransferases (CGTase; EC 2.4.1.19) are able to convert starch
into cyclodextrins (CDs), closed-ring structures in which six or more
glucose units are joined by means of -1,4 glucosidic bonds
(15). Depending to the number of glucose units (six, seven,
or eight), they are named -, -, or -CDs, respectively. They
are able to form inclusion complexes with many organic and inorganic
molecules, thereby changing the physical and chemical properties of the
included compounds. Therefore, CGTase is an important enzyme for the
food, cosmetic, and pharmaceutical industries. CGTase are known to
catalyze four different transferase reactions: cyclization, coupling,
disproportionation, and hydrolysis. CGTase are classified in the
-amylase family, which includes -amylase, isoamylase,
pullulanase, amylopullulanase, neopullulanase, and the branching enzyme
(28, 34, 46). The -amylase family enzymes include the
four highly conserved amino acid sequences containing the substrate
binding and active sites. The three-dimensional structure of CGTase
closely resembles the (/)8-barrel structure of
-amylase (43).
CGTase is produced by many Bacillus strains,
Klebsiella pneumoniae, Klebsiella oxytoca, a
Brevibacterium sp., a Thermoanaerobacter sp., and
Thermoanaerobacterium thermosulfurigenes (11, 21, 22,
26, 33, 38, 49). The thermophilic anaerobic bacteria Thermoanaerobacter sp. and T. thermosulfurigenes
produce the thermostable CGTase. The optimum temperatures of these
enzymes are 90 to 95°C and 80 to 90°C, respectively. Thermostable
CGTase is very useful for industrial utilization. Liquefaction, which
is the first step in the production of CDs from starch, is performed by
jetcooking, where a starch slurry is treated with thermostable
-amylase at 105 to 110°C. Thermostable CGTase can be used in
liquefaction and then in CD formation without being replaced
(38).
Recently, hyperthermophilic archaea which can grow at temperatures
higher than 90°C have been isolated, and their enzymes have been
shown to be extremely thermostable. Extremely thermostable -amylase
family enzymes including -amylase, pullulanase, and amylopullulanase
have been characterized from the genera Thermococcus, Pyrococcus, Sulfolobus, Thermofilum,
Desulfurococcus, and Staphylothermus (4-8,
12, 17, 24, 25, 29, 41, 45). However, CGTase has not been
isolated from archaea. We have initiated a program to isolate
hyperthermophiles which can secrete CGTases. In this paper, we report
on the purification and characterization of an extremely thermostable
CGTase from a newly isolated hyperthermophilic archaeon,
Thermococcus sp. strain B-1001.
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MATERIALS AND METHODS |
Isolation of the hyperthermophile.
Hot sedimentary materials
and venting water were collected at a thermal vents and hot spring
environments in Japan. Samples were put into 50-ml screw cap bottles
and brought back to the laboratory while being held at about 80°C for
the transportation. The samples were inoculated immediately into
anaerobic gas-flushed 15-ml test tubes containing 5 ml of medium. A
mixture of anaerobic gases,
N2-CO2-H2 (90:5:5 [vol/vol/vol]),
was used. The tubes were closed tightly and incubated at 85°C until
cell growth was observed. To obtain pure cultures, a
dilution-to-extinction technique was used. After the cell density of an
enrichment culture reached approximately 108 cells per ml,
five serial transfers into same medium were carried out by performing
10 1:10 dilutions per transfer. Each dilution in the
dilution-to-extinction series was incubated for at least 4 days. The
culture in the tube showing growth at the highest dilution was chosen
as the pure culture, which usually grew up to about 108
cells per ml in 2 days. Strain B1001 was isolated from the hot spring
at Tottori prefecture, Japan.
Culture conditions.
