Eur. J. Biochem.

253, 2512262 (1998)

 FEBS 1998

Molecular and enzymatic characterization of a maltogenic amylase

that hydrolyzes and transglycosylates acarbose
Hyun-Ju CHA 1, Hyun-Geun YOON 1 , Young-Wan KIM 1, Hee-Seob LEE 1 , Jung-Wan KIM 2 , Ki-Sung KWEON 1, Byung-Ha OH 3
and Kwan-Hwa PARK 1
1
Department of Food Science & Technology and Research Center for New Bio-Materials in Agriculture, Seoul National University, Korea
2
Department of Biology, University of Inchon, Inchon, Korea
3
Department of Life Science, Pohang University of Science and Technology, Pohang, Korea

(Received 24 October 1997) 2 EJB 97 1513/4

A gene encoding a maltogenic amylase of Bacillus stearothermophilus ET1 was cloned and expressed
in Escherichia coli. DNA sequence analysis indicated that the gene could encode a 69627-Da protein
containing 590 amino acids. The predicted amino acid sequence of the enzyme shared 47270% identity
with the sequences of maltogenic amylase from Bacillus licheniformis, neopullulanase from B. stearother-
mophilus, and cyclodextrin hydrolase (CDase) I-5 from an alkalophilic Bacillus I-5 strain. In addition to
starch, pullulan and cyclodextrin, B. stearothermophilus could hydrolyze isopanose, but not panose, to
glucose and maltose. Maltogenic amylase hydrolyzed acarbose, a competitive inhibitor of amylases, to
glucose and a trisaccharide. When acarbose was incubated with 10% glucose, isoacarbose, containing an
A-1,6-glucosidic linkage was produced as an acceptor reaction product. B. stearothermophilus maltogenic
amylase shared four highly similar regions of amino acids with several amylolytic enzymes. The β-
cyclodextrin2hydrolyzing activity of maltogenic amylase was enhanced to a level equivalent to the activ-
ity of CDase when its amino acid sequence between the third and the fourth conserved regions was made
more hydrophobic by site-directed mutagenesis. Enhanced transglycosylation activity was observed in
most of the mutants. This result suggested that the members of a subfamily of amylolytic enzymes,
including maltogenic amylase and CDase, could share similar substrate specificities, enzymatic mecha-
nisms and structure/function relationships.
Keywords : Bacillus stearothermophilus; maltogenic amylase ; acarbose ; transglycosylation; site-directed
mutagenesis.

Many types of amylases with unique properties have been megaterium [7], and A-amylase of Thermoactinomyces vulgaris
isolated and characterized for various applications in the starch [8] have been reported to hydrolyze the A-1,4 linkages of pullu-
industry [1, 2]. These proteins share many structural and mecha- lan to produce panose. Amylolytic enzymes, such as cyclo-
nistic characteristics. However, amylases can be divided into dextrin glucanotransferases (CGTase) and CDase exhibit their
several groups according to substrate specificities, patterns of highest levels of activity on cyclomaltodextrins [9211]. Some
starch cleavage, transglycosylation or cyclization activities, and amylolytic enzymes, including debranching enzymes and
structural features. Classical A-amylases (e.g. 1,4-A-D-glucan CGTase, catalyze transglycosylation by forming A-1,4 or A-1,6
glucanohydrolase) catalyze hydrolysis of A-1,4-glucosidic link- linkages.
ages in starch, and different amylases give rise to oligosaccha- Jespersen et al. [12] used sequence alignments and structure-
rides with specific lengths of glucose as major product [2]. De- prediction models to predict the presence of A-amylase-type
branching enzymes are capable of hydrolyzing A-1,6-glucosidic (β/A)8-barrel domains and the positions of the β-strands and A-
linkages in starch and/or pullulan [1, 325] to produce maltotri- helices found in 47 amylolytic and related enzymes. An evolu-
ose. Other amylolytic enzymes catalyze the hydrolysis of pullu- tionary distance tree constructed from 37 residues representing
lan in a different way. Neopullulanase [6], A-amylase of Bacillus the four most highly conserved β-strands of these related en-
Correspondence to K.-H. Park, Department of Food Science, Seoul zymes revealed several clusters of enzymes harboring similar
National University, 103 Seo-Dun Dong, Kweon-Sun Ku, Suwon, reaction specificities. This correlation strongly suggests that the
441-744, Korea conserved β-strands may determine substrate and/or product
Fax: 182 331 294 1336. specificity. Thus, modification of certain residues in these con-
E-mail: [email protected] served regions could improve the activities of the amylolytic
Abbreviations. CDase, cyclomaltodextrin hydrolase; CGTase, cyclo- enzymes and this, in turn, could be useful in the commercial
maltodextrin glucanotransferase; MALDI-TOF, matrix-assisted laser-de- application of these proteins.
sorption/ionization time of flight; HPIC, high-performance ion chroma-
We have isolated previously several amylolytic enzymes and
tography.
Enzymes. Cyclomaltodextrin glucanotransferase (EC 2.4.1.19) ; cy-
their corresponding genes from various Bacillus species. These
clomaltodextrinase (EC 3.2.1.54). include a typical thermostable A-amylase [13] and maltogenic
Note. The nucleotide sequence data presented here have been sub- amylase [14] from Bacillus licheniformis, and a cyclodextrinase
mitted to the GenBank database and are available under accession (CDase I-5; unpublished data) and a CGTase (unpublished data)
number U50744. from the alkalophilic Bacillus sp. I-5. Among the amylolytic
252 Cha et al. (Eur. J. Biochem. 253)

