A thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 shows a novel domain organization among glycoside hydrolase family 13 enzymes

Article abstract of DOI:10.1271/bbb.130284

α-Amylases (EC 3.2.1.1) hydrolyze internal α-1,4- glucosidic linkages of starch and related glucans. Bacillus sp. AAH-31 produces an alkalophilic thermophilic α-amylase (AmyL) of higher molecular mass, 91 kDa, than typical bacterial %α-amylases. In this study, the AmyL gene was cloned to determine its primary structure, and the recombinant enzyme, produced in Escherichia coli, was characterized. AmyL shows no hydrolytic activity towards pullulan, but the central region of AmyL (Gly395-Asp684) was similar to neopullulanase- like α-amylases. In contrast to known neopullulanase- like %α-amylases, the N-terminal region (Gln29-Phe102) of AmyL was similar to carbohydratebinding module family 20 (CBM20), which is involved in the binding of enzymes to starch granules. Recombinant AmyL showed more than 95% of its maximum activity in a _pH range of 8.2-10.5, and was stable below 65 °C and from _pH 6.4 to 11.9. The k_cat values for soluble starch, γ-cyclodextrin, and maltotriose were 103 s^-1, 67.6 s^-1, and 5.33 s^-1, respectively, and the K _m values were 0.100mg/mL, 0.348mM, and 2.06mM, respectively. Recombinant AmyL did not bind to starch granules. But the substitution of Trp45 and Trp84, conserved in site 1 of CBM20, with Ala reduced affinity to soluble starch, while the mutations did not affect affinity for oligosaccharides. Substitution of Trp61, conserved in site 2 of CBM20, with Ala enhanced hydrolytic activity towards soluble starch, indicating that site 2 of AmyL does not contribute to binding to soluble long-chain substrates.

Full text of DOI:10.1271/bbb.130284

Biosci. Biotechnol. Biochem., 77 (9), 1867–1873, 2013
A Thermophilic Alkalophilic ꢀ-Amylase from Bacillus sp. AAH-31 Shows
a Novel Domain Organization among Glycoside Hydrolase Family 13 Enzymes
2;
1
1
2
Wataru SABURI,1; Naoki MORIMOTO, Atsushi MUKAI, Dae Hoon KIM, Toshihiko TAKEHANA,
*
*
y
Seiji KOIKE,2 Hirokazu MATSUI,1 and Haruhide MORI
1;
1Research Faculty of Agriculture, Hokkaido University, N-9, W-9, Sapporo 060-8589, Japan
2Adeka Corporation, 7-2-34 Higashi-ogu, Arakawa-ku, Tokyo 116-8553, Japan
Received April 3, 2013; Accepted May 30, 2013; Online Publication, September 7, 2013
[doi:10.1271/bbb.130284]
ꢀ-Amylases (EC 3.2.1.1) hydrolyze internal ꢀ-1,4-
glucosidic linkages of starch and related glucans.
Bacillus sp. AAH-31 produces an alkalophilic thermo-
philic ꢀ-amylase (AmyL) of higher molecular mass,
91 kDa, than typical bacterial ꢀ-amylases. In this study,
the AmyL gene was cloned to determine its primary
structure, and the recombinant enzyme, produced in
Escherichia coli, was characterized. AmyL shows no
hydrolytic activity towards pullulan, but the central
region of AmyL (Gly395-Asp684) was similar to neo-
pullulanase-like ꢀ-amylases. In contrast to known neo-
pullulanase-like ꢀ-amylases, the N-terminal region
(Gln29-Phe102) of AmyL was similar to carbohydrate-
binding module family 20 (CBM20), which is involved in
the binding of enzymes to starch granules. Recombinant
AmyL showed more than 95% of its maximum activity
in a pH range of 8.2–10.5, and was stable below 65 ꢀC
and from pH 6.4 to 11.9. The kcat values for soluble
applications, and the de-sizing of textiles and paper.
Alkalophilic liquefying enzymes are particularly useful
as additive compounds for dishwashing and laundry
detergents to remove food residues on dishes and food
stains from clothes.1)
According to a sequence-based classification of
glycoside hydrolases (GHs),2) ꢀ-amylases are classified
into GH families 13, 57, and 119, while most enzymes
are found in GH family 13. GH family 13 is the largest
of the GH families. It is composed of various retaining
GHs and glycosyltransferases that act on ꢀ-glucans
such as starch, maltooligosaccharides, and sucrose.3)
Enzymes of this family show three-dimensional struc-
tures similar to enzymes belonging to GH families 70
and 77, and hence these families are categorized into
clan GH-H, regarded as the ꢀ-amylase family.
GH family 13 enzymes share three domains (A, B,
and C).3) Domain A is the catalytic domain formed by a
ðꢁ=ꢀÞ8-barrel. Domain B is a long loop connecting the
third ꢁ-strand and the third ꢀ-helix, and a substrate
binding cleft is formed at the interface of domains A and
B. Domain C, following domain A, is formed by an
antiparallel ꢁ-sheet. GH family 13 enzymes have been
further classified into 40 subfamilies based on amino
acid sequences.4) Most subfamilies contain enzymes that
utilize the same substrate and produce the same product.
ꢀ-Amylases are found in subfamilies 1, 2, 5, 6, 7, 15, 19,
20, 24, 27, 28, and 32.
Bacillus sp. AAH-31, first isolated from Japanese
paddy soil, produces extracellular liquefying ꢀ-amylase
(AmyL), which is highly stable at high pH and temper-
atures.5) Many alkaline ꢀ-amylases have been identified
in various alkalophilic bacteria,6–11) but AmyL is more
stable at high temperature than the known alkalophilic
enzymes. Furthermore, this enzyme is stable in the
presence of chelating reagents, including EDTA, nitri-
lotriacetic acid, and sodium tripolyphosphate. These
properties of AmyL are useful in automatic dishwashing
detergents, because an automatic dishwashing machine
generally operates at high pH at temperatures above
60 ꢀC. On SDS–PAGE analysis, the molecular mass of
AmyL was estimated to be 91 kDa, considerably higher
starch, ꢁ-cyclodextrin, and maltotriose were 103 sÀ1
,
67.6 sÀ1, and 5.33 sÀ1, respectively, and the Km values
were 0.100 mg/mL, 0.348mM, and 2.06 mM, respectively.
Recombinant AmyL did not bind to starch granules. But
the substitution of Trp45 and Trp84, conserved in site 1
of CBM20, with Ala reduced affinity to soluble starch,
while the mutations did not affect affinity for oligosac-
charides. Substitution of Trp61, conserved in site 2 of
CBM20, with Ala enhanced hydrolytic activity towards
soluble starch, indicating that site 2 of AmyL does not
contribute to binding to soluble long-chain substrates.
Key words: ꢀ-amylase; neopullulanase; glycoside hy-
drolase family 13; carbohydrate-binding
module family 20; starch-binding domain
ꢀ-Amylases (EC 3.2.1.1), widely distributed in living
organisms including animals, plants, and microorgan-
isms, catalyze the hydrolysis of internal ꢀ-1,4-glucosidic
linkages of starch, glycogen, and related glucans with
net retention of the anomeric configuration. Fungal and
bacterial ꢀ-amylases are utilized in various industries,
including the production of isomerized sugar (liquefac-
tion and saccharification of starch granules), bakery
y
*
To whom correspondence should be addressed. Tel/Fax: +81-11-706-2497; E-mail: hmori@chem.agr.hokudai.ac.jp
These authors contributed equally to this work.
