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Journal of Bacteriology, September 2001, p. 5050-5057, Vol. 183, No. 17
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.17.5050-5057.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Extracellular Synthesis, Specific Recognition, and
Intracellular Degradation of Cyclomaltodextrins by the
Hyperthermophilic Archaeon Thermococcus sp. Strain
B1001
Yoshiteru
Hashimoto,1,
Tomoko
Yamamoto,2
Shinsuke
Fujiwara,2
Masahiro
Takagi,2,
and
Tadayuki
Imanaka3,*
CREST1 and
Department of Biotechnology, Graduate School of Engineering,
Osaka University, 2-1 Yamadaoka, Osaka
565-0871,2 and Department of Synthetic
Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Sakyo-ku, Kyoto 606-8501,3
Japan
Received 11 December 2000/Accepted 4 June 2001
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ABSTRACT |
A unique extracellular and thermostable cyclomaltodextrin
glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly
(>85%)
-cyclomaltodextrin (
-CD) from starch (Y. Tachibana, et
al., Appl. Environ. Microbiol. 65:1991-1997, 1999). Nucleotide
sequencing of the CGTase gene (cgtA) and its flanking
region was performed, and a cluster of five genes was found, including
a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the
degradation of CDs (cgtB), the gene encoding CGTase
(cgtA), a gene homolog for a CD-binding protein (CBP)
(cgtC), and a putative CBP-dependent ABC transporter
involved in uptake of CDs (cgtDE). The CDase was expressed
in Escherichia coli and purified. The optimum pH and
temperature for CD hydrolysis were 5.5 and 95°C, respectively. The
molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed
-CD most efficiently among other CDs. Maltose
and pullulan were not utilized as substrates. Linear maltodextrins with
a small glucose unit were very slowly hydrolyzed, and starch was
hydrolyzed more slowly. Analysis by thin-layer chromatography revealed
that glucose and maltose were produced as end products. The purified
recombinant CBP bound to maltose as well as to
-CD. However, the CBP
exhibited higher thermostability in the presence of
-CD. These
results suggested that strain B1001 possesses a unique metabolic
pathway that includes extracellular synthesis, transmembrane uptake,
and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.
 |
INTRODUCTION |
Cyclomaltodextrins (CDs) are cyclic
oligosaccharides consisting of
-1,4-linked 6-, 7-, or
8-glucopyranose units, usually referred to as
-,
-, or
-CDs,
respectively. CDs possess a unique torus shape and the polar hydroxyl
groups are oriented toward the outside, keeping the interior cavity
relatively hydrophobic. Therefore, CDs are soluble in water and the
hydrophobic environment of the cavity enables them to form inclusion
complexes with many organic and inorganic molecules, thereby changing
the physical and chemical properties of the included compounds. This is
the basis of broad applications in the food, cosmetic, and
pharmaceutical industries (2, 28).
CDs are formed enzymatically from starch by the action of
cyclomaltodextrin glucanotransferase (CGTase) [EC 2.4.1.19;
1,4-
-D-glucan 4-
-D-(1,4-glucano)-transferase (cyclizing)]
(39). Cyclomaltodextrinase (CDase) [EC 3.2.1.54;
cyclomaltodextrin dextrin-hydrolase (decyclizing)] is a unique enzyme
which can hydrolyze CD and release the substance from CD inclusion
complexes. After the first report of the CDase from Bacillus
maceranse (8), CDases from various microorganisms such as Bacillus coagulans (23), Bacillus
sphaericus E-244 (34, 35), Clostridium
thermohydrosulfuricum strain 39E (recently reclassified as
Thermoanaerobacter ethanolicus strain 39E) (38, 40), alkalophilic Bacillus sp. (50),
Bacillus subtilis strain H-17 (24, 25),
Flavobacterium sp. (3), B. sphaericus strain ATCC 7055 (15), Klebsiella
oxytoca strain M5a1 (11, 12), and alkalophilic
Bacillus sp. strain I-5 (22) were studied.
In K. oxytoca M5a1, the novel starch degradation pathway via
CD, including the extracellular conversion of starch into CDs by CGTase
and uptake of the CDs by a specific system followed by intracellular
linearization by a CDase, was proposed. In addition, the genes
responsible for starch metabolism were clustered on the chromosome.
Until now, no other organisms possessing both CGTase and CDase for the
synthesis and degradation of CDs have been found.
Recently, we have isolated a hyperthermophile,
Thermococcus sp. strain B1001, which produces a unique
CGTase that can catalyze predominantly the formation of
-CD
(>85%), with small amounts of
- and
-CDs, from starch
(45). The cgtA gene encoding CGTase has been
cloned and sequenced (48). In the present paper, we report
the sequence analysis of the adjacent region of the CGTase gene
containing the genes involved in transport and degradation of CDs. We
also describe the purification and characterization of an extremely
thermostable CDase and a CD-binding protein (CBP) from
Thermococcus sp. strain B1001. Furthermore, a unique
metabolic pathway of starch involving synthesis, transport, and
degradation of CDs is proposed.
 |
MATERIALS AND METHODS |
Microorganisms, media, and growth conditions.
Thermococcus sp. strain B1001 was cultured as previously
described (45). Escherichia coli strain JM109
(49) was used as a host for transformation and plasmid
preparation and grown at 37°C in Luria-Bertani medium
(30). E. coli BL21(DE3) and E. coli
BL21-CodonPlus(DE3)-RIL (Novagen, Madison, Wis.) were used for
heterologous expression of the target protein and were grown at 37°C
in NZCYM medium (21, 30). Ampicillin (100 µg/ml) was added to the medium when necessary to select plasmid carrier.
Chemicals.
Soluble starch was purchased from Nacalai Tesque
Co., Inc. (Kyoto, Japan). Maltose, maltotriose, maltotetraose,
maltopentaose, and maltohexaose were obtained from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan), and maltoheptaose was obtained from
Sigma (St. Louis, Mo.). CDs were obtained from Nihon Shokuhin Kako Co., Inc. (Tokyo, Japan), and pullulan was obtained from Hayashibara Co.,
Inc. (Okayama, Japan). Other biochemicals were standard commercial preparations.