Enrichments and isolation were carried
out in a modified SME medium (2) containing the following
components (per liter): 2 g of yeast extract (Difco), 2 g of
NaCl, 3.5 g of MgSO4 · 2H2O, 2.75 g of MgCl2 · 6H2O, 1 g of
CaCl2 · 2H2O, 0.5 g of
KH2PO4, 0.325 g of KCl, 50 mg of NaBr, 15 mg of
H3BO3, 7.5 mg of SrCl2 · 6H2O, 2 mg of
(NH4)2Ni(SO4)2 · 2H2O, 2.5 µg of KI, 10 ml of trace minerals mixture,
10 g of elemental sulfur, 0.48 g of Na2S · 9H2O, and 1 mg of resazurine. The pH was adjusted to 7.0 with KOH. The trace minerals mixture (1) contained (per
liter) 1.5 g of nitrilotriacetic acid, 3 g of
MgSO4 · 7H2O, 0.5 g of
MnCl2 · 4H2O, 1 g of NaCl, 0.1 g of FeSO4 · 7H2O, 0.1 g of
CoSO4, 0.1 g of CaCl2 · 2H2O, 0.1 g of ZnSO4, 10 mg of
CuSO4 · 5H2O, 10 mg of
AlK(SO4)2, 10 mg of
H3BO3, and 10 mg of
Na2MoO4 · 2H2O.
Physiological characteristics.
The physiological
characteristics of the isolated strain were investigated in anaerobic
gas-flushed 15-ml test tubes containing 10 ml of medium supplemented
with 0.2% peptone. To check the optimal growth conditions, the
incubation temperature, pH, and NaCl concentration of the culture
medium were varied over a wide range of conditions. The temperature was
tested from 40 to 110°C. Temperatures were maintained with aluminum
heating blocks in an anaerobic incubator (EAN-140; Tabai Espec Corp.,
Osaka, Japan). The pH of the medium was adjusted from 4 to 10 by the
addition of H2SO4 or KOH. The NaCl
concentration ranged from no NaCl added to 6% NaCl. Samples were
collected every 2 h under anaerobic and high-temperature conditions, and the cells were counted. To determine substrate utilization, substrates were added at 0.2% (wt/vol) to yeast extract- and peptone-free medium.
Sensitivity to the antibiotics benzylpenicillin, vancomycin,
streptomycin, chloramphenicol, and rifampin was examined by the addition of 100 µg of antibiotic per ml of medium under the same conditions as those reported for Thermococcus fumicolans
(13) and Thermococcus celer (50).
H2S was qualitatively detected by adding 10 µl of a
saturated lead acetate solution to 1 ml of culture sample based on the procedure of Williams (48). A blackish-brown precipitate
indicated the presence of H2S.
Determination of cell numbers.
The number of cells in liquid
cultures was determined by direct cell counting in a Thoma counting
chamber (depth, 0.02 mm) under a phase-contrast microscope. Generation
times were calculated by linear regression analysis from three points
along the logarithmic part of the resulting growth curves.
Electron microscopy.
For scanning electron microscopy, the
cells were fixed in 0.1 M phosphate-buffered glutaraldehyde (2.5%
final concentration) at pH 7.0. The fixed samples were dehydrated
through 25, 50, 75, and 100% ethanol and t-butanol, dried
to the critical point, and sputter-coated with palladium-gold alloy.
The samples were examined under an S-3200N scanning electron microscope
(Hitachi, Tokyo, Japan).
DNA preparation and DNA base composition.
The genomic DNA
was isolated with a GenomicPrep kit (Pharmacia, Uppsala, Sweden). The
G+C content of the genomic DNA was determined by direct analysis of the
mononucleoside content of the decomposed DNA (31) with a
DNA-GC kit (Yamasa Shoyu, Tokyo, Japan). Calf thymus DNA (42 mol% G+C)
(Sigma) was used as reference. The value used was the average of the
results of three separate experiments.
Analysis of the 16S rRNA gene.
The 16S rRNA gene of strain
B1001 was amplified by PCR with forward primer
5'-TTCCGGTTGATCCYGCCGGA-3' and reverse primer
5'-GGTTACCTTGTTACGACTT-3', which correspond to nucleotides 2 to 21 and 1510 to 1492 of the Escherichia coli 16S rRNA gene
sequence, respectively (39). The amplified PCR product was
cloned in pBluescript II SK(+), and both strands were sequenced by the
dideoxy-chain termination method with fluorescent primer (A.L.F DNA
sequencer; Pharmacia).