Abbreviations: GH, glycoside hydrolase; AmyL, Bacillus sp. AAH-31 ꢀ-amylase; PVDF, polyvinylidene difluoride; CD, cyclodextrin; TVAI,
Thermoactinomyces vulgaris R-47 ꢀ-amylase I; TVAII, Thermoactinomyces vulgaris R-47 ꢀ-amylase II; CBM20, carbohydrate-binding module
family 20; rAmyL, recombinant AmyL; pNPG5, p-nitrophenyl ꢀ-maltopentaoside; GA SBD, starch-binding domain of Aspergillus niger
glucoamylase

1868
W. SABURI et al.
isopropyl 1-thio-ꢁ-D-galactopyranoside (Wako Pure Chemical Indus-
tries, Osaka, Japan) was added to induce production of the recombinant
protein. The induction culture was carried out at the same temperature
for 3 h. Bacterial cells harvested by centrifugation were suspended in
10 mM Tris–HCl buffer (pH 7.0) containing 0.5 M NaCl and disrupted
by sonication. After the cell debris was removed by centrifugation, the
cell-free extract was applied to a Ni-chelating column by Chelating
Sepharose Fast Flow (GE Healthcare, Uppsala, Sweden). The column
was thoroughly washed with 10 mM Tris–HCl buffer (pH 7.0)
containing 0.5 M NaCl, and the adsorbed protein was eluted with
0.15 M imidazole in the same buffer. Highly purified fractions,
confirmed by SDS–PAGE, were collected and dialyzed against
10 mM Tris–HCl buffer (pH 7.0). The protein concentration was
determined from the concentrations of amino acids after complete acid
hydrolysis of the purified enzyme with 6 N HCl at 110 ꢀC for 24 h.
Amino acids were measured by the ninhydrin colorimetric method
using a JLC-500/V (JEOL, Tokyo).14)
than those of the typical bacterial ꢀ-amylases of GH
family 13 (approximately 60 kDa) and close to enzymes
possessing starch-binding domains.11,12) In the present
study, the gene encoding AmyL was cloned to elucidate
its primary structure, and the recombinant enzyme,
produced in Escherichia coli, was characterized. Fur-
thermore, a site-directed mutational study at the starch-
binding site, which was identified through sequence
analysis, was carried out.
Materials and Methods
Analysis of N-terminal and partial internal amino acid sequences.
Ten mg of AmyL purified from the culture supernatant of Bacillus sp.
AAH-315) was further separated by SDS–PAGE and transferred to a
polyvinylidene difluoride (PVDF) membrane (Sequi-Blot PVDF
Membrane, Bio-Rad, Hercules, CA) by electroblotting with a semi-
dry blotting apparatus.13) The band of AmyL, detected by staining with
Coomassie Brilliant Blue, was cut off from the membrane and
subjected to N-terminal sequence analysis with a Procise 492HT
protein sequencer (Perkin Elmer, Waltham, MA). To analyze partial
internal amino acid sequences of AmyL, the enzyme was digested by
endoproteinase Asp-N (Takara Bio, Otsu, Japan). Twenty-five mg of
AmyL was transferred to a PVDF membrane as described above, and
the band of AmyL was incubated in 100 mL of 50 mM sodium
phosphate buffer (pH 8.0) containing 0.65 ng/mL of endoproteinase
Asp-N for 4 h at 37 ꢀC. Ten mL of the resulting supernatant was
subjected to separation by HPLC under the following conditions:
column, Capcell Pak C8 (ꢂ4:6 ꢁ 250 mm, Shiseido, Tokyo); column
temperature, room temperature; elution, 0–60% acetonitrile; flow rate,
0.5 mL/min; detection, absorbance at 280 nm. The N-terminal amino
acid sequences of the purified peptides were analyzed as described
above.
Standard enzyme assay. A reaction mixture (100 mL) consisting of
an appropriate concentration of the enzyme, 100 mM Tris–HCl buffer
(pH 9.5), 5 mg/mL of soluble starch (Nacalai Tesque, Kyoto, Japan),
and 0.025 mg/mL of Triton X-100 was incubated at 50 ꢀC for 10 min,
and the resulting reducing sugar was measured by the dinitrosalicylic
acid method,15) in which 0–5 mM maltose (Nacalai Tesque) was used as
the standard reducing sugar. One U of enzyme activity was defined as
the amount of enzyme producing 1 mmol of reducing sugar per min.
Effects of pH and temperature on the activity and stability of
recombinant AmyL. Enzyme activities at various pH values were
measured to determine the optimum pH of AmyL. One hundred mM
Britton-Robinson buffer (pH 6.0–11.0, composed of a mixture of
100 mM acetate, phosphate, and glycine, pH adjusted with 5 N NaOH)
was used as the reaction buffer. The other reaction conditions were
same as for the standard enzyme assay.
To evaluate pH and temperature stabilities, the enzyme solution was
incubated at various pHs and temperatures, and residual activity was
measured by the standard enzyme assay. For the pH studies, the
enzyme solution was incubated in 100 mM Britton-Robinson buffer
(pH 4–13) at 4 ꢀC for 24 h. For heat treatment, the enzyme solution
was incubated at 30–80 ꢀC for 15 min, and then cooled down
immediately on ice. The enzyme was considered to be stable in those
ranges of pH and temperature over which the enzyme maintained more
than 90% of its original activity.
Gene cloning of AmyL and construction of its expression plasmid.
To obtain partial AmyL, PCR was carried out with LA Taq DNA
polymerase (Takara Bio), the genomic DNA of Bacillus sp. AAH-31,
and primers designed from the N-terminal amino acid sequence and an
internal amino acid sequence (P2, described below) of AmyL, S: 50-
CARGAYTAYGARAAYATHGT-30 (sense), and R: 50-TCYTCIACR-
TAYTGYTCRTC-30 (antisense). The amplified DNA fragment was
cloned into the EcoRV site of the pBluescript II SK (þ) vector
(Stratagene, La Jolla, CA), and sequenced with an ABI Prism 310
Genetic Analyzer (Applied Biosystems, Foster City, CA). Two mg of
the genomic DNA digested with HindIII (Takara Bio) was cyclized
through self-ligation with T4 DNA Ligase (Takara Bio), and used as
the template for inverse PCR. The regions upstream and downstream
of the section obtained by the PCR, as described above, were amplified
with cyclized genomic DNA as template. Primers, HS1: 50-ACCA-
TCGGCTCCAGTTTG-30 (sense) and HR1: 50-TGGATGATTGTTT-
GAACTCCAG-30 (antisense), were used for the upstream region, and
primers, HS2: 50-ACCAGTGGCAGATTGAGC-30 (sense) and HR2:
50-GGCACTTTCTACTTTGGAAAG-30 (antisense), for the down-
stream region. The further downstream region was amplified by
inverse PCR, in which the genomic DNA cyclized after digestion by
BamHI was used as template. The primers used for this PCR were BS:
50-TGTCCCAAAGTGAGGACCAT-30 (sense) and BR: 50-CTCTG-
TTCCGTAATAGATGGC-30 (antisense).
Kinetic parameters. The reaction velocities for the hydrolysis of
various concentrations of soluble starch, ꢃ-cyclodextrin (ꢃ-CD), and
maltotriose were measured. In the hydrolysis of soluble starch, a
reaction mixture (500 mL) consisting of 1.07–2.14 nM enzyme, 100 mM
Tris–HCl buffer (pH 9.5), 0.05–0.3 mg/mL of soluble starch, and
0.025 mg/mL of Triton X-100 was incubated at 50 ꢀC. In the reaction
to ꢃ-CD, 0.1–1.0 mM ꢃ-CD (Nacalai Tesque) was added to the reaction
mixture. Fifty mL of the reaction mixture was removed every 2 min
up to 10 min, and the reducing sugar produced was measured by
the copper bicinchoninate acid method,16) in which 0–300 mM maltose
was used as the standard. Initial reaction velocities, determined
from the slopes of the progress curves, at various substrate concen-
trations were fitted to the Michaelis-Menten equation by nonlinear
regression with Grafit version 7.0.2 (Erithacus Software, West Sussex,
UK). Data shown are mean ꢂ standard deviation for three independent
experiments.