DNA manipulation.
Restriction endonucleases, DNA polymerase,
and T4 DNA ligase were purchased from Toyobo Co., Ltd. (Osaka, Japan).
Southern blot analysis was performed according to established
procedures (48). A Thermo Sequenase fluorescent-labeled
primer cycle sequencing kit with 7-deaza dGTP was purchased from
Amersham Pharmacia Biotech UK Ltd., (Little Chalfont, Buckinghamshire,
England). Plasmids for DNA sequencing were extracted and purified by
using a Wizard Plus Minipreps DNA Purification System (Promega,
Madison, Wis.). Unless otherwise stated, DNA manipulations were
performed essentially as described by Maniatis et al.
(30).
Construction of plasmids pET-cgtB1 and
pET-cgtB2.
The cgtB gene was amplified by
PCR with three kinds of primers: primer 1, 5'-CATATGTATAAAATTTTCGGCTTTAAAGACAATGACTACC-3'; primer 2, 5'-CATATGCGTGAAAAGGGCGATCGTTGGTATATCAAGGTAGAGC-3';
and primer 3, 5'-CAGGAAACAGCTATGAC-3' (M13 reverse
primer). Primers 1 and 2 possess an additional NdeI site at
the 5'-terminal regions, shown in italics in the sequences. In order to
achieve efficient expression (20, 29), several rare codons
which are not efficiently utilized in E. coli for N-terminal
amino acids were replaced with codons which are frequently used in
E. coli. GGA for Gly6 and Gly50, AGG
for Arg47, AGA for Arg52, and ATA for
Ile55 were replaced with GGC, CGT, CGT, and ATC,
respectively. First, to construct pTY-331, plasmid pTY-33
(48) was digested with SacI (position 1) and
BamHI (position 2521), and then the 2.5-kbp fragment
harboring the entire coding region of CDase was inserted into the
respective site of pUC19. As for pET-cgtB1 construction, PCR
amplification was carried out with primers 1 and 3 using pTY-331 as a
template. The amplified DNA was subcloned into vector pBluescript II
SK(+) (Stratagene, La Jolla, Calif.) and checked by DNA sequencing. The
insert DNA was digested with NdeI and BamHI and
then inserted into the respective site of pET-25b(+) (Novagen). The
resultant plasmid was designated pET-cgtB1. As for
pET-cgtB2 construction, primer 2 was used instead of primer
1. Other procedures were carried out as for pET-cgtB1 construction.
Enzyme assay.
CDase activity was measured in the reaction
mixture that contained 1%
-CD in 50 mM Britton-Robinson buffer (pH
5.5) and appropriately diluted enzyme. After incubation at 90°C for 5 min, the reducing sugar was measured by the dinitrosalicylic acid
method (4). One unit of activity was defined as the amount
of enzyme which released 1 µmol of reducing sugar equivalent to
glucose per min. Protein concentration was measured by the BCA Protein
Assay Kit (Pierce, Rockford, Ill.) with bovine serum albumin as the standard.
Purification of recombinant CDase.
CDase production by
E. coli BL21(DE3) carrying pET-cgtB1 was induced
by 1 mM isopropyl-
-D-thiogalactopyranoside (IPTG) at mid-exponential growth phase and incubation continued for 4 h at
37°C. The cells (from 5 liters of culture) were centrifuged and the
pellet was washed with buffer A (50 mM Tris-HCl buffer [pH 8.0]).
Cells were disrupted by sonication and supernatant fraction was
recovered by centrifugation at 48,384 × g for 30 min
at 4°C. The supernatant was heat-treated at 85°C for 15 min and
centrifuged again at 48,384 × g for 30 min at 4°C.
The obtained supernatant was brought to 60% ammonium sulfate
saturation and kept at 4°C overnight. The precipitate was collected
by centrifugation at 48,384 × g for 30 min, dissolved
in 40 ml of buffer A, and dialyzed overnight against the same buffer.
The dialysate was applied to a weak anion-exchange column (DEAE
Sepharose Fast Flow; Pharmacia, Uppsala, Sweden) previously
equilibrated with buffer A, and CDase was eluted by a linear gradient
of NaCl using a fast-performance liquid chromatography (FPLC) system
(Pharmacia). The active fraction was loaded onto a high-resolution
anion-exchange column (Mono Q HR 5/5; Pharmacia) equilibrated with
buffer A and eluted with a linear gradient of NaCl.
TLC.
A mixture of oligosaccharides was analyzed by silica
gel thin-layer chromatography (TLC). Aliquots (3 µl) of the sample
were developed on a silica gel plate (Kieselgel 60; Merck Co., Berlin, Germany) with isopropyl alcohol-acetone-water (2:2:1 [vol/vol/vol]), and the oligosaccharides were detected by spraying the plate with aniline diphenylamine reagent (4 ml of aniline, 4 g of
diphenylamine, 200 ml of acetone, and 30 ml of 85% phosphoric acid)
and baking it.
Construction of plasmid pET-CBP.
In order to construct
pTY-401, plasmid pTY-40 was digested with PstI (position
4158) and EcoRI (position 6425) and then the 2.3-kbp
fragment was inserted into respective site of pUC18. As for pET-CBP
construction, the cgtC gene was amplified by PCR with primer
3 and primer 4 (5'-CATATGAAGAAGGCACTGTTTGCTTTATTGTTG-3') using
pTY-401 as a template. The amplified DNA was subcloned into vector
pBluescript II SK(+) and checked by DNA sequencing. The insert DNA was
digested with EcoRI completely and then with NdeI partially. The 1.4-kbp fragment harboring the entire coding region of
CBP was inserted into the respective site of pET-25b(+). The resultant
plasmid was designated pET-CBP.
Purification of recombinant CBP.