The 16S rRNA gene sequence was aligned with a representative group of
archaeal sequences obtained from GenBank and EMBL. The
phylogenetic
tree was constructed based on the neighbor-joining
(NJ) method
(
42). Alignment of sequences and estimations of
the numbers
of nucleotide substitutions were performed with the
CLUSTAL W program
(
47).
Assay of CGTase activity.
Potato starch (1 g) was suspended
in 20 ml of distilled water and then dissolved by adding 5 ml of 2 N
NaOH in boiling water. Then 2 N CH3COOH (5 ml) and
distilled water (25 ml) were added to neutralize the solution. The
solution was adjusted to the appropriate pH with 0.1 N
CH3COOH at 80°C and made up to 100 ml with distilled water as the substrate.
Two assay methods for determining CGTase activity were used. The
starch-degrading activity assay was based on the decrease
in absorbance
(blue value) of the iodine-amylose complex as a
result of the starch
degradation. The enzyme solution (6 µl) was
incubated at 90°C for
10 min with 60 µl of substrate (1% starch),
and the reaction was
terminated by cooling the solution in ice-water.
The reaction mixture
(10 µl) was removed and mixed with 100 µl
of 0.1 N HCl. This
solution (100 µl) was mixed with 2 ml of an
iodine solution (0.005%
I
2, 0.05% KI), and the absorbance of the
mixture at 660 nm
was measured. One unit of starch-degrading activity
was defined as the
amount of enzyme which can decrease the absorbance
at 660 nm of the
amylose-iodine complex by 1% per
min.
The CD synthesis activity was examined by measurement of liberated
-CD by the methyl orange method (
30) with slight
modifications.
The reaction mixture (50 µl) was mixed with 6 N HCl
(7.5 µl) and
52.5 µM methyl orange solution (100 µl), and then
incubated at
25°C for 30 min. The absorbance of the mixture at 505 nm
was measured.
One unit of CD synthesis activity was defined as the
amount of
enzyme which released 1 µmol of
-CD per
min.
The amount of liberated reducing sugar was determined by a
dinitrosalicyclic acid method (
3) with slight modifications
(on a 1/10 scale). The protein concentration was measured with
a
NanoOrange protein quantitation kit (Molecular Probes, Eugene,
Oreg.).
Purification of CGTase.
Culture broth (70 liters) of strain
B1001 was filtered through Toyo no. 2 filter paper (Toyo Roshi Co.,
Tokyo, Japan) to remove elemental sulfur and then centrifuged
(8,000 × g for 10 min) to obtain the supernatant. The
supernatant was concentrated to 500 ml by an ultrafiltration system
(AIP-1010; Asahikasei Co., Tokyo, Japan), brought to 80% ammonium
sulfate saturation, and kept at 4°C overnight. The precipitate was
collected by centrifugation (27,000 × g for 30 min),
dissolved in 25 mM sodium acetate buffer (pH 5.0), and dialyzed
overnight against the same buffer. The dialysate was applied to a
Resource Q column (Pharmacia) equipped with a fast protein liquid
chromatography system (Pharmacia), equilibrated with 25 mM sodium
acetate buffer (pH 5.0), and eluted with a linear gradient of NaCl (0 to 1.0 M) at a flow rate of 1 ml/min. The eluted fractions containing
CGTase activity were pooled and then brought to 80% ammonium sulfate
saturation. The precipitate was collected by centrifugation
(27,000 × g for 30 min), dissolved in 25 mM Tris-HCl
buffer (pH 7.0) containing 12% ammonium sulfate saturation, and
applied to a phenyl-Superose HR 5/5 column (Pharmacia) previously
equilibrated with the same buffer. The column was washed with the same
buffer and eluted with 25 mM Tris-HCl buffer (pH 7.0) at a flow rate of
0.3 ml/min. The eluted fractions containing CGTase activity were pooled
and applied to an -CD-(epoxy)-Sepharose 6B affinity column
(Pharmacia) previously equilibrated with 25 mM Tris-HCl buffer (pH 7.0)
containing 100 mM NaCl. The column was washed with 25 mM Tris-HCl
buffer (pH 7.0) containing 1.0 M NaCl, and the CGTase was eluted at a flow rate of 0.2 ml/min with 25 mM Tris-HCl buffer (pH 7.0)
supplemented with 1% -CD. The eluted fractions containing CGTase
activity were pooled and dialyzed against distilled water for 3 days.