AmyL was inserted into pET-21a vector (Novagen, Darmstadt,
Germany) to overexpress it in E. coli. The DNA fragment amplified by
PCR was cloned into pET-21a via the NdeI and XhoI sites, using
Primestar HS DNA polymerase (Takara Bio), the genomic DNA of
Bacillus sp. AAH-31, and primers 50-ACGCATATGCAGGATTAT-
GAGAACATTGTCCTAAGA-30 (sense, NdeI site underlined) and 50-
GTTCCCCTCGAGTTTGTTAATGTTGCCCAGGACC-30 (antisense,
XhoI site underlined).
For the analysis of maltotriose as substrate, a reaction mixture
(100 mL) consisting of 49.5–94.5 nM enzyme, 100 mM Tris–HCl buffer
(pH 9.5), 1–8 mM maltotriose (Wako Pure Chemical Industries), and
0.025 mg/mL of Triton X-100 was incubated at 50 ꢀC for 10 min, and
heated at 100 ꢀC for 10 min to terminate the reaction. After the addition
of 200 mL of 2 M Tris–HCl buffer (pH 7.0), the D-glucose released was
measured by the glucose-oxidase peroxidase method.17) Kinetic
parameters were determined as described above.
Preparation of recombinant AmyL. The transformant of E. coli
BL21 (DE3) harboring the expression plasmid for AmyL was cultured
at 37 ꢀC in 300 mL of Luria-Bertani medium supplemented with
100 mg/mL of ampicillin until A600 reached 0.5, and 0.3 mL of 0.1 M
Estimation of the molecular mass of recombinant AmyL. To
determine the molecular mass of recombinant AmyL, gel filtration
column chromatography was carried out. A Superose 12 column
(ꢂ10 ꢁ 300 mm, GE Healthcare) was used, and column chromatog-

Primary Structure of Bacillus sp. AAH-31 ꢀ-Amylase
1869
program.19) The deduced amino acid sequence of mature
AmyL is comprised of 793 amino acid residues, and the
molecular mass estimated from the sequence, 91,274 Da,
coincided well with the molecular mass of 91 kDa for
native AmyL.5) The P1 sequence was also found in
the deduced sequence of AmyL (Asp354-Lys357). A
BLAST search of the mature AmyL sequence revealed
that Gly395-Asp684 was assigned as the catalytic
domain of ꢀ-amylase (PF00128),20) and the four con-
served sequences of GH family 13 enzymes were found
in this region, as follows: region I, 464-YDFVPNH-470;
region II, 542-GFRLDYAKG-550; region III, 571-
FIFGEIWD-578; and region IV, 635-FLDNHD-640.
Asp546, Glu575, and Asp640 are predicted to be the
catalytic nucleophile, the general acid/base catalyst, and
the second catalytic aspartic acid fulfilling multiple
functions essential for catalysis, including elevating the
pKa of the general acid/base catalyst and promoting
substrate distortion.21) The catalytic domain of AmyL
showed high sequence identity to neopullulanase-like
ꢀ-amylases belonging to subfamily 20 of GH family
13,4) although AmyL has no hydrolytic activity towards
pullulan, unlike the other enzymes in this subfamily.
The sequence identities of the catalytic domain of AmyL
to Geobacillus stearothermophilus neopullulanase,
Thermoanaerobacter pseudethanolicus cyclodextrinase,
Thermoactinomyces vulgaris R-47 ꢀ-amylase I (TVAI),
T. vulgaris R-47 ꢀ-amylase II (TVAII), and Bacillus
acidpullulyticus maltogenic amylase were 37%, 37%,
33%, 36%, and 37%, respectively.
raphy was performed in 10 mM Tris–HCl buffer (pH 8.5) containing
100 mM KCl at 0.5 mL/min. The eluted protein was detected by the
absorbance at 280 nm. A set of gel filtration standards (Bio-Rad) was
used to calibrate the molecular mass.
Analysis of the action pattern of recombinant AmyL on p-nitro-
phenyl ꢀ-maltopentaoside (pNPG5). One mL of a reaction mixture
consisting of 5.6 nM recombinant AmyL, 1 mM pNPG5 (Calbiochem,
Bad Soden, Germany), and 20 mM Tris–HCl buffer (pH 9.5) was
incubated at 50 ꢀC. At 2, 5, 10, and 15 min, 100 mL of the reaction
mixture was removed and mixed with 10 mL of 5 M acetic acid to
terminate the reaction. The reaction products were analyzed by HPLC
under the following conditions: injection volume, 10 mL; column,
YMC C18 Pack Pro (ꢂ6:0 mm ꢁ 250 mm, YMC, Kyoto, Japan);
column temperature, 40 ꢀC; eluent, 10% acetonitrile; flow rate,
1 mL/min; detection, absorbance at 313 nm.
Preparation of W45A, W61A, and W84A AmyL mutants. Site-
directed mutagenesis was carried out with a Primestar Mutagenesis
Basal Kit (Takara Bio). The expression plasmid for wild-type AmyL
was used as template. The sequences of the primers used in the PCRs
were as follows: for W45A, 50-CTTGACGCGAGTTCAAACAAT-
CATCCA-30 (sense, nucleotides mutated in boldface) and 50-TGAA-
CTCGCGTCAAGGGGGGCCAAGCT-30 (antisense); for W61A,
50-GGAACTGCGAAAAGCAATCCCATCCCT-30 (sense) and 50-
GCTTTTCGCAGTTCCATCTGACTCATC-30 (antisense); for W84A,
50-GGGCAAGCGCTGCCGGGAGAGAATCTC-30 (sense) and 50-
CGGCAGCGCTTGCCCATCCATCACATA-30
(antisense). The
expression plasmid of each of the mutant enzymes was introduced
into E. coli BL21 (DE3), and the enzymes were prepared in the same
fashion as the wild type.
Results
Cloning and sequence analysis of AmyL
In contrast to neopullulanase-like ꢀ-amylases, the
N-terminal region of AmyL (Gln29-Phe102) showed
similarity to Carbohydrate-Binding Module family 20
(CBM20, PF00686), involved in binding to starch
granules.22) CBM20 is formed by a ꢁ-sandwich fold,
and most CBM20s have two sugar-binding sites, site 1
and site 2. Two Trp residues, Trp45 and Trp84, of
AmyL corresponded to the Trp residues conserved in
site 1 of CBM20, which formed a carbohydrate-binding
platform (Fig. 2). Lys and Asn, forming direct hydrogen
bonding interactions,23) were also found at the equiv-
alent positions of AmyL (Lys77 and Asn89). Trp61 of
AmyL corresponded to the Trp residue conserved in
site 2. Other amino acid residues responsible for binding
to the carbohydrate in site 2 were not easily predicted,
because this site is structurally more diverse than site 1.
When the sequence of AmyL was compared with the
starch-binding domain of Aspergillus niger glucoamy-
lase (GA SBD), the best characterized enzyme in CBM
family 20,23) no other amino acid residues in site 2 that
contribute to the interaction with the carbohydrate were
found at the corresponding positions of AmyL.
The N-terminal and partial internal amino acid
sequences of AmyL purified from the culture super-
natant of Bacillus sp. AAH-31 were analyzed. The
N-terminal sequence of AmyL was QDYENIVLRG-
SLAPLDW, and the sequences of the peptides derived
from AmyL by Asn-N endoprotease digestion were P1,
DWAK; P2, DEQYVE; and P3, DWSSNNHPLTK.