CBP synthesis by E. coli BL21-CodonPlus(DE3)-RIL carrying pET-CBP was induced by 1 mM
IPTG at mid-exponential phase and incubation continued for 6 h at
25°C. The cells (from 1 liter of culture) were centrifuged and the
pellet was washed with buffer A. Cells were disrupted by sonication and
the supernatant fraction was recovered by centrifugation at
48,384 × g for 30 min at 4°C. The supernatant was
heat-treated at 80°C for 10 min and centrifuged again at
48,384 × g for 30 min at 4°C. The obtained
supernatant was applied to a strong anion-exchange column (HiTrap Q;
Pharmacia) previously equilibrated with buffer A, and CBP was eluted by
the linear gradient of NaCl using an FPLC system.
Binding assay of CBP.
Purified CBP was applied to an
-CD-(epoxy)-Sepharose 6B affinity column (Pharmacia) previously
equilibrated with buffer A. The column was washed with buffer A
containing 1.0 M NaCl, and bound protein was eluted with buffer A
supplemented with 1%
-CD. As another binding assay method, purified
CBP was loaded onto an amylose resin (New England Biolabs, Inc.,
Beverly, Mass.) and then the column was washed with buffer A containing
1.0 M NaCl. Bound protein was eluted with buffer A containing 1%
maltose. The washed and eluted fractions were applied to sodium dodecyl sulfate (SDS)-polyacrylamide gels and protein bands were detected by
staining with Coomassie brilliant blue.
Nucleotide sequence accession number.
The nucleotide
sequence data reported in this paper appear in the GenBank database
with the accession number AB034969.
 |
RESULTS |
Nucleotide sequence of the CDase gene and identification of the
gene product.
The cgtA gene encoding CGTase of
Thermococcus sp. strain B1001 was previously cloned and
sequenced (48). Sequence analysis of the upstream region
of cgtA revealed that another open reading frame (ORF)
clustered with cgtA, as shown in Fig.
1. Computer inspection revealed that the
ORF contained two putative ATG start codons at base positions 248 and
383 and a TAG termination codon at position 2228. The deduced amino
acid sequence of the ORF contains four conserved regions identified
with amylolytic enzymes as belonging to the
-amylase family
(27, 46) (Fig. 2). A search
with the BLAST program (43) revealed that the protein
encoded by the ORF is homologous to neopullulanases (19,
26), CDases (22, 35), maltogenic amylase
(6), and other amylolytic enzymes. It was suggested that
the ORF encoded a kind of amylolytic enzyme. In order to specify the
type of amylolytic characteristics and to identify an essential
methionine as a translation start site, two types of plasmids,
pET-cgtB1 and pET-cgtB2, for the ORF starting from each of two methionines, were constructed as explained above and the respective recombinant plasmids were used to transform E. coli BL21(DE3). The crude recombinant protein prepared from E. coli carrying the ORF translated from the first ATG
hydrolyzed
-CD as a substrate, but the protein could not hydrolyze
soluble starch and pullulan. By contrast, another recombinant protein translated from the second ATG did not exhibit hydrolytic activity. Furthermore, the first ATG is preceded by a putative ribosome-binding site complementary to the 3' end of 16S rRNA from strain B1001 (45). Therefore, the larger ORF starting at position 248 (1,980 nucleotides coding for a protein of 660 amino acids, with a
calculated molecular mass of 78,839 Da) was considered to be the gene
encoding the amylolytic enzyme. The most putative type of the enzyme is CDase, because it hydrolyzed CDs efficiently but apparently did not
hydrolyze soluble starch and pullulan in a 5-min reaction.

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FIG. 1.
Structure of the gene cluster containing genes for
synthesis, transport, and degradation of CDs in Thermococcus
sp. strain B1001. Arrows show the localization of each gene and the
orientation of the coding sequences. pTY-33, pTY-40, pTY-41, and pTY-48
indicate the regions cloned by the respective plasmids.
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FIG. 2.
Comparison of the deduced amino acid sequences for the
CDase proteins. The four regions conserved among the -amylase family
enzymes are boxed. Asterisks represent identical amino acid residues in
all five polypeptides, and bullets indicate identical amino acid
residues among organisms belonging to the domain Bacteria.
Putative catalytic amino acid residues are indicated by circles.
Polypeptides: B1001, Thermococcus sp. strain B1001
(AB034969); 39E, T. ethanolicus 39E (M88602); I-5,
Bacillus sp. strain I-5 (U49646); E-244, B. sphaericus E-244 (X62576); and M5a1, K. oxytoca M5a1
(X86014).
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Comparison of amino acid sequences.
The deduced amino acid
sequence of the CDase from B1001 was compared with those of other
CDases reported previously (Fig. 2), and a distinct region homologous
with CDases from various bacteria was found (12, 22, 35,
38). In addition to the four highly conserved regions,
Asp416, Glu442, and Asp507 residues
as catalytic sites corresponding to Asp206,
Glu230, and Asp297 of Taka-amylase A are very
well conserved. Based on the results from site-directed mutagenesis,
these three residues have been recognized as the catalytic sites in
Taka-amylase A (33) and CDase (37).
Therefore, it was suggested that the Asp416,
Glu442, and Asp507 residues of the CDase from
B1001 are directly involved in the catalytic site. Moreover, the CDase
from B1001 possesses a unique N-terminal extension containing about 60 amino acid residues. Deletion of this N-terminal extended region caused
a lack of CDase activity, suggesting that this extended region is
important for the enzymatic activity of the CDase, although the
mechanism is not clear.
Purification and characterization of the recombinant CDase.
CDase was purified from E. coli BL21(DE3) cells carrying
plasmid pET-cgtB1 encoding the ORF from the first ATG codon.
The recombinant CDase was purified by the following steps: preparation of cell lysate, heat treatment (85°C, 15 min), and ammonium sulfate fractionation (60% saturation), followed by two ion-exchange column chromatographies with DEAE Sepharose Fast Flow and Mono Q HR 5/5. The
purity of the CDase was confirmed by migration of the protein as a
single band with a molecular mass of approximately 79 kDa in an
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (data not shown).
Hydrolysis of various substrates was analyzed with the purified enzyme
(Table 1). The CDase showed a specific
activity of 1,940 U/mg of protein.