All enzyme purification procedures were performed at 4°C.
Gel electrophoresis.
Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and native PAGE were carried out with
the Phastsystem (Pharmacia) by the procedures recommended in the
Phastsystem manual. Protein bands were detected by staining with 0.1%
Coomassie brilliant blue R-250. For amylase activity staining, after
gel electrophoresis the gel was incubated at 85°C for 1 h in 100 mM sodium acetate buffer (pH 5.0) containing 5% soluble starch. After
being washed with distilled water, the gel was subjected to staining
with 50% methanol solution containing 1% iodine and 10% potassium iodide.
N-terminal analysis.
The NH2-terminal amino acid
sequence was determined by using the automated Edman degradation system
of ABI protein sequencer 473A (Applied Biosystems Japan, Tokyo, Japan).
The sequences of other CGTases were obtained from the NBRF database
(accession numbers are as follows:
Bacillus macerans,
S31281;
B. licheniformis,
S15920;
Bacillus sp.,
S26399;
B. stearothermophilus,
S26588) and the EMBL database
(accession
numbers are as follows:
Thermoanaerobacterium
thermosulfurigenes,
M57580;
Thermoanaerobacter sp.,
Z35484).
Determination of the amounts of -, -, and -CD.
Reaction mixtures (100 µl) containing 2.5% (wt/vol) soluble starch
and 50 mM sodium acetate buffer (pH 5.0) were incubated with the
purified enzyme (8 mU as CD synthesis activity) at 90°C. Samples were
taken at convenient time intervals, and the reaction was terminated by
cooling the mixtures on ice. The reaction mixture (50 µl) was removed
and mixed with 1 µl (2 U) of glucoamylase (Toyobo Co., Osaka, Japan),
9 µl of 2 M sodium acetate buffer (pH 5.0), and 40 µl of distilled
water. The mixture was incubated at 40°C for 60 min, mixed with 150 µl of acetonitrile, and then filtered through a membrane filter (pore
size, 0.45 µm). The filtered sample (10 µl) was analyzed by
high-performance liquid chromatography (HPLC) with a TSKgel Amide-80
column (TOSOH, Tokyo, Japan) and eluted with acetonitrile-water (60:40
[vol/vol]) at 1 ml/min. The flow cell was set at 30°C, and products
were detected with a refractive index detector.
The amount of total sugar in the reaction mixtures was assayed by the
phenol-sulfuric acid method (
9) with glucose as a
standard.
Nucleotide sequence accession number.
The nucleotide
sequence data of the gene for 16S rRNA reported in this paper has been
submitted to the DDBJ, EMBL, and GenBank DNA databases under accession
no. AB016298.
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RESULTS |
Isolation of the CGTase-producing hyperthermophile.
To isolate
the CGTase-producing hyperthermophile, various hyperthermophilic
strains were isolated by enrichment of the water and sediment samples
of hot spring environments and serial dilutions of positive
enrichments. These strains were incubated in medium containing 0.2%
soluble starch, and the extracellular supernatants were prepared. After
incubation of starch with the supernatant, the degradation of starch
(the decrease in the blue value) and the liberated reducing power were
measured. When the starch is incubated with CGTase, which produces
mainly CD (no reducing sugar), the blue value decreases rapidly and the
reducing power increases slightly. One strain, named B1001, for which
the blue value decreased but the reducing power increased only
slightly, was selected. The -, -, and -CD-specific synthesis
activity measurement (see Materials and Methods) (18, 19)
and the HPLC of reaction products suggested the formation of CDs (data
not shown).