From PCR using primers, based on the sequence of the
N-terminal and P2 and genomic DNA as template, a
488 bp DNA fragment was obtained (Fig. 1). The
sequence of P3 was confirmed in the deduced amino
acid sequence of the DNA fragment. To obtain the
regions upstream and downstream of the DNA fragment
obtained, inverse PCRs were carried out successively,
and a 3,533-bp portion of the DNA sequence, including
the full-length of AmyL (2,466 bp) encoding 821 amino
acid residues, was determined. The nucleotide sequence
has been deposited in the DDBJ database under
accession no. AB775769 (Fig. 1). No ATG codon was
found in the upstream region encoding the N-terminal
amino acid sequence of AmyL. The TTG codon found
7 bp downstream of a putative ribosome binding site
(50-AGGAGG-30) with good complementarity to the 30-
terminal of Bacillus subtilis 16S rRNA18) was regarded
as the initiation codon of the AmyL gene. There were
putative ꢃ35 and ꢃ10 regions, sequences of which are
50-TTGTCA-30 and 50-TATAAT-30, respectively, up-
stream of the ribosome binding site, and these regions
were separated by 18 bp. An inverted-repeat sequence
was found 6 bp downstream of the stop codon.
Pro234-Thr301 and Tyr714-Gly780 were assigned
to the chitobiose/ꢁ-hexosaminidase C-terminal domain
(PF13290) and
a domain of unknown function
(DUF3459, PF11941), respectively, and their functions
are not yet understood.
Production and characterization of recombinant
AmyL
Recombinant AmyL with a His-tag at the C-terminal
(rAmyL) was produced in E. coli BL21 (DE3), and
purified to homogeneity by Ni-chelating column chro-
matography. Sixteen mg of the purified enzyme, with a
Twenty-eight amino acid residues at the N-terminal,
which were not found in the native enzyme, are
predicted to be a signal peptide by the Signal-P 4.0

1870
W. SABURI et al.
AAGCTTGGACGG@-676
CCGGCTTAAaAGCGCTGGGTATCAATGATCCTGACTATGGCCACTATCTGGCAGAACGCTTTCCTGCTGAACGGC@-601
GCAAACGTTCTATCGTTTATGATGAAACTTTTGAGGTGCTGGATGAACTCAAAGAAAATTACAGATTACTGCTTT@-526
TAACCAACGGCTCACCTGACTTGCAACGGGAGAAACTTTCCACCCTTCCCGACCTGACACCATACTTTGAACATA@-451
TAGTCATTTCCGGTGATTTCGGTAAAGGAAAGCCTGATCCGTCTATCTTTGAACATGCGCTGAACTTGATGGCTC@-376
TAGTTCCTGATGAAGCCATCATGGTAGGTGATAACTTGATGACGGATATCCTCGGTTCCTCACGGGCAGGCATGA@-301
AAAATGTGTGGATCAACAGGAATGGAAAACCCCCTCATCCGGAAGTGATTCCAGACTTTGAGATCAAAAGTTTAA@-226
CCGAACTTCCGCCACTGGTCCAAAAGTTGTCGTAATGATGTTAGAAAGATGTTAGAAAACTGAAAGCGGCTATCA@-151
AAGAGGATGGCTGCTTTTTTATTTGGATTTTGTTCAAACAAAGTTTATTTTTCAAAATTTTTATATCCAAATTCC@-76
TTGTCAACATTCTCCTTGATCAAGTATAATTAATGCAATCGGTTTCATAAAAAATTAAGACAAGGAGGTTTAGAA@-1
-35
-10
RBS
TTGAAGAAATATTTATCTATTATCTTATCACTTTCCTTTGTTCTATCTTTTTTTGTGGTACCGTTTGCACAAAAT@75
@L@@K@@K@@Y@@L@@S@@I@@I@@L@@S@@L@@S@@F@@V@@L@@S@@F@@F@@V@@V@@P@@F@@A@@Q@@N@@
1
ACGGAGGCTCAGGATTATGAGAACATTGTCCTAAGAGGTAGCTTGGCCCCCCTTGACTGGAGTTCAAACAATCAT@150
S
26 @T@@E@@A@@Q@@D@@Y@@E@@N@@I@@V@@L@@R@@G@@S@@L@@A@@P@@L@@D@@W@@S@@S@@N@@N@@H@@
CCATTAACAAAAGATGAGTCAGATGGAACTTGGAAAAGCAATCCCATCCCTCTTCCAGGAGGACAACGCCTGGAA@225
HS2
51 @P@@L@@T@@K@@D@@E@@S@@D@@G@@T@@W@@K@@S@@N@@P@@I@@P@@L@@P@@G@@G@@Q@@R@@L@@E@@
TTCAAATATGTGATGGATGGGCAATGGCTGCCGGGAGAGAATCTCGTCTTTGATATTCCCCAAACCGGCAACTAC@300
76 @F@@K@@Y@@V@@M@@D@@G@@Q@@W@@L@@P@@G@@E@@N@@L@@V@@F@@D@@I@@P@@Q@@T@@G@@N@@Y@@
ATCTTTATCTTTCACCCGGACAACCAGCGTAAAGTGGATGTCATTCTGGTTGAAGCTGATGGTAAAGTCACGCTG@375
101 @I@@F@@I@@F@@H@@P@@D@@N@@Q@@R@@K@@V@@D@@V@@I@@L@@V@@E@@A@@D@@G@@K@@V@@T@@L@@
CTCTTGACCGTGCCGGACAACACACCGTCACGCATTGTGCCCACCATCGGCTCCAGTTTGAACAACTTTAATTAT@450
HS1
126 @L@@L@@T@@V@@P@@D@@N@@T@@P@@S@@R@@I@@V@@P@@T@@I@@G@@S@@S@@L@@N@@N@@F@@N@@Y@@
TCCGTGACAAAGCTTTCCAAAGTAGAAAGTGCCGAGAACCAGTGGCAGATTGAGCTTTCCGGTGAACCAGGGCAG@525
HR2
HS2
151 @S@@V@@T@@K@@L@@S@@K@@V@@E@@S@@A@@E@@N@@Q@@W@@Q@@I@@E@@L@@S@@G@@E@@P@@G@@Q@@
GAGTTTTCATATCTTTATGCCCTGGGGGATGAACAGTACGTAGAAGACAGAGATGCCCCCCGTACAGCCACTTTC@600
R
176 @E@@F@@S@@Y@@L@@Y@@A@@L@@G@@D@@E@@Q@@Y@@V@@E@@D@@R@@D@@A@@P@@R@@T@@A@@T@@F@@
TTAGAAGATCATCTCGTTATTGAAGATGTGGTAGAATCCTGGAAAGCTGTGCCCATTGCCAAAAATGTGTCTCAC@675
201 @L@@E@@D@@H@@L@@V@@I@@E@@D@@V@@V@@E@@S@@W@@K@@A@@V@@P@@I@@A@@K@@N@@V@@S@@H@@
AATTTTAATCATGAGCCCTTTATCCCCGGTTCGCAAGACGACGTGAACATCACTGTTCATGTTGATCATTACGGC@750
226 @N@@F@@N@@H@@E@@P@@F@@I@@P@@G@@S@@Q@@D@@D@@V@@N@@I@@T@@V@@H@@V@@D@@H@@Y@@G@@