The enzyme activities at various temperatures were compared by
measuring the formation of the reducing sugar after 5 min of incubation
of the mixture of the enzyme and
-CD in Britton-Robinson buffer (pH
5.5). The maximum enzyme activity was observed at 95°C, and even at
120°C almost 40% of the maximum activity was detected. The optimum
pH for the reaction of the CDase on
-CD at 90°C was 5.5. In order
to study the thermal stability of the CDase in the absence of
substrate, the enzyme (in 50 mM Britton-Robinson buffer [pH 5.5]) was
incubated at 90°C for various time periods, and the remaining
activity was measured by the standard method. At 90°C, the half-life
of the CDase was 2 h.
A number of linear maltodextrins and three types of CDs (
-,
-,
and
-CDs) were incubated with CDase for a prolonged time and samples
were taken periodically. The spectrum of reaction products was assessed
by TLC. Figure 3 shows the results when
-CD, maltoheptaose, and maltotriose were used as substrates. The
analysis of hydrolyzed products suggested that these three CDs were
initially hydrolyzed to the linear maltodextrins of each size and then
they were subsequently degraded to the small maltodextrins. The
relative reaction velocity decreased when maltodextrins with shorter
chain lengths were used. Indeed, maltotetraose and maltotriose were
very slowly hydrolyzed and maltose was not utilized as a substrate of
the CDase. The end products of the CDase reaction were, therefore,
glucose and maltose. Starch was hydrolyzed more slowly than maltotriose
and an extremely small amount of degraded maltodextrins was detected
after 18 h, whereas no increase in reducing ends was observed in
the case of pullulan even after 24 h of incubation (results not
shown).

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FIG. 3.
Thin-layer chromatograms of hydrolysis products of
-CD (A), maltoheptaose (B), and maltotriose (C). Enzyme reactions
were performed at the indicated periods and generated products were
analyzed. Standards given are (from top): G1, glucose; G2, maltose; G3,
maltotriose; G4, maltotetraose; G5, maltopentaose; G6, maltohexaose;
G7, maltoheptaose.
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Nucleotide sequence of the gene for a putative CBP.
Gene
walking towards the downstream region from the position of the
cgtA was performed, a 4.2-kbp
HindIII-HindIII (positions 2639 and 6840, respectively) fragment was cloned into pUC19, and the resultant plasmid
was named pTY-40. Sequence analysis of the downstream region of
cgtA revealed the existence of another ORF (ORF3) forming a
gene cluster with cgtB-cgtA as shown in Fig. 1. ORF3,
starting from ATG at nucleotide position 4779, was in the same
direction as the cgtA. A possible ribosome-binding site (GAGG) was located 9 nucleotides upstream from the ATG. ORF3 ends with
a TGA stop codon at position 6102. The nucleotide sequence of ORF3
consists of 1,323 nucleotides, encoding 441 amino acid residues with a
calculated molecular mass of 48,513 Da. The deduced amino acid sequence
of ORF3 exhibited 34, 32, and 34% identity with those of the
periplasmic maltose-binding protein (MalE) of E. coli
(9), the periplasmic CBP (CymE) of K. oxytoca
M5a1 (12, 36), and the trehalose-maltose-binding protein
of the hyperthermophilic archaeon Thermococcus litoralis
(18), respectively. Archaea do not possess an
outer membrane and periplasmic space. Therefore, these binding proteins
were suggested to exist as soluble lipoproteins which allowed the
anchorage of the binding proteins to the external surface of the
cytoplasmic membrane. We found that the 25 amino acids in the
N-terminal region of ORF3 show typical characteristics of signal
peptides found in precursors of secretory proteins: (i) a hydrophobic
core adjacent to the N terminus and (ii) the sequence VASGCIG,
corresponding to consensus amino acid sequence of lipoprotein signal
peptidase cleavage sites (LAAGCSS) (47). Thus, ORF3 is
considered to encode a solute-specific binding protein. Following the
putative signal cleavage site, a Gly-rich region (amino acids 25 to 28)
and a remarkable Thr- and Ser-rich region (amino acids 29 to 63), which
may function as a flexible linker, were found.
Identification of the binding protein.
An expression vector
for the putative binding protein (ORF3) was constructed and introduced
into E. coli BL21-CodonPlus(DE3)-RIL. Heat treatment of the
crude extract at 80°C for 10 min resulted in considerable purity of
the target protein. The putative solute-binding protein was purified by
anion-exchange column chromatography and found to migrate as a single
protein band with a molecular mass of approximately 49 kDa in SDS-PAGE
(Fig. 4, lane 2). Considering that ORF3
is located downstream of the cgtB-cgtA genes, the protein might be involved in the degradation process for CDs synthesized extracellularly. Thus, it was speculated that ORF3 encodes a CBP. The
binding specificity of a protein derived from ORF3 was analyzed by
measuring the binding to the
-CD affinity column. As shown in Fig.
4, the protein bound to the
-CD affinity column and was eluted by
buffer containing
-CD. Furthermore, the protein bound to not only
-CD but also amylose resin, and it was eluted by maltose. When the
bound protein fraction was eluted by
-CD and was heat-treated in an
SDS-gel loading buffer at 100°C for 5 min, two bands were observed
(lane 4). The smaller band disappeared when the eluted protein sample
was treated under highly denaturing conditions (120°C, 5 min) (lane
5). In the fraction eluted by maltose (lane 9), a single band was
observed even when treated in SDS-gel loading buffer at 100°C for 5 min, indicating that the structure of the putative CBP in the presence
of
-CD is more thermostable than that bound to maltose. These
results indicated that ORF3 might encode a CBP rather than a
maltose-binding protein.

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FIG. 4.