Characterization of isolate B1001.
Cells of B1001 were
irregular cocci of 0.5 to 1.0 µm in diameter (Fig.
1) and grew between 60 and 95°C; the
optimum growth temperature was 85°C (Fig.
2A). The pH range for growth was 5.0 to
9.0, and the optimum pH was around 7.0 (Fig. 2B). Growth was observed
in medium containing 1 to 6% NaCl, with the optimum at 2% (Fig. 2C).
The presence of elemental sulfur stimulated growth, which was
accompanied by H2S production. The generation time for strain B1001 was 44 min under the optimal cultivation conditions, and
the cell density reached a maximum of 3 × 108
cells/ml after 16 h of incubation (Fig.
3). Cells were also grown in the absence
of elemental sulfur; however, growth was very slow. The cell density in
medium without elemental sulfur reached a maximum of 5 × 107 cells/ml after 24 h of incubation (Fig. 3). Cell
growth was not observed in the absence of Na2S (oxidized
conditions).
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FIG. 2.
Influence of temperature (A), pH (B), and NaCl
concentration (C) on the growth of Thermococcus sp. strain
B1001. The generation times were calculated from the slopes of the
growth curves. All values are the average of two separate
experiments.
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FIG. 3.
Growth of strain B1001. Cultivations were carried out
under optimal conditions ( ) and in medium lacking elemental sulfur
( ).
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Strain B1001 was capable of growing on proteinaceous complex substrates
such as yeast extract, peptone, and tryptone, but
it did not grow on
Casamino Acids. No growth was observed on glucose,
maltose, fructose,
sucrose, lactose, soluble starch,
-CD, ethanol,
or methanol as the
sole carbon source. Growth of strain B1001
was not influenced by
benzylpenicillin, vancomycin, or streptomycin.
It was partly inhibited
by chloramphenicol and completely inhibited
by
rifampin.
The genomic DNA of strain B1001 had a G+C content of 43.0 mol% as
calculated by direct analysis of the nucleotides. The 16S
rRNA gene
sequence (1,416 bases) of strain B1001 was determined.
The sequence of
B1001 was aligned and compared to the 16S rRNA
gene sequences of
various species of the domain
Archaea. It showed
high
sequence similarity to the 16S rRNA from
Thermococcus
litoralis (97.7%),
T. fumicolans (97.0%), and
T. celer (96.6%). For a more
precise classification, the
phylogenetic tree was inferred by
the NJ method (Fig.
4). The result indicates that strain
B1001
belongs to the genus
Thermococcus and is most closely
related
to
Thermococcus litoralis.
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FIG. 4.
Phylogenetic tree based on 16S rRNA gene sequences. The
tree was constructed by the NJ method. Numbers indicate bootstrap
values of 1,000 times trial. The scale bar represents 10 nucleotide
substitutions per 100 nucleotides. The accession numbers of 16S rRNA
sequences used for the unrooted tree are as follows:
Sulfurishaera ohwakuensis, D85507; Sulfolobus
solfataricus, X03235; Sulfolobus acidocaldarius,
D14053; Halogeometricum borinquense, AF002984;
Haloferax denitrificans, D14128; Halobacterium
sodomense, D13379; Methanobrevibacter smithii, U55234;
Methanobacterium formicicum, AF028689; Methanococcus
maripaludis, AF005049; Methanococcus deltae, U38485;
T. profundus, Z75233; T. stetteri, Z75240;
T. hydrothermalis, Z70244; T. celer, M21529;
T. zilligii, U76534; T. fumicolans, Z70250;
T. litoralis, Z70252; Pyrococcus horikoshii,
D45214; Pyrococcus abyssi, Z70246; T. chitonophagus, X99570; and Pyrococcus furiosus,
U20163.
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Purification of the CGTase.