enzymes isolated in our laboratory, B. licheniformis maltogenic strands were sequenced using universal primers (Amersham).
amylase and CDase I-5 exhibited A-1,6-transglycosylation and Protein sequencing of purified B. stearothermophilus maltogenic
A-1,4-hydrolysis activities that were likely to be coupled. Both amylase was carried out using an analyzer manufactured by Ap-
hydrolyzed starch, mainly to maltose, and pullulan to panose. plied Biosystems Inc. (Model 473A).
These enzymes differ from other amylases in that they prefer Enzyme purification. The E. coli transformant harboring
cyclodextrin to starch or pullulan as a substrate and are localized the maltogenic amylase gene was cultured in a 5-l jar fermentor
in the cytoplasm. While B. licheniformis maltogenic amylase (Korea Fermentor Inc.) containing 3 l Luria-Bertani medium
hydrolyzes cyclodextrin to maltotriose and maltose [14], CDase [1% (mass/vol.) Bacto tryptone, 0.5 % (mass/vol.) yeast extract,
I-5 hydrolyzes these substrates mainly to maltose. Many of these 0.5% (mass/vol.) NaCl] and ampicillin (50 µg/ml) at 37°C for
properties, if not all, are shared by other amylolytic enzymes 9 h. The cells were harvested and suspended in 300 ml 50 mM
[10, 15219]. Tris/HCl pH 7.5, and lysed using a sonicator (Ultrasonics & Ma-
A CDase is defined as an enzyme that degrades cyclodextrin terials; Model VC-600) with 60% duty cycle at output 7 for
faster than it degrades starch [8], and many CDase have been 10 min over a dry ice/ethanol bath. After cell debris was re-
isolated from various sources [22226]. B. licheniformis malto- moved by centrifugation at 10 000 rpm for 20 min in a VS-
genic amylase could be classified as a CDase, although it gen- 21SMT centrifuge (Vision Scientific Co.), protein was precipi-
erally has higher transglycosylation and pullulan-hydrolyzing tated once with 20% (mass/vol.) saturated ammonium sulfate
activities than a CDase (unpublished data). Podkovyrov and Zei- for 2 h at 4°C and once with 50% (mass/vol.) saturated ammo-
kus [10] reported that there is an additional conserved sequence nium sulfate for 2 h at 4 °C. The precipitate was collected by
between the third and fourth conserved regions of various centrifugation at 10 000 rpm for 20 min, suspended in 80 ml
CDase. This finding is highly relevant since the cyclodextrin- 50 mM Tris/HCl pH 7.5, and dialyzed against the same buffer
binding site is known to be situated in these regions [27]. Char- for 728 h at 4 °C. The dialyzed solution was applied to FPLC
acterization of the structures and biochemical properties of amy- (Pharmacia) as follows. The solution was loaded onto a DEAE-
lolytic enzymes that exhibit transglycosylation and/or cyclo- Toyopearl 650 (3.0 cm315 cm) column equilibrated with
dextrin-hydrolyzing activity would be useful to understand more 20 mM Tris/HCl pH 7.5, and proteins were eluted with a linear
precisely the catalytic activities and substrate-binding patterns. gradient of NaCl from 0.15 M to 0.4 M in the same buffer at a
Recently, a maltogenic amylase was isolated from Bacillus flow rate of 7 ml/min. The fractions with enzymatic activity
stearothermophilus in our laboratory [28]. The enzyme readily were concentrated using a YM-10 membrane (Amicon Co.) un-
hydrolyzed β-cyclodextrin, pullulan and soluble starch with rela- der nitrogen atmospheric conditions and dialyzed against 20 mM
tive activities of 8.7, 1 and 0.3, respectively. β-cyclodextrin and Tris/HCl pH 7.5, for 728 h at 4 °C. The concentrated enzyme
starch were hydrolyzed mainly to maltose, and pullulan to solution was applied to a Mono-Q HR 5/5 column and eluted
panose, maltose and glucose. It exhibited A-1,6-transglycosyl- with a linear gradient of NaCl from 0.15 M to 0.3 M in 20 mM
ation activity in addition to the hydrolyzing activity on A-1,4- Tris/HCl pH 7.5, at a flow rate of 1 ml/min. Active fractions