Binding assay of CBP. Proteins were electrophoresed on a
12% acrylamide gel and stained with Coomassie brilliant blue. Lanes:
1, 6, and 11, molecular mass standards (namely, rabbit muscle
phosphorylase b [94 kDa], bovine serum albumin [67 kDa], egg white
ovalbumin [43 kDa], bovine erythrocyte carbonic anhydrase [30.1
kDa], and soybean trypsin inhibitor [20.1 kDa]); 2 and 7, purified
CBP used for binding experiment; 3, unbound fraction from -CD
affinity column; 4 and 5, bound fraction from -CD affinity column
(eluted fraction with -CD); 8, unbound fraction from amylose resin;
9 and 10, bound fraction from amylose resin (eluted fraction with
maltose). Bound protein to -CD affinity column was eluted by -CD
and incubated at 100°C (lane 4) or 120°C (lane 5) for 5 min in an
SDS-gel loading solution. Bound protein to amylose resin was eluted by
the buffer containing maltose and incubated at 100°C (lane 9) or
120°C (lane 10) for 5 min in an SDS-gel loading buffer.
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Nucleotide sequence of the genes for putative CBP-dependent
permease proteins.
Starting from an ATG at position of 6151, there
is another ORF (ORF4), but a putative stop codon could not be found in
the cloned fragment. To obtain the DNA region encoding the entire ORF4,
subsequent gene walking was performed, a 2.2-kbp
EcoRI-SphI (positions 6425 and 8576, respectively) fragment was cloned into pUC19, and the resultant plasmid
was named pTY-48 (Fig. 1). The nucleotide sequence of the entire ORF4
could be determined. ORF4 encodes a protein of 300 amino acids with
molecular weight of 33,915. Immediately downstream of ORF4, ORF5, which
encodes a protein of molecular weight 49,706 (447 amino acids), is
located in the same direction. A search with the BLAST program revealed that the deduced amino acid sequences of ORF4 and ORF5 were homologous to the permease proteins of the maltose transport system encoded by
malF and malG, respectively. MalF and MalG are
inner membrane components of the maltose-binding protein-dependent
maltose-maltodextrin ABC transporter (5). The deduced
amino acid sequence of ORF4 shared 27, 31, and 36% identity with those
of the maltose transport protein (MalF) of E. coli
(13), the cyclomaltodextrin transport protein (CymF) of
K. oxytoca M5a1 (12), and the trehalose-maltose transport protein (MalG) of T. litoralis (18),
respectively. The deduced amino acid sequence of ORF5 shared 34, 36, and 34% identity with those of MalG of E. coli
(7), CymG of K. oxytoca M5a1 (12),
and MalG of T. litoralis (18), respectively.
The predicted amino acid sequences of both ORF4 and ORF5 contain the consensus sequence EAAX2L/DGAX8IXLP that is
found in membrane components of ABC transporters (41). In
this conserved region, the amino acids are predominantly hydrophobic,
with a consistent location at the C terminus. ORF4 and ORF5 may encode
putative CBP-dependent membrane transporter proteins. Accordingly, the three genes (ORF3, ORF4, and ORF5) were designated cgtC,
cgtD, and cgtE, respectively.
 |
DISCUSSION |
We have shown the presence of a cgtBACDE gene cluster
on the Thermococcus sp. strain B1001 chromosome (Fig. 1).
Respective genes encode functional proteins necessary for intracellular
degradation (CgtB) of CDs, extracellular synthesis (CgtA), specific
recognition (CgtC), and transmembrane transport (CgtD and CgtE).
Purification and characterization of recombinant CDase (CgtB) and CBP
(CgtC) were performed in the present study.
The CDase is very thermostable, with an optimum temperature for
activity of 95°C. The CDases from other thermophilic sources, B. subtilis H-17 and T. ethanolicus 39E, have
optimum temperatures of 65 to 68°C (24) and 65°C
(38, 40), respectively. The strain B1001 CDase exhibited a
half-life of 2 h at 90°C and the hydrolytic activity was
maintained in the presence of substrates even after 18 h at 90°C
(Fig. 3). The B1001 CDase is the most thermostable among the enzymes
previously reported and characterized.
The cgtC gene was suggested to encode a kind of
solute-binding protein by sequence similarity. Characterization of the
purified recombinant CgtC revealed that the gene product is functional as a CBP. The CBP contains a long stretch of putative signal sequence in the N-terminal region, and the possible cleavage site might be
modified by lipid, which may function as an anchor to the cytoplasmic membrane. The Thr- and Ser-rich region (SSPTQTTTTT repeat) was observed
between the putative anchor domain and CD binding domain. It was
reported for a pullulanase of T. thermosulfurigenes EM1 that
the Thr-, Ser-, and Gly-rich region, due to the probable extended,
flexible nature of its structure, would allow optimal orientation of
the enzyme's catalytic site toward the substrate (31).
Interestingly, several S-layer anchoring enzymes exhibit variations of
Thr-rich regions, which are usually described as O-glycosylation
regions. Indeed, in the case of an archaeal hyperthermostable type II
pullulanase from Thermococcus hydrothermalis, the Thr-rich region has been suggested to be a target for intensive O glycosylation (10). It has also been reported that glucoamylase from
Aspergillus awamori possesses this region for O
glycosylation and the region is important for efficient degradation of
insoluble starch granules (42). The Thr- and Ser-rich
region forms a rather extended and flexible conformation susceptible to
proteolytic digestion (32). Intense O glycosylation of
this region would reduce the vulnerability of this structure. Based on
these reports, the Thr- and Ser-rich region of strain B1001 may serve
as a linker to maintain the flexibility of the CD binding domain on the
surface of the cell membrane for efficient uptake of extracellular CDs.
This is the first report of a solute-binding protein that includes this
unique linker.