Strain B1001 culture broth (70 liters) was concentrated to a final volume of 500 ml. The purification
steps are summarized in Table 1. By
applying anion-exchange chromatography, hydrophobic interaction
chromatography, and affinity chromatography, CGTase was purified
1,750-fold with a yield of 10%. The purified enzyme exhibited a
specific CD synthesis activity of 1,400 U/mg of protein.
The purified enzyme was analyzed by native PAGE and SDS-PAGE. After
native PAGE, the mobility of the active band as determined
by
starch-degrading activity staining coincided with that of the
single
protein band stained with Coomassie brilliant blue (Fig.
5A). It indicated that the purification
of B1001 CGTase had been
accomplished. Furthermore, after heating at
100°C for 40 min,
the same result was observed, suggesting that the
enzyme was highly
stable to heating. However, unusual mobility was
observed when
the purified CGTase was subjected to SDS-PAGE (Fig.
5B).
When
the enzyme was incubated in denaturing buffer (2.3% SDS, 5%
2-mercaptoethanol)
at 60°C for 5 min, a single protein band with a
molecular mass
of 72 kDa was observed, and this protein band was
visualized by
starch-degrading activity staining. The 72-kDa band was
considered
to be detected due to incomplete denaturation. Incubation at
100
or 110°C for 5 min caused the appearance of another protein band,
with a molecular mass of 83 kDa, which was not detected by activity
staining. Furthermore, the 72-kDa band visible with activity staining
became weaker with increasing temperature during heating treatment.
After incubation at 120°C for 5 min, the 72-kDa band completely
disappeared and only the 83-kDa band was observed. The CGTase
was
considered to be switched from the active form of 72 kDa to
the
unfolded, denatured form of 83 kDa in SDS-PAGE. Hence, the
size of
B1001 CGTase was estimated to be approximately 83 kDa
by SDS-PAGE.
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FIG. 5.
Behavior of CGTase in native PAGE (A) and SDS-PAGE (B).
(A) Enzyme samples were visualized by Coomassie brilliant blue staining
(lanes 1 and 2) and activity staining (lanes 3 and 4). The purified
samples (lanes 1 and 3) were treated in 0.1 M sodium acetate buffer (pH
5.0) at 100°C for 40 min (lanes 2 and 4). (B) The purified samples
were visualized by Coomassie brilliant blue staining (lanes 1 to 4) and
activity staining (lanes 5 to 8). The samples were treated at 60°C
(lanes 4 and 8), 100°C (lanes 1 and 5), 110°C (lanes 2 and 6), or
120°C (lanes 3 and 7) for 5 min in denaturing buffer containing 2.3%
SDS and 5% 2-mercaptoethanol. Protein molecular mass standards are
shown in lane M and are listed on the left.
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Effects of temperature and pH on activity, and thermal stability of
the enzyme.
The optimum starch-degrading activity of CGTase was
observed at 110°C, and the optimum CD synthesis activity was noted at 90 to 100°C. Almost 70% starch-degrading activity and 50% (CD synthesis) of the maximum enzyme activity were detected at 120°C (Fig. 6A). The pH optimum of CGTase was
5.0 to 5.5 at 110°C for both the starch-degrading activity and the CD
synthesis activity. At 90°C, the optimum pH curve changed to a broad
curve in the range of pH 4.5 to 6.0 (Fig. 6B). The stability of CGTase
at various temperatures (at pH 5.0) was examined. Significant loss of
CGTase activity was not observed after heat treatment at 90°C for 40 min. About 5% of the initial activity was lost after boiling (100°C) for 40 min. Further treatment at 110°C for 40 min induced activity loss, resulting in 50% of the maximal activity. These results indicate
that CGTase from strain B1001 is extremely thermostable in comparison
with other known bacterial CGTases.
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FIG. 6.
Effects of temperature (A) and pH (B) on enzyme
activity. (A) Starch-degrading activity () and CD synthesis activity
() were measured at pH 5.0 and various temperatures. (B)
Starch-degrading activity (,  ) and CD synthesis activity (,
 ) were measured at various pHs and 90°C (open symbols) or 110°C
(solid symbols). The values are shown as relative activity. The
activity under the optimized condition is indicated as 100%. Each
experiment was performed in triplicate.