glucosidic and A-1,6-glucosidic linkages, when reacted with a were concentrated three times using a PM-10 membrane (Ami-
5% (mass/vol.) starch solution. con Co.) as described above. B. licheniformis maltogenic amy-
In this paper, we report the nucleotide sequence of a malto- lase was prepared as reported previously [14].
genic amylase from B. stearothermophilus ET1. The mechanism Enzyme assay. Maltogenic amylase activity was assayed at
of the enzyme action was characterized by catalysis and inhibi- 55°C in 50 mM sodium citrate pH 6.0, using 3,5-dinitrosalicylic
tion studies using acarbose, an amylase inhibitor, and site-di-
acid and β-cyclodextrin (Miwon Inc.), soluble starch (Showa
rected mutagenesis. Models of maltogenic amylase action
Chemicals Inc.), pullulan (Sigma Co.) or acarbose as substrates
pattern are proposed based on the data obtained in the present
according to methods described by Miller [31]. 1 U enzyme ac-
and previous studies. The results of this study indicated that B.
tivity was defined as the amount of enzyme giving an increase
stearothermophilus maltogenic amylase was an enzyme with
in absorbance at 575 nm of 1 when the reaction was carried out
properties similar to those of B. licheniformis maltogenic amy-
lase, but with at least one unique property, and its catalytic prop- at 55°C for 30 min [14]. Protein concentration was measured by
erties could be changed by site-directed mutagenesis. the Bradford method [32] with BSA as a standard.
Determination of molecular mass. Molecular mass of puri-
fied maltogenic amylase was determined by SDS/PAGE using
4% (mass/vol.) stacking and 12% (mass/vol.) resolving gels as
MATERIALS AND METHODS described by Laemmli [33]. Matrix-assisted laser-desorption/
Gene cloning. B. stearothermophilus ET1, isolated from ionization time-of-flight (MALDI-TOF) MS was performed
Korean soil (KCTC 0114BP), was the source of B. stearo- using a Lasermat 2000 (Finnigan). Samples were prepared by
thermophilus maltogenic amylase and its corresponding gene. mixing 1 µl enzyme solution (2.0 mg in 1 ml 0.1 % trifluoro-
Chromosomal DNA of B. stearothermophilus ET1 isolated acetic acid) with 1 µl saturated sinapinic acid. 1 µl sample was
by the spool method [29] was partially cleaved with HindIII spotted into a well of the sample plate and allowed to dry in air.
before ligation to the same restriction site of pUC18. A genomic Hydrolytic and transferring activity. Purified maltogenic
DNA library was constructed by transforming the ligation amylase (400 U/g substrate) was incubated with 1 ml 1% (mass/
mixture into an Escherichia coli strain, DH5A [supE44, vol.) substrate (β-cyclodextrin, pullulan, soluble starch, panose
∆lacU69(f80lacZ∆M15), hsdR17, recA1, endA1, gyrA96, thi-1, or isopanose) in 50 mM sodium citrate, pH 6.0, at 55°C for 12 h
relA1], and the resulting transformants were screened for starch- to determine the hydrolytic mode of action. To determine the
hydrolyzing activity by an iodine test after treatment with mechanism of the transferring activity, 0.5 % or 5% (mass/vol.)
D-cycloserine as described previously [14]. maltotriose, maltotetraose, maltopentaose, maltohexaose or mal-
Sequencing analysis. Plasmid DNA sequencing was carried toheptaose (Sigma Co.) in 50 mM sodium citrate pH 6.0 were
out by the chain-termination method of Sanger [30] using the incubated with maltogenic amylase (400 U/g substrate) under
Sequenase kit (United States Biochemical Co.). Serial deletion the same conditions. Resulting products were analyzed by TLC
of the insert was carried out using exonuclease III (Amersham) and high-performance ion chromatography (HPIC) as described
to generate overlapping fragments for sequencing. Both DNA previously [21].
 Cha et al. (Eur. J. Biochem. 253) 253