The cgtDE genes are located downstream of the
cgtC gene, in the same direction. The deduced amino acid
sequence of cgtD (CgtD) and cgtE (CgtE) showed
similarity to those of MalF and MalG, parts of the maltose-binding
protein-dependent ABC transport systems of E. coli. In
addition, the organization of genes for CBP and the membrane
transporting apparatus cluster (cgtCDE) is similar to that
of E. coli and other bacterial binding protein-dependent ABC
transport systems. In the case of the E. coli
maltose-maltodextrin transport system, the maltose-binding protein
(MalE) is located in the periplasmic space, two hydrophobic membrane
proteins (MalF and MalG) form the translocation pore, and two
additional subunits (MalK) are peripherally associated with the
membrane proteins at the inner face of the membrane. Recently, an ABC
transporter for maltose-trehalose was reported in the hyperthermophilic
archaeon T. litoralis (17, 18). These recent
reports and our experimental results strongly suggest that strain B1001
possesses the CBP-dependent ABC transport system for specific uptake
and utilization of CD as a carbon source. In the organization of the
T. litoralis ABC transporter for maltose-trehalose, the
malEFG operon was very similar to that of E. coli
but did not contain the E. coli malK homolog. The location
of the malK gene is close to the malEFG gene in
T. litoralis (17). The malK homolog
was not found directly adjacent and distal to cgtE in strain
B1001. The possibility that strain B1001 has the malK
homolog encoding the ATPase subunit should be considered, though it is
unclear that its gene is also a member of the cgtBACDE cluster.
Typical signal peptide sequences (a positively charged N terminus
followed by a stretch of hydrophobic residues) as secretory signals
were found in the precursors of the CGTase (48) and CBP
from strain B1001 and the
-amylase of Pyrococcus
kodakaraensis strain KOD1 (44). It is generally
assumed that signal peptides of Archaea are similar to those
of Bacteria and Eucarya (1). However, comparison of the amino acid sequence of the CDase revealed that the gene does not encode a putative signal peptide, suggesting that the CDase of strain B1001 is an intracellular enzyme. Previously, we reported that strain B1001 produced a thermostable CGTase in the
extracellular fraction (45). In the present study, the
existence of a CBP-dependent ABC transport system for CD was implied.
It has been reported that K. oxytoca M5a1 utilizes starch as
a sole carbon and energy source via two metabolic pathways. The first pathway involves extracellular degradation of starch into linear maltodextrins by hydrolysis of the
-1,6-glycosidic bonds via the
pullulanase and subsequent cleavage of the
-1,4-glycosidic linkages
by disproportionation activity of the CGTase. Maltodextrins are
transported and assimilated via a binding protein-dependent ABC
transporter and intracellular hydrolytic enzymes. The second pathway, a
novel starch degradation pathway, involves the extracellular conversion
of starch into CDs by CGTase and uptake of the CDs by a specific
binding and transporting system following intracellular linearization
by a CDase (12). The genes involved in this starch utilization pathway are organized in two divergently oriented clusters
in the chromosome of K. oxytoca M5a1. The existence of the
CGTase, CBP, and CDase in strain B1001 indicates that a mechanism similar to that of the second pathway of K. oxytoca M5a1
exists as a starch assimilation system (Fig.
5). In addition, indirect evidence for
this starch metabolism only through CDs is supported by the fact that
no remarkable enzymatic activity to produce reducing sugar was observed
in the culture of B1001. The unique starch metabolic pathway via CDs
may be more advantageous for strain B1001 among hyperthermophiles
growing in a high-temperature environment because synthesis of CDs
allows effective and competitive exploitation of starch as a carbon
source. CDs are not hydrolyzed by exo-type amylases such as
glucoamylases and
-amylases because CDs have no nonreducing ends.
The raw starch-binding domain of A. awamori exhibits binding
affinity not only to raw starch but also to
-,
-, and
-CDs,
although the enzyme cannot hydrolyze CDs. Thus, the activity of the
glucoamylase will be competitively inhibited by binding of CDs to the
raw starch binding site of the enzyme (14, 16). CDs show
various degrees of resistance and inhibition to hydrolytic enzymes
produced by competitors, indicating that strain B1001 converts starch
to CDs as inhibitors of amylolytic enzymes produced by competitors. The
production of linear maltodextrins as a carbon source by competitors is
inhibited by CDs. In addition, CBP binds not only to CDs but also to
linear maltodextrins, although strain B1001 does not secrete a
maltodextrin-producing enzyme. The fact that a CBP-dependent ABC
transporter system also functions as the classical maltose-maltodextrin
transporter indicates that strain B1001 might assimilate even
maltodextrins produced by competitors as well as CDs. The cluster
encoding extracellular CGTase, the CBP-dependent ABC transport system
for CD, and intracellular CDase would provide benefits to the cell in
the exploitation of carbon sources in a high-temperature environment
where starch substrates are in alpha form (Fig. 5).
 |
ACKNOWLEDGMENTS |
This work was supported in part by the Core Research for
Evolutional Science and Technology (CREST) Program of the Japan Science and Technology Corporation.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto, University, Sakyo-ku, Kyoto 606-8501, Japan. Phone:
(81) 75-753-5568. Fax: (81) 75-753-4703. E-mail:
imanaka{at}sbchem.kyoto-u.ac.jp.
Present address: Institute of Applied Biochemistry, The University
of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
Present address: School of Materials Science, Japan Advanced
Institute of Science and Technology, Hokuriku, 1-1 Asahidai
Tatsunokuchi, Ishikawa 923-1292, Japan.
 |
REFERENCES |
| 1.
|
Albers, S.-V.,
W. N. Kornings, and A. J. M. Driessen.
1999.
A short signal sequence in membrane-anchored proteins of Archaea.
Mol. Microbiol.
31:1595-1597[CrossRef][Medline].
|
| 2.
|
Bender, H.
1986.
Production, characterization, and application of cyclodextrins.
Adv. Biotechnol. Proc.
6:31-71.
|
| 3.
|
Bender, H.
1993.
Purification and characterization of a cyclodextrin-degrading enzyme from Flavobacterium sp.
Appl. Microbiol. Biotechnol.
39:714-719[CrossRef].
|
| 4.
|
Bernfeld, H.
1955.
Amylases and .
Methods Enzymol.
1:149-150[CrossRef].
|
| 5.
|
Boos, W., and H. Shuman.
1998.
Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation.
Microbiol. Mol. Biol. Rev.