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CD production.
Production of CDs was analyzed by HPLC (Fig.
7). -, -, and -CDs produced from
starch by purified enzyme are clearly indicated. The profiles of CD
production with time were analyzed (Fig.
8). B1001 CGTase produced -CD as the
major compound. The yield of 12% conversion of soluble starch into CDs
could be obtained after 1 h; the production ratio at that point
was 92% -CD, 4% -CD, and 4% -CD. After 24 h, the
conversion yield was 30% and the production profile was still biased
toward -CD formation (79% -CD, 14% -CD, and 7% -CD).
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FIG. 7.
HPLC analysis of the formation of CDs. The reaction
mixture containing 2.5% soluble starch was incubated with 8 mU of the
enzyme (as CD synthesis activity). Samples were taken at various times
during incubation (90°C) after addition of the purified enzyme. The
amounts of CDs were determined by HPLC as described in Materials and
Methods. The CD standard is a mixture of -, -, and -CDs.
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FIG. 8.
Time course of CD production by purified CGTase. CDs
produced at the indicated reaction times were separated by HPLC as
described in the text. All values are the average of two separate
experiments. Symbols: , -CD;  , -CD; , -CD; , total
of -, -, and -CDs.
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Under industrial CD production conditions, CGTase is incubated with
high concentrations of starch. Thus, B1001 CGTase was
incubated at
96°C for 24 h with 30% (wt/vol) cornstarch (with
no pH
adjustment; the pH was 4.5). The enzyme converted 34.4%
of the starch
into CDs, and the production ratios were 69%
-CD,
20%
-CD, and
11%
-CD (data not
shown).
N-terminal sequence.
The N-terminal amino acid sequence of the
purified B1001 CGTase was determined. The sequence is shown in
comparison with the N-terminal sequences of CGTase from various sources
in Fig. 9. Interestingly, the sequence
(positions 13 to 20) of B1001 CGTase shows significant similarity to
those of known CGTases, suggesting that archaeal CGTase has an
evolutionary relationship to bacterial CGTase.
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|
FIG. 9.
Comparison of the N-terminal sequences between B1001
CGTase and other CGTases. The highly homologous regions are shaded.
Abbreviations: B. ma, Bacillus macerans; B. li, B. licheniformis; B. sp, Bacillus sp.; B. st, B. stearothermophilus; T. th, Thermoanaerobacterium
thermosulfurigenes; T. sp, Thermoanaerobacter sp.;
B1001, Thermococcus sp. strain B1001.
|
|
| |
DISCUSSION |
On the basis of its G+C content and 16S rRNA sequence, strain
B1001 clearly belongs to the genus Thermococcus. This
classification is consistent with the morphological and physiological
characteristics of strain B1001, which are similar to the
characteristics of this genus. The genus Thermococcus
includes nine described species: T. celer (50),
T. litoralis (37), T. stetteri
(32), T. profundus (23), T. peptonophilus (14), T. chitonophagus
(16), T. fumicolans (13), T. alcaliphilus (20), and T. zilligii
(40). All species and strain B1001 are cocci or irregular
cocci, 0.5 to 2 µm in diameter, and are anaerobes that grow
preferentially on peptides and yeast extract. Elemental sulfur has
always been found to considerably stimulate growth, with the
concomitant formation of H2S. Typical optimal temperatures
are 80 to 88°C (75°C for T. stetteri and T. chitonophagus), and the G+C contents are approximately 40 to 50 mol%, except for those of T. celer (57 mol%) and T. litoralis (38 mol%). Thermococcus strains show a
unique bias for antibiotic sensitivity. Strain B1001 resembles T. litoralis, T. stetteri, T. profundus,
T. peptonophilus, and T. fumicolans in its
susceptibility to 100 µg of rifampin per ml (T. celer and
T. zilligii have no sensitivity).
The effects of pH on the starch-degrading activity and the CD synthesis
activity were virtually identical, but the effects of temperature on
these two activities were not. To explain this different behavior,
temperature, the reaction mechanism of CGTase has to be taken into
account. Since the catalytic residues of CGTase are proposed to be
equivalent to those of -amylases, CGTase will cleave the
-1,4-glucosidic bond of amylose in the same way as -amylases do.