Fig. 1. Restriction map of the insert cloned in pSG18 and sequence of the maltogenic amylase coding region. Insert of a 4.7-kb HindIII
fragment containing the maltogenic amylase gene is shown as a box and the portion of cloning vector, pUC18, as a line. The coding region and
the direction of the gene are indicated with a black box and an arrow, respectively. The nucleotide sequence of the maltogenic amylase gene and
its flanking regions are shown below the map. The deduced protein sequence of an ORF responsible for maltogenic amylase starts with a methionine
and is presented under the nucleotide sequence. Sequences for a putative promoter and a ribosome-binding site (rbs) are underlined. Three restriction
enzyme sites that were shown on the restriction map are marked above the corresponding sequences. Asterisks represent the termination codon.

Inhibition studies using acarbose. Acarbose (O-4,6-di- 2.5 U enzyme/mg acarbose were used. The reaction was stopped
deoxy-4-{[4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-1- by boiling for 5 min. Reactions without either of the substrates
yl]amino}-A- D -glucopyranosyl-(1→4)-O-A- D -glucopyranosyl- were carried out as controls. Samples (10 µl) were loaded onto
(1→4)-D-glucose), a pseudo-tetrasaccharide inhibitor, was ob- Silica gel 60 TLC plates (Merck) and developed as described
tained from Bayer through Dr Y. Konishi at Osaka City Univer- previously [14].
sity, Japan. B. stearothermophilus maltogenic amylase in 10 mM Transglycosylation of acarbose. Isoacarbose was prepared
sodium citrate pH 6.0, or B. licheniformis maltogenic amylase by mixing 10% glucose, 3.3 % acarbose and B. stearothermophi-
in 10 mM maleate pH 6.8 was mixed with 3.8 mM acarbose and lus maltogenic amylase (10 U/mg acarbose) in 5 mM sodium
3.8 mM β-cyclodextrin in 400 µl. The reaction was carried out citrate pH 6.0. The reaction was carried out at 50°C for 48 h.
at 50°C and aliquots (80 µl) were sampled every 8 h. The final Excessive glucose in the reaction was removed through fermen-
concentration of reaction buffers was approximately 5 mM, and tation (30°C, overnight) by Saccharomyces cerevisiae var. ellip-
254 Cha et al. (Eur. J. Biochem. 253)

Fig. 1. (continued).