62:204-29[Abstract/Free Full Text].
|
| 6.
|
Cha, H. J.,
H. G. Yoon,
Y. W. Kim,
H. S. Lee,
J. W. Kim,
K. S. Kweon,
B. H. Oh, and K. H. Park.
1998.
Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose.
Eur. J. Biochem.
253:251-262[Medline].
|
| 7.
|
Dassa, E., and M. Hofnung.
1985.
Sequence of gene malG in E. coli K12: homologies between integral membrane components from binding protein-dependent transport systems.
EMBO J.
4:2287-2293[Medline].
|
| 8.
|
DePinto, J. A., and L. L. Campbell.
1968.
Purification and properties of the cyclodextrinase of Bacillus macerans.
Biochemistry
7:121-125[CrossRef][Medline].
|
| 9.
|
Duplay, P.,
H. Bedouelle,
A. Fowler,
I. Zabin,
W. Saurin, and M. Hofnung.
1984.
Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12.
J. Biol. Chem.
259:10606-10613[Abstract/Free Full Text].
|
| 10.
|
Erra-Pujada, M.,
P. Debeire,
F. Duchiron, and M. J. O'Donohue.
1999.
The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain.
J. Bacteriol.
181:3284-3287[Abstract/Free Full Text].
|
| 11.
|
Feederle, R.,
M. Pajatsch,
E. Kremmer, and A. Böck.
1996.
Metabolism of cyclodextrins by Klebsiella oxytoca M5a1: purification and characterisation of a cytoplasmically located cyclodextrinase.
Arch. Microbiol.
165:206-212[Medline].
|
| 12.
|
Fiedler, G.,
M. Pajatsch, and A. Böck.
1996.
Genetics of a novel starch utilisation pathway present in Klebsiella oxytoca.
J. Mol. Biol.
256:279-291[CrossRef][Medline].
|
| 13.
|
Frosshauer, S., and J. Beckwith.
1984.
The nucleotide sequence of the gene for malF protein, an inner membrane component of maltose transport system of Escherichia coli.
J. Biol. Chem.
259:10896-10903[Abstract/Free Full Text].
|
| 14.
|
Fukuda, K.,
Y. Teramoto,
M. Goto,
J. Sakamoto,
S. Mitsuiki, and S. Hayashida.
1992.
Specific inhibition by cyclodextrins of raw starch digestion by fungal glucoamylase.
Biosci. Biotechnol. Biochem.
56:556-559[Medline].
|
| 15.
|
Galvin, N. M.,
C. T. Kelly, and W. M. Fogarty.
1994.
Purification and properties of the cyclomaltodextrinase of Bacillus sphaericus ATCC 7055.
Appl. Microbiol. Biotechnol.
42:46-50[CrossRef].
|
| 16.
|
Goto, M.,
K. Tanigawa,
W. Kanlayakrit, and S. Hayashida.
1994.
The mechanism of binding of glucoamylase I from Aspergillus awamori var. kawachi to cyclodextrins and raw starch.
Biosci. Biotechnol. Biochem.
58:49-54.
|
| 17.
|
Greller, G.,
R. Horlacher,
J. DiRuggiero, and W. Boos.
1999.
Molecular and biochemical analysis of MalK, the ATP-hydrolyzing subunit of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis.
J. Biol. Chem.
274:20259-20264[Abstract/Free Full Text].
|
| 18.
|
Horlacher, R.,
K. B. Xavier,
H. Santos,
J. DiRuggiero,
M. Kossmann, and W. Boos.
1998.
Archaeal binding protein-dependent ABC transporter: molecular and biochemical analysis of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis.
J. Bacteriol.
180:680-689[Abstract/Free Full Text].
|
| 19.
|
Igarashi, K.,
K. Ara,
K. Saeki,
K. Ozaki,
S. Kawai, and S. Ito.
1992.
Nucleotide sequence of the gene that encodes a neopullulanase from an alkalophilic Bacillus.
Biosci. Biotechnol. Biochem.
56:514-516[Medline].
|
| 20.
|
Izumi, M.,
S. Fujiwara,
M. Takagi,
S. Kanaya, and T. Imanaka.
1999.
Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: analysis of an ATPase-deficient mutant enzyme.
Appl. Environ. Microbiol.
65:1801-1805[Abstract/Free Full Text].
|
| 21.
|
Jeon, S.-J.,
S. Fujiwara,
M. Takagi, and T. Imanaka.
1999.
Pk-cdcA encodes a CDC48/VCP homolog in the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1: transcription and enzymatic characterization.
Mol. Gen. Genet.
262:559-567[CrossRef][Medline].
|
| 22.
|
Kim, T. J.,
J. H. Shin,
J. H. Oh,
M. J. Kim,
S. B. Lee,
S. Ryu,
K. Kwon,
J. W. Kim,
E. H. Choi,
J. F. Robyt, and K. H. Park.
1998.
Analysis of the gene encoding cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 and characterization of enzymatic properties.
Arch. Biochem. Biophys.
353:221-227[CrossRef][Medline].
|
| 23.
|
Kitahata, S.,
M. Taniguchi,
S. D. Beltran,
T. Sugimoto, and S. Okada.
1983.
Purification and some properties of cyclodextrinase from Bacillus coagulans.
Agric. Biol. Chem.
47:1441-1447.
|
| 24.
|
Krohn, B. M., and J. A. Lindsay.
1991.
Purification and characterization of a thermostable -glucosidase from Bacillus subtilis high-temperature growth transformant.
Curr. Microbiol.
22:273-278.
|
| 25.
|
Krohn, B. M., and J. A. Lindsay.
1992.
Reclassification of a thermostable glucosidase from Bacillus subtilis H-17 as a cyclomaltodextrinase.
Enzyme Microb. Technol.
14:194-196[CrossRef].
|
| 26.
|
Kuriki, T., and T. Imanaka.
1989.
Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus.
J. Gen. Microbiol.
135:1521-1528[Medline].
|
| 27.
|
Kuriki, T., and T. Imanaka.
1999.
The concept of the -amylase family: structural similarity and common catalytic mechanism.