ACAGTAGATTCAGGGGCCATTTACTTTACCACTGACGGCTCCTCGCCGCTTGGAAAAAGAGGCGATGCCGCCAAT@825
251 @T@@V@@D@@S@@G@@A@@I@@Y@@F@@T@@T@@D@@G@@S@@S@@P@@L@@G@@K@@R@@G@@D@@A@@A@@N@@
GGCAATGTGGTGCCGTTACAAGTGACATCCTCCTCTGAAAACAGTGACGGAACCATTCGTTCTGTGTTGACGGGA@900
276 @G@@N@@V@@V@@P@@L@@Q@@V@@T@@S@@S@@S@@E@@N@@S@@D@@G@@T@@I@@R@@S@@V@@L@@T@@G@@
ACAATTCCCAAGCAGCCCGATTATACACCGGTGAAGTACCGCATAGATGTCTGGGACAGTCAAAGTAACGATGAC@975
301 @T@@I@@P@@K@@Q@@P@@D@@Y@@T@@P@@V@@K@@Y@@R@@I@@D@@V@@W@@D@@S@@Q@@S@@N@@D@@D@@
CACTCCCAATTTGCTGACAATAACAGCCTTGTGCCCGAACAGGCCACTGAGTTTGCTTACTACGTGGAAGAATTT@1050
326 @H@@S@@Q@@F@@A@@D@@N@@N@@S@@L@@V@@P@@E@@Q@@A@@T@@E@@F@@A@@Y@@Y@@V@@E@@E@@F@@
AAATCTCCAGACTGGGCCAAAGATGCGGTGATCTATCACATCTTTGTGGATCGTTTTAAAGACGGCGATCCATCC@1125
351 @K@@S@@P@@D@@W@@A@@K@@D@@A@@V@@I@@Y@@H@@I@@F@@V@@D@@R@@F@@K@@D@@G@@D@@P@@S@@
AATAACGAACCTGTCGATGAATCGCTTCCCTATGCAGAACGATTGAAAGGCTGGATGGGCGGAGACCTGGAAGGT@1200
376 @N@@N@@E@@P@@V@@D@@E@@S@@L@@P@@Y@@A@@E@@R@@L@@K@@G@@W@@M@@G@@G@@D@@L@@E@@G@@
GTCAAACAGAAGCTGGACTATTTGGAGGAACTGGGCGTTAACGTCATTTGGCTCTCTCCCGTATTTGAAGGCCCA@1275
401 @V@@K@@Q@@K@@L@@D@@Y@@L@@E@@E@@L@@G@@V@@N@@V@@I@@W@@L@@S@@P@@V@@F@@E@@G@@P@@
TACTCGCATGGCTACCATCCCACAGACTTTAAACAAGTGGATCCTCGCTTCGGAGACAAGGAACTGTTGCAATCT@1350
426 @Y@@S@@H@@G@@Y@@H@@P@@T@@D@@F@@K@@Q@@V@@D@@P@@R@@F@@G@@D@@K@@E@@L@@L@@Q@@S@@
CTGATTGAGGAAGCCCATCAACGGGACATGAAAGTGATTTATGACTTTGTCCCAAATCATTCATCTAACCGGCAT@1425
451 @L@@I@@E@@E@@A@@H@@Q@@R@@D@@M@@K@@V@@I@@Y@@D@@F@@V@@P@@N@@H@@S@@S@@N@@R@@H@@
CCGTTCTTTCAAGACGCTTTCAACCGCGGGAAGGACAGCCCTTACTATTACTGGTACACCTTTACCAACTGGCCT@1500
476 @P@@F@@F@@Q@@D@@A@@F@@N@@R@@G@@K@@D@@S@@P@@Y@@Y@@Y@@W@@Y@@T@@F@@T@@N@@W@@P@@
TACGAATATAAGACATTTTATGGAATCGATGAACTTCCCCAATTAAACAATGATTATCCCGAAACCCGTGAATAT@1575
501 @Y@@E@@Y@@K@@T@@F@@Y@@G@@I@@D@@E@@L@@P@@Q@@L@@N@@N@@D@@Y@@P@@E@@T@@R@@E@@Y@@
ATGCTGTATGATGTGGTGCCCTTTTGGTTGTTAGAGTTGGACTTTGACGGATTCAGATTAGACTATGCCAAAGGC@1650
526 @M@@L@@Y@@D@@V@@V@@P@@F@@W@@L@@L@@E@@L@@D@@F@@D@@G@@F@@R@@L@@D@@Y@@A@@K@@G@@
CCAAGCTACAGTTTCTGGGTAGACTTCCGTCACGCCGTCAAAAAAATGAAGCCAGATGCCTTTATATTCGGCGAA@1725
551 @P@@S@@Y@@S@@F@@W@@V@@D@@F@@R@@H@@A@@V@@K@@K@@M@@K@@P@@D@@A@@F@@I@@F@@G@@E@@
ATTTGGGACAACCGGGACAAGATTAATTCCTATGCCGGCAAGCTGGATGGGGCTTTGGATTTCCCGCTGCAAAGC@1800
576 @I@@W@@D@@N@@R@@D@@K@@I@@N@@S@@Y@@A@@G@@K@@L@@D@@G@@A@@L@@D@@F@@P@@L@@Q@@S@@
GCCTTAGTGGACACATTTGCCTATAATCACCCAATGTCCAGGGTGGCCAATACGATTAGAGACAATCTGAACACT@1875
601 @A@@L@@V@@D@@T@@F@@A@@Y@@N@@H@@P@@M@@S@@R@@V@@A@@N@@T@@I@@R@@D@@N@@L@@N@@T@@
TACCATCCTGAATACATGATGGTGACGTTCCTGGATAACCATGATCTCCCCCGCTTCCTATTCCAGTCCAGAGGG@1950
626 @Y@@H@@P@@E@@Y@@M@@M@@V@@T@@F@@L@@D@@N@@H@@D@@L@@P@@R@@F@@L@@F@@Q@@S@@R@@G@@
GATAGTGCCAAACTGAAACTGGCCGCAACAGCCCAGTTTACCTTACCGGGGATCCCGGCCATCTATTACGGAACA@2025
651 @D@@S@@A@@K@@L@@K@@L@@A@@A@@T@@A@@Q@@F@@T@@L@@P@@G@@I@@P@@A@@I@@Y@@Y@@G@@T@@
GAGATAGGCTTGTCCCAAAGTGAGGACCATAATCAATACACAGATTGGAGAGACCGGTGGTTCCGTGAAATGATG@2100
BS
BR
676 @E@@I@@G@@L@@S@@Q@@S@@E@@D@@H@@N@@Q@@Y@@T@@D@@W@@R@@D@@R@@W@@F@@R@@E@@M@@M@@
CCTTGGGATGAAAAGCAACAGGACTTGAAATTGAAAGCTTACTACAAACAGCTTATCGATCTCAGACACCGGGAA@2175
701 @P@@W@@D@@E@@K@@Q@@Q@@D@@L@@K@@L@@K@@A@@Y@@Y@@K@@Q@@L@@I@@D@@L@@R@@H@@R@@E@@
GAAGCCTTCAAAACAGGAGAGTACAATGAACTGTACGTTGATCAAGACCTGTTTGTCTTTGAACGTAAACACAAA@2250
726 @E@@A@@F@@K@@T@@G@@E@@Y@@N@@E@@L@@Y@@V@@D@@Q@@D@@L@@F@@V@@F@@E@@R@@K@@H@@K@@
AACAAACACTTCATTGTCATCATCAACAAAGGCAACACCACACAAGAGGTGGCTATTGACGAGTTGCTTAACTTA@2325
751 @N@@K@@H@@F@@I@@V@@I@@I@@N@@K@@G@@N@@T@@T@@Q@@E@@V@@A@@I@@D@@E@@L@@L@@N@@L@@
AGACGTACCAACGGCATTTCCCTTCAAGACGCCCTGAACAAAGGGAAAGTGATCAAAGCAAATCGCCAGGGAGAA@2400
776 @R@@R@@T@@N@@G@@I@@S@@L@@Q@@D@@A@@L@@N@@K@@G@@K@@V@@I@@K@@A@@N@@R@@Q@@G@@E@@
CTTAAAGTGAAGAGTTCTCCAGCTTCAGCCACCATTTTACGGGTCCTGGGCAACATTAACAAATAATTTGGGAAC@2475
801 @L@@K@@V@@K@@S@@S@@P@@A@@S@@A@@T@@I@@L@@R@@V@@L@@G@@N@@I@@N@@K@@*@@@@@@@@@@@
CTCCCATGTACCTCTAAAAACATGGGAGGTTTTTTGAGCTTGGAAATCCATGAACTTAACACCAACCATAGTGAA@2550
TATCATGGATATGTGAGTGATCCTATAAACATTTGCTGGGCAAAATTCCCTCAGGGGTGATCGAAAGTGCGAGAT@2625
GTATCTCATGTTGCTTGGGCTGATATCAAAGGAAGTCCTCTTGCTCCAAACATCAAAAGGAGTGTCATCTTTAAA@2700
TCGGTTCCGGGAGGCACCAATGTGTCCGTTGAAGTGTCCGGATTACCCCCTTATCGTCCAGGAGGAAAAAAACGT@2775
CCCCCCATTGGCCCCCATGGATTCCACATCCATGAATTTGGAACATGTGAAATCGGAAATCCTAAGGATCC@@@@@2846
Fig. 1. DNA and Amino Acid Sequences of AmyL.