The transglycosylation reaction of CGTase is operated by a
"ping-pong" mechanism (35). In this mechanism, the
transglycosylation occurs after the reducing side of the cleaved amylose is released from the enzyme. Then the enzyme transfers the
newly formed reducing end of the substrate either to the nonreducing end of a separate linear acceptor molecule or glucose (the
disproportionation reaction) or to its own nonreducing end (the
cyclization reaction or CD synthesis reaction). The hydrolysis reaction
(the starch-degrading reaction) will occur when this intermediate is
attacked nucleophilically by a water molecule. For preferential CD
synthesis, the efficient formation of the helical structure of amylose
in the active-site cleft of enzyme is required (10, 36). In
a crystal structure, amylose can occur as a single helix with six to
eight glucose molecules in one helical turn (27). The most
widely accepted hypothesis describes amylose in solution having an
interrupted coil-like structure composed of helical and nonhelical
segments (44). Therefore, the formation of CD by CGTase can
be explained as a consequence of a preferential helical structure of
the amylose in solution. However, the high temperature will destabilize
the helical structure of the amylose, resulting in a shift to random structure. Accordingly, it is considered that the reaction at high
temperature by B1001 CGTase resulted in a shift toward the starch-degrading reaction.
There are several processing difficulties in the present CD production
system. The first step in the industrial production of CDs from starch
is liquefaction. This is carried out by a bacterial thermostable
-amylase in a jetcooking process at 105 to 110°C and pH 6.0 to
6.5. However, further -amylase treatment reduces the CD yield,
because the maltodextrins, oligosaccharides, and glucose formed during
this process act as acceptors, so that the coupling reaction (the
degradation of CD) is accelerated. Another problem relating to the pH
of liquefaction is the need to raise the pH of the 30 to 35% (wt/vol)
starch slurry (the pH in general is 4.5 to 5.0) to pH 6.0 to 6.5. This
pH adjustment requires the costly addition of acid-neutralizing
chemicals. Moreover, in the next CD formation step, the pH must be
adjusted to the optimum for CGTase. To overcome these two
complications, thermostable CGTase of Thermoanaerobacter sp.
as the liquefying enzyme can be applied applicable (38). In
addition, B1001 CGTase is the most thermoactive and thermostable enzyme
in reported CGTases and can react at acidic pH ranges (pH 4 to 5).
Therefore, B1001 CGTase is more applicable than that of
Thermoanaerobacter sp. for industrial application as a
liquefying and CD-forming enzyme without pH adjustment prior to
reaction by jetcooking.
Depending on the most abundant type of CD produced, CGTase is sometimes
classified into three types (-, -, and -CGTase). The B1001
enzyme is the -CGTase, which was reported from B. macerans, B. stearothermophilus, and K. pneumoniae (22). In general, -CD formed mainly at
early stage of the reaction is decomposed and -, -, and -CD
are produced from decomposition products. Interestingly, B1001 CGTase
produced predominantly -CD in the later reaction as well as in the
initial reaction. B1001-derived CGTase, which produced mainly -CD
with a small amount of -CD and -CD from starch, can provide
advantages in -CD manufacturing.
We are currently working on isolation of the CGTase gene by using an
oligonucleotide mixture as a probe that corresponds to the N-terminal
sequence of the CGTase.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Research and
Development Center, Nagase Co., Ltd., 2-2-3 Murotani, Nishi-ku, Kobe
651-2241, Japan. Phone: (81)-78-992-3164. Fax: (81)-78-992-1050.
E-mail: yoshihisa.tachibana{at}nagase.co.jp.
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Abstract
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