J. Biosci. Bioeng.
87:557-565[CrossRef][Medline].
|
| 28.
|
Loftsson, T., and M. E. Brewster.
1996.
Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization.
J. Pharm. Sci.
85:1017-1025[CrossRef][Medline].
|
| 29.
|
Makrides, S. C.
1996.
Strategies for achieving high-level expression of genes in E. coli.
Microbiol. Rev.
60:512-538[Abstract/Free Full Text].
|
| 30.
|
Maniatis, T.,
E. F. Fritsch, and J. Sambrook.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 31.
|
Matuschek, M.,
G. Burchhardt,
K. Sahm, and H. Bahl.
1994.
Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface.
J. Bacteriol.
176:3295-3302[Abstract/Free Full Text].
|
| 32.
|
Moens, S., and J. Vanderleyden.
1997.
Glycoproteins in prokaryotes.
Arch. Microbiol.
168:169-175[CrossRef][Medline].
|
| 33.
|
Nagashima, T.,
S. Tada,
K. Kitamoto,
K. Gomi,
C. Kumagai, and H. Toda.
1992.
Site-directed mutagenesis of catalytic active-site residues of Taka-amylase A.
Biosci. Biotechnol. Biochem.
56:207-210[Medline].
|
| 34.
|
Oguma, T.,
M. Kikuchi, and K. Mizusawa.
1990.
Purification and some properties of cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus.
Biochim. Biophys. Acta
1036:1-5[Medline].
|
| 35.
|
Oguma, T.,
A. Matsuyama,
M. Kikuchi, and E. Nakano.
1993.
Cloning and sequence analysis of the cyclomaltodextrinase gene from Bacillus sphaericus and expression in Escherichia coli cells.
Appl. Microbiol. Biotechnol.
39:197-203[Medline].
|
| 36.
|
Pajatsch, M.,
M. Gerhart,
R. Peist,
R. Horlacher,
W. Boos, and A. Böck.
1998.
The periplasmic cyclodextrin binding protein CymE from Klebsiella oxytoca and its role in maltodextrin and cyclodextrin transport.
J. Bacteriol.
180:2630-2635[Abstract/Free Full Text].
|
| 37.
|
Podkovyrov, S. M.,
D. Burdette, and J. G. Zeikus.
1993.
Analysis of the catalytic center of cyclomaltodextrinase from Thermoanaerobacter ethanolicus 39E.
FEBS Lett.
317:259-262[CrossRef][Medline].
|
| 38.
|
Podkovyrov, S. M., and J. G. Zeikus.
1992.
Structure of the gene encoding cyclomaltodextrinase from Clostridium thermohydrosulfuricum 39E and characterization of the enzyme purified from Escherichia coli.
J. Bacteriol.
174:5400-5405[Abstract/Free Full Text].
|
| 39.
|
Pulley, O. A., and D. French.
1961.
Studies on the schardinger dextrins. XI. The isolation of new schardinger dextrins.
Biochem. Biophys. Res. Commun.
5:11-15[CrossRef][Medline].
|
| 40.
|
Saha, B. C., and J. G. Zeikus.
1990.
Characterization of thermostable cyclodextrinase from Clostridium thermohydrosulfuricum 39E.
Appl. Environ. Microbiol.
56:2941-2943[Abstract/Free Full Text].
|
| 41.
|
Saurin, W.,
W. Köster, and E. Dassa.
1994.
Bacterial binding protein-dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins.
Mol. Microbiol.
12:993-1004[Medline].
|
| 42.
|
Semimaru, T.,
M. Goto,
K. Furukawa, and S. Hayashida.
1995.
Functional analysis of the threonine and serine-rich Gp-I domain of glucoamylase I from Aspergillus awamori var. kawachi.
Appl. Environ. Microbiol.
61:2885-2890[Abstract].
|
| 43.
|
Stephen, F. A.,
G. Warren,
M. Webb,
W. M. Eugene, and J. L. David.
1990.
Basic local alignment search tool.
J. Mol. Biol.
215:403-410[CrossRef][Medline].
|
| 44.
|
Tachibana, Y.,
M. M. Leclere,
S. Fujiwara,
M. Takagi, and T. Imanaka.
1996.
Cloning and expression of the -amylase gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1, and characterization of the enzyme.
J. Ferment. Bioeng.
82:224-232[CrossRef].
|
| 45.
|
Tachibana, Y.,
A. Kuramura,
N. Shirasaka,
Y. Suzuki,
T. Yamamoto,
S. Fujiwara,
M. Takagi, and T. Imanaka.
1999.
Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic archaeon, a Thermococus sp.
Appl. Environ. Microbiol.
65:1991-1997[Abstract/Free Full Text].
|
| 46.
|
Takata, H.,
T. Kuriki,
S. Okada,
Y. Takesada,
M. Iizuka,
N. Minamiura, and T. Imanaka.
1992.
Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at -(1 4)- and -(1 6)-glucosidic linkages.
J. Biol. Chem.
267:18447-18452[Abstract/Free Full Text].
|
| 47.
|
Tam, R., and M. H. Saier.
1993.
Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria.
Microbiol. Rev.
57:320-346[Abstract/Free Full Text].
|
| 48.
|
Yamamoto, T.,
S. Fujiwara,
Y. Tachibana,
M. Takagi,
K. Fukui, and T. Imanaka.
2000.
Alteration of product specificity of cyclodextrin glucanotransferase from Termococcus sp. B1001 by site directed mutagenesis.
J. Biosci. Bioeng.
89:206-210.
|
| 49.
|
Yanisch-Perron, C.,
J. Yieira, and J. Messing.
1985.
Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors.
Gene
33:103-119[CrossRef][Medline].
|
| 50.
|
Yoshida, A.,
Y. Iwasaki,
T. Akiba, and K. Horikoshi.
1991.
Purification and properties of cyclomaltodextrinase from alkalophilic Bacillus sp.
J. Ferment. Bioeng.
71:226-229[CrossRef].
|
Journal of Bacteriology, September 2001, p. 5050-5057, Vol. 183, No. 17
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.17.5050-5057.2001
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