Numbers of amino acids and nucleotides are shown to the left and right, respectively. Asterisk indicates the stop codon. The N-terminal and
partial internal amino acid sequences of AmyL purified from the culture supernatant of Bacillus sp. AAH-31 are shown in bold face. The
ribosome binding site (RBS), inverted repeat, and ꢃ35 and ꢃ10 regions are underlined. HindIII and BamHI sites are boxed. Conserved regions
of GH family 13 enzymes are indicated by double underlining. Arrows indicate primers used to obtain AmyL.
specific activity of 60.9 U/mg, was obtained from a
300-mL culture. The molecular mass of rAmyL was
estimated to be 107 kDa, close to the value obtained by
SDS–PAGE (91 kDa), by gel filtration column chroma-
tography, indicating that rAmyL is a monomer in
solution. rAmyL retained more than 90% of its maximum
activity in a broad pH range, 7.8–10.5, as observed for
the native enzyme (Fig. 3).5) At high pH values, rAmy
was less active than the native enzyme. It retained more
than 90% of its original activity below 65 ꢀC (for 15 min
at pH 9.5) and from pH 6.4 to 11.9 (for 24 h at 4 ꢀC). At
70 ꢀC and higher temperatures, rAmy was more rapidly
inactivated than the native enzyme. The native enzyme is
stable in a pH range of 6.4–10.3, and thus rAmy was
more stable at alkaline pH values.
The kcat values of rAmyL for soluble starch, ꢃ-CD,
and maltotriose were 103 ꢂ 9 sꢃ1, 67:6 ꢂ 2:0 sꢃ1, and
5:33 ꢂ 0:43 sꢃ1, respectively, and the Km values were
0:100 ꢂ 0:028 mg/mL, 0:348 ꢂ 0:019 mM, and 2:06 ꢂ
0:33 mM, respectively.
To determine the predominant productive binding
mode, the product distribution from the hydrolysis of
pNPG5 was analyzed by measuring the releasing
velocity of each aglycone having a p-nitrophenyl group.
The releasing velocity of p-nitrophenyl ꢀ-glucoside was
the most prominent at 53% of the entire reaction

Primary Structure of Bacillus sp. AAH-31 ꢀ-Amylase
1871
AmyL
AnGA
BcBA
29: -------------------QDYENIVLRGSLAPL-DWSSN-NHPLTKDES------DGT
543: ----------FDLTATT--TYGENIYLVGSISQLGDWETSDGIALSADKY---TSSDPL
448: ----TPVMQTIVVKNVP-TTIGDTVYITGNRAELGSWDTK-QYPIQLYYD----SHSND
Bc251CGT 612: -----QVSVRFVVNNAT-TALGQNVYLTGSVSELGNWDP--AKAIGPMYN-QVVYQYPN
Bc8CGT
GsCGT
TtCGT
617: ----DQVTVRFVVNNAS-TTLGQNLYLTGNVAELGNWSTG-STAIGPAFN-QVIHQYPT
609: ----DQVSVRFVVNNAT-TNLGQNIYIVGNVYELGNWDT--SKAIGPMFN-QVVYSYPT
609: ----NQICVRFVVNNAS-TVYGENVYLTGNVAELGNWDT--SKAIGPMFN-QVVYQYPT
613: ----TQTSVVFTVKSAPPTNLGDKIYLTGNIPELGNWSTDTSGAVNNAQGPLLAPNYPD
445: GEPGALVSVSFRCDNGA-TQMGDSVYAVGNVSQLGNWSPAAALRLTDTSG------YPT
Novamyl
G4-Amy
AmyL
AnGA
BcBA
61: WKSNPIPLPGGQRLEFKYVM----DGQ----WLPGENLVFDIPQTG-----NYIFIFH-
587: WYVT-VTLPAGESFEYKFIR---IESDDSVEWESDPNREYTVPQACGTSTATVTDTW--
499: WRGN-VVLPAERNIEFKAFIK--SKDGTVKSWQTIQQSWNPVPLKT----TSHTSSW--
Bc251CGT 662: WYYD-VSVPAGKTIEFKFLK---KQGS-TVTWEGGSNHTFTAPSSG---TATINVNW--
Bc8CGT
GsCGT
TtCGT
669: WYYD-VSVPAGKQLEFKFFK---KNGS-TITWESGSNHTFTTPASG---TATVTVNW--
660: WYID-VSVPEGKTIEFKFIK---KDSQGNVTWESGSNHVYTTPTNT---TGKIIVDW--
660: WYYD-VSVPAGTTIQFKFIK---KNGN-TITWEGGSNHTYTVPSSS---TGTVIVNW--
668: WFYV-FSVPAGKTIQFKFFI---KRADGTIQWENGSNHVATTPTGA---TGNITVTWQN
497: WKGS-IALPAGQNEEWKCLIRNEANATQVRQWQGGANNSLTPSEGA---TTVGRL----
Novamyl
G4-Amy
Fig. 2. Comparison of the N-Terminal Sequence of AmyL with CBM20.
The amino acid sequences of the N-terminal region of AmyL and CBM20s, the three-dimensional structures of which are known, were aligned
with the Clustal W program (http://clustalw.ddbj.nig.ac.jp/). Amino acid residues conserved in sites 1 and 2 are shown in boldface, and the circle
and triangle indicate site 1 and site 2, respectively. AnGA, A. niger glucoamylase (Genbank ID: CAK38411.1); BcBA, B. cereus ꢁ-amylase
(BAA34650.1); Bc251CGT, B. circulans 251 cyclodextrin glucanotransferase (CGTase, CAA55023.1); Bc8CGT, B. circulans 8 CGTase
(CAA48401.1); GsCGT, G. stearothermophilus NO. 2 CGTase (CAA41770.1); TtCGT, Thermoanaerobacterium thermosulfurigenes EM1
CGTase (AAB00845.1); Novamyl, G. stearothermophilus C599 maltogenic ꢀ-amylase (ABA38666.1); G4-Amy, Pseudomonas stutzeri MO-19
maltotetraose-forming ꢀ-amylase (AAA25707.1).
velocity (the sum of the reaction velocities of the
aglycone-releasing), followed by p-nitrophenyl ꢀ-malto-
side (34%). This result indicates that pNPG5 is hydro-
lyzed mainly through two productive binding modes
covering subsites ꢃ4 to þ2 and subsites ꢃ3 to þ3.
A binding assay using raw cornstarch was carried out
because a CBM20-like region was identified in the
deduced amino acid sequence of AmyL. No decrease in
enzyme activity of the supernatant was observed after
incubation of 0.94–9.4 mM AmyL at 4 ꢀC for 1 h with
gentle shaking in the presence of 100 mg/mL of starch
granules (data not shown), and thus AmyL did not
adsorb onto the starch granules.