soideus entrapped in 3 % sodium alginate. Isoacarbose was puri- respectively. The primers were synthesized by Gene Assembler
fied by FPLC (Pharmacia) using a Bio-Gel P2 column Special for Primers (Pharmacia). The introduced mutations were
(2.0 cm390 cm; Bio-Rad) and a Waters differential refractome- confirmed by DNA sequencing.
ter (Waters, R401). The sample was eluted by deionized water
at a flow rate of 0.3 ml/min.
The structure of isoacarbose (20 mg/ml) was analyzed by RESULTS
NMR (JEOL JNM-LA-400, FT NMR, 400 MHz) using D2O as
solvent at 90°C. Cloning and DNA sequencing analysis of the B. stearother-
Site-directed mutagenesis. Seven maltogenic amylase mu- mophilus maltogenic amylase gene. A positive clone for amy-
tants were derived by oligonucleotide-mediated site-directed lolytic activity was obtained from the E. coli DH5A cells trans-
mutagenesis according to methods reported by Kunkel et al. formed with a B. stearothermophilus ET1 genomic DNA library
[34, 35]. The oligonucleotides used to create the mutations by treating with D-cycloserine and iodine solution on a starch
Asn381→Leu, Gly382→Ala/Thr383→Val, Gly421→Asp, agar plate. The cloned plasmid was designated as pSG18 and
Asn331→Ser/Glu332→Val, Gly382→Met/Thr383 →Leu, contained a 4.7-kb insert. Southern analysis of B. stearother-
Gly382→Ala and Thr383→Val were 5′-TTACACTTGGGA- mophilus ET1 genomic DNA using the insert as a probe revealed
CCCTT-3′ (CHJ381), 5′-TACAAATGCGGTCCTTCGTT-3′ that a 9.4-kb BamHI fragment, 8-kb and 10-kb EcoRI fragments,
(CHJA382), 5′-TTACTCGACAGTCACGAT-3′ (CHJ421), 5′- 2.0-kb and 2.3-kb HindIII fragments hybridized to the HindIII
GTCGCTAGCGTGGTCGAT-3′ (CHJ331), 5′-ACAAATATG- fragment (data not shown). These results correlated well with
CTCCTTCG-3′ (CHJM383), 5′-TACAAATGCGACCCTTC-3′ restriction analysis in that the HindIII fragment had an EcoRI
(CHJ382), and 5′-CAAATGGGGTACTTCGTase-3′ (CHJ383), and two internal HindIII sites.
 Cha et al. (Eur. J. Biochem. 253) 255
Table 1. Comparison of amino acid sequences in the regions conserved among various amylases.

Amylolytic enzymes Amino acid of conserved domain Reference

I II III IV

Maltogenic amylase of B. stearothermophilus DAVFNH GWRLDVANE EIWH LLGSHD this study

Maltogenic amylase of B. licheniformis DAVFNH GWRLDVANE EIWH LLDSHD [14]
Neopullulanase of B. stearothermophilus DAVFNH GWRLDVANE EIWH LLGSHD [36]
Amylase II of T. vulgaris DAVFNH GWRLDVANE EIWH LLDSHD [37]
Pullulanase of K. aerogenes DVVYNH GFRFDLASV EGWD YVSKHD [38]
CDase I-5 of Bacillus DAVFNH GWRLDVANE EVWH LLDSHD unpublished data
Thermostable A-amylase of B. licheniformis DVVINH GFRLDAVKH EYWQ FVDNHD [13]
CGTase of B. macerans DFAPNH GIRFDAVKH EWFL FIDNHD [39]
CGTase of B. stearothermophilus DFAPNH GIRMDAVKH EWFL FIDNHD [40]
Consensus DAVINH GFRLDAAKH EVID FVDNHD

Fig. 2. Comparison of the spacing between the domains conserved among various amylolytic enzymes. The enzymes are cited from the
references listed in Table 1 except CDase of B. spaericus [25]. NPL, neopullulanase ; TVAII, T. vulgaris amylase II; TA, thermostable A-amylase.
The numbers represent the first amino acid residues of each conserved domain.

DNA sequencing was carried out to analyze the structure of with CDase I-5 (unpublished data), and 69.9% with neopullula-
the maltogenic amylase gene (Fig. 1). An ORF initiated by a nase [36] at the predicted amino acid sequence level. However,
methionine codon was found in the sequenced region, encoding B. stearothermophilus maltogenic amylase showed even higher
a protein of 590 amino acids. The predicted gene product has a levels of identity in the four domains known to be conserved
molecular mass of 69627 Da as calculated from the deduced among various amylolytic enzymes (Table 1). The amino acid
amino acid sequence. N-terminal-sequence analysis of malto- sequence of B. stearothermophilus maltogenic amylase was
genic amylase showed that the first seven amino acids were Met- identical to that of neopullulanase in