AmyL, and the function of the N-terminal domain. The
amino acid sequence of the central region of AmyL
showed high similarity to the catalytic domains of
neopullulanase-like ꢀ-amylases of GH family 13 sub-
family 20 (Fig. 1). The calcium ion bound to the
conserved binding site of typical ꢀ-amylases is not
observed in neopullulanase-like ꢀ-amylases.24) Similarly
to neopullulanase-like ꢀ-amylases, AmyL also presum-
ably does not have a calcium ion bound to the conserved
binding site. The absence of a conserved calcium site in
AmyL is thought to be the reason this enzyme showed
high tolerance to chelating reagents. The amino acid
residues responsible for thermostability and high resist-
ance at alkaline pH were not easily predicted from the
amino acid sequence of AmyL. Pro551 and Pro642,
which were not found at the corresponding positions of
the related enzymes, were predicted to be situated at the
loops connecting the 4th ꢁ-strand and the 4th ꢀ-helix,
and the 7th ꢁ-strand and the 7th ꢀ-helix, respectively.
These residues might be important for high thermo-
stability, because Pro residues situated at loops con-
necting secondary structures were postulated to be
important for high thermostability.25)
In contrast to AmyL, which is a monomer in solution,
many neopullulanase-like ꢀ-amylases are homodimers.
The N-terminal domain of neopullulanase-like ꢀ-amy-
lase is involved in the formation of the active site
together with domain A of the other subunit.26–28) The
active-site clefts of dimeric neopullulanase-like ꢀ-
amylases are narrow and deep, suitable for binding
CDs.26) The N-terminal domain of TVAI, also a
monomer similar to AmyL, interacts strongly with
domains A and B, unlike other neopullulanase-like ꢀ-
amylases, to create the globular structure.24) These
structural differences are responsible for the variations
in substrate specificity. TVAI has higher activities
towards starch and pullulan, and lower activities towards
ꢀ- and ꢁ-CDs than TVAII (a typical neopullulanase-like
ꢀ-amylase).29,30) AmyL shows high activity towards
starch and ꢃ-CD, and low activity towards ꢀ- and ꢁ-
Site-directed mutagenesis to three conserved Trp
residues in the N-terminal CBM20-like sequence of
AmyL
To confirm the function of the CBM-like region in the
process of binding to soluble substrates, three conserved
Trp residues (Trp45 and Trp84 from site 1 and Trp61
from site 2) were replaced with Ala. All the mutant
enzymes were prepared as for wild-type AmyL, and the
kinetic parameters for soluble starch, ꢃ-CD, and malto-
triose were measured (Table 1). The kinetic parameters
for ꢃ-CD were only minimally impacted by the
mutations. Using maltotriose as substrate, W45A and
W84A showed 1.7 to 1.9-fold lower Km values, and
W61A had a Km similar to the wild type. In contrast to
the reactions with oligosaccharides, W45A and W84A
showed 2.2 to 2.4-fold lower kcat values and 2.5-fold
higher Km values for soluble starch than the wild type. In
contrast to the other two mutated AmyLs, W61A had a
0.8-fold and a 2.3-fold lower kcat and Km for soluble
starch, respectively, resulting in a 1.9-fold higher
kcat=Km than the wild type.
Discussion
In this study, we analyzed the primary structure of
AmyL, the enzymatic characteristics of recombinant

1872
W. SABURI et al.
Table 1. Comparison of Kinetic Parameters between Trp Mutated AmyL and the Wild Type
Soluble starch
ꢃ-CD
Maltotriose
Enzyme
kcat
(sꢃ1
Km
(mgꢄmLꢃ1
kcat=Km
(sꢃ1ꢄmgꢃ1ꢄmL)
kcat
(sꢃ1
Km
kcat=Km
kcat
(sꢃ1
Km
kcat=Km
ꢃ1
ꢃ1
)
)
)
(mM)
(sꢃ1ꢄmM
)
)
(mM)
(sꢃ1ꢄmM
)
Wild type 103 ꢂ 9
0:100 ꢂ 0:028
0:251 ꢂ 0:028
1030
171
67:6 ꢂ 2:0 0:348 ꢂ 0:019
76:9 ꢂ 6:0 0:503 ꢂ 0:032
69:5 ꢂ 2:2 0:353 ꢂ 0:039
73:0 ꢂ 7:3 0:381 ꢂ 0:038
194
153
197
192
5:33 ꢂ 0:43 2:06 ꢂ 0:33
4:26 ꢂ 0:20 1:21 ꢂ 0:18
4:71 ꢂ 0:32 1:82 ꢂ 0:44
4:05 ꢂ 0:10 1:10 ꢂ 0:09
2.59
3.52
2.59
3.68
W45A
W61A
W84A
42:9 ꢂ 4:1
84:6 ꢂ 7:0 0:0439 ꢂ 0:0151
1930
192
47:7 ꢂ 6:8
0:248 ꢂ 0:026
Data are mean ꢂ standard deviation for three independent experiments.
to dimeric neopullulanase-like ꢀ-amylases.31) It shows
hydrolytic activity towards raw starch, and hence
N-terminal CBM34 is considered to behave like a
starch-binding domain. The N-terminal region of AmyL
showed similarity to the portion of CBM20 involved in
binding to starch granules. No adsorption of AmyL to
starch granules was observed, but our site-directed
mutational study revealed that the two Trp residues
(Trp45 and Trp84) conserved in site 1 of CBM20
contribute to binding to a soluble long-chain substrate
(Table 1), suggesting that the CBM20-like region of
AmyL plays a role as substrate-binding domain. Both
AmyL and TVAI have N-terminal sugar-binding
domains followed by the catalytic domains, but AmyL
has a substrate-binding domain structurally different
from that of TVAI. To our knowledge, there is no report
that enzymes belonging to GH family 13 subfamily20
have N-terminal CBM20, and hence AmyL is an
enzyme having a novel domain organization. The
W61A AmyL mutant showed higher catalytic efficiency
for the hydrolysis of soluble starch than the wild type,
and thus Trp61 of AmyL, corresponding to the con-
served Trp residue in site 2 of CBM20, is not thought to
be involved in sugar binding. This mutant enzyme
showed a lower Km value for soluble starch than the wild
type, while its kcat value was similar to that of the wild
type, suggesting that Trp61 disturbs the formation of
the Michaelis complex. AmyL does not have a Tyr
residue at the position corresponding to Tyr556 of
GA SBD, which interacts mostly with carbohydrates,23)
and site 2 of AmyL presumably does not act as a
sugar-binding site. In the complex structure of Bacillus
cereus ꢁ-amylase and maltose, maltose was observed to
be bound only at site 1 of CBM20 at the C-terminal of
the enzyme,32) and hence site 2 of CBM20 of this
enzyme does not appear to possess binding affinity for
carbohydrates.
a
100
80
60
40
20
0
5
7
9
11
13
pH
b
100
80
60
40
20
0
3
5
7
9
11
13
pH
c
100
80
60
40
20
0
20
40
60
80
Temperature (°C)
Neopullulanase-like ꢀ-amylases hydrolyze pullulan,
producing panose, but AmyL shows no hydrolytic
activity towards this polysaccharide.5) This is an
essential difference in substrate specificity between
AmyL and neopullulanase-like ꢀ-amylases. On compar-
ison of the amino acid sequences of AmyL and
neopullulanase-like ꢀ-amylases for which the three-
dimensional structures have been solved, the loop
connecting the 8th ꢁ-strand and the 8th ꢀ-helix in
AmyL is longer than that in neopullulanase-like ꢀ-
amylases, in which the conserved Asp on this loop is
involved in the formation of subsite ꢃ2 (Fig. 4).31) In
neopullulanase-like ꢀ-amylases, short loop connecting
the 8th ꢁ-strand and the 8th ꢀ-helix appears to make
space for binding to pullulan, and thus this long loop of
Fig. 3. Comparison of Physicochemical Properties between Native
and Recombinant AmyL.
The pH activity curve (a), pH stability (b), and thermal stability
(c) of the native ( ) and recombinant ( ) AmyL were compared.
Data for the native enzyme are from reference 5.
CDs,5) similarly to TVAI. Although the N-terminal
region of AmyL does not show any similarity to that of
TVAI, AmyL is also a monomeric enzyme like TVAI,
and the active-site cleft of AmyL might be shallow and
more suitable for binding to starch, as was observed for
TVAI.
TVAI has CBM34, which contains two maltooligo-
saccharide binding sites, at the N-terminal, in contrast

Primary Structure of Bacillus sp. AAH-31 ꢀ-Amylase
β-strand 8
1873
α-strand 8
AmyL 668: IPAIYYGTEIGLSQSEDHNQYTDWRDRWFREMMPWDEKQQDLKLKAYYKQLIDLRHRE
TVA1 529: TPTIYYGDEYGMQGGADP---------DNRRSFDWSQATPSNSAVALTQKLITIRNQY
TVA2 449: TPLIYYGDEIGMAGATDP---------DCRRPMIWEEKEQNRGLFEFYKELIRLRHRL
NPU 452: SPCIYYGDEIGMTGGNDP---------ECRKCMVWDPMQQNKELHQHVKQLIALRKQY
TMA 452: SPCIYYGDEIGMTGGNDP---------ECRKCMVWDPEKQNKELYEHVKQLIALRKQY
BCD 449: TPCIYYGDEVGLDGGHDP---------GCRKCMEWDETKHDKDLFAFYQTVIRLRQAH
Fig. 4. Comparison of Partial Amino Acid Sequences between ꢁ-Strand 8 and ꢀ-Helix 8 of AmyL and Related Enzymes.
The amino acid sequences of AmyL and related enzymes were aligned with the Clustal W program. Amino acid residues completely
conserved in the enzymes are shown in bold face. TVAI, T. vulgaris R-47 ꢀ-amylase I (Genbank ID: BAA02471.1); TVAII, T. vulgaris ꢀ-
amylase II (BAA02473.1); NPU, G. stearothermophilus neopullulanase (AAA22622.1); TMA, Thermus sp. IM6501 (AAC15072.1); BCD,
Bacillus sp. cyclomaltodextrinase (AAA92925.1).
11) Murakami S, Nishimoto H, Toyama Y, Shimamoto E, Takenaka
S, Kaulpiboon J, Prousoontorn M, Limpaseni T, Pongsawasdi P,
AmyL might result in steric hindrance that disturbs the
process of binding to pullulan.
In this study, we found that AmyL has a catalytic
domain similar to neopullulanase-like ꢀ-amylases, al-
though it has no hydrolytic activity towards pullulan. In
and Aoki K, Biosci. Biotechnol. Biochem., 71, 2393–2401
(2007).
12) Shirokizawa O, Akiba T, and Horikoshi K, FEMS Microbiol.
Lett., 58, 131–135 (1990).
addition, we found that the N-terminal CBM20-like
domain contributed to the process of binding to soluble
long-chain substrates. Mutational analysis revealed that
two Trp residues conserved at site 1 of CBM20 were
important for high activity towards soluble long-chain
substrates. Structural insight into the substrate binding
mode and specificity should be pursued through the
three-dimensional structure analysis of AmyL.
13) Pluskal MG, Przekop MB, Kavonian MR, Vecoli C, and Hicks
DA, BioTechniques, 4, 272–283 (1986).
14) Moore S and Stein WH, J. Biol. Chem., 176, 367–388 (1948).
15) Miller GL, Anal. Chem., 31, 426–428 (1959).
16) McFeeters RF, Anal. Biochem., 103, 302–306 (1980).
17) Miwa I, Okuda J, Maeda K, and Okuda G, Clin. Chim. Acta, 37,
538–540 (1972).
18) Murray CL and Rabinowitz JC, J. Biol. Chem., 257, 1053–1062
(1982).
19) Petersen TN, Brunak S, von Heijne G, and Nielsen H, Nat.
Methods, 8, 785–786 (2011).
Acknowledgments
20) Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell
C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L,
Sonnhammer EL, Eddy SR, Bateman A, and Finn RD, Ncleic
Acids Res., 40, D290–D301 (2012).
We thank Mr. Tomohiro Hirose of the Instrumental
Analysis Division, Equipment Management Center,
Creative Research Institution, Hokkaido University for
amino acid analysis.
21) Uitdehaag JCM, Mosi R, Kalk KH, van der Veen BA,
Dikhuizen L, Withers SG, and Dijkstra BW, Nat. Struct. Biol.,
6, 432–436 (1999).
22) Christiansen C, Abou Hachem M, Janecek S, Viksø-Nielsen A,
Blennow A, and Svensson B, FEBS J., 276, 5006–5029 (2009).
References
1) Ito S, Kobayashi T, Ara K, Ozaki K, Kawai S, and Hatada Y,
Extremophiles, 2, 185–190 (1998).
23) Sormachi K, Le Gal-Coeffet MF, Williamson G, Archer DB,
¨
and Williamson MP, Structure, 5, 647–661 (1997).
24) Kamitori S, Abe A, Ohtaki A, Kaji A, Tonozuka T, and Sakano
Y, J. Mol. Biol., 318, 443–453 (2002).
2) Cantarel B, Coutinho PM, Rancurel C, Bernard T, Lombard V,
and Henrissat B, Nucleic Acids Res., 37, D233–D238 (2009).
3) MacGregor EA, Janecek S, and Svensson B, Biochim. Biophys.
Acta, 1546, 1–20 (2001).
25) Watanabe K, Chishiro K, Kitamura K, and Suzuki Y, J. Biol.
Chem., 266, 24287–24294 (1991).
4) Stam MR, Danchin EG, Rancurel C, Coutinho PM, and
Henrissat B, Protein Eng. Des. Sel., 19, 555–562 (2006).
5) Kim DH, Morimoto N, Saburi W, Mukai A, Imoto K, Takehana
T, Koike S, Mori H, and Matsui H, Biosci. Biotechnol.
Biochem., 76, 1378–1383 (2012).
26) Kim JS, Cha SS, Kim HJ, Kim TJ, Ha NC, Oh ST, Cho HS, Cho
MJ, Kim MJ, Lee HS, Kim JW, Choi KY, Park KH, and Oh BH,
J. Biol. Chem., 274, 26279–26286 (1999).
27) Kamitori S, Kondo S, Okuyama K, Yokota T, Shimura Y,
Tonozuka T, and Sakano Y, J. Mol. Biol., 287, 907–921 (1999).
28) Hondoh H, Kuriki T, and Matsuura Y, J. Mol. Biol., 326, 177–
188 (2003).
6) Horikoshi K, Agric. Biol. Chem., 35, 1783–1791 (1971).
7) Hayashi T, Akiba T, and Horikoshi K, Agric. Biol. Chem., 52,
443–448 (1988).
29) Ibuka A, Tonozuka T, Matsuzawa H, and Sakai H, J. Biochem.,
123, 275–282 (1998).
8) Boyer EW and Ingle MB, J. Bacteriol., 110, 992–1000 (1972).
9) Igarashi K, Hatada Y, Hagihara H, Saeki K, Takaiwa M,
Uemura T, Ara K, Ozaki K, Kawai S, Kobayashi T, and Ito S,
Appl. Environ. Microbiol., 64, 3282–3289 (1998).
30) Shimura Y, Tonozuka T, and Sakano Y, J. Appl. Glycosci., 45,
393–395 (1998).
31) Abe A, Tonozuka T, Sakano Y, and Kamitori S, J. Mol. Biol.,
335, 811–822 (2004).
10) Hagihara H, Igarashi K, Hayashi Y, Endo K, Ikawa-Kitayama
K, Ozaki K, Kawai S, and Ito S, Appl. Environ. Microbiol., 67,
1744–1750 (2001).
32) Mikami B, Adachi M, Kage T, Sarikaya E, Nanmori T, Shinke
R, and Utsumi S, Biochemistry, 38, 7050–7061 (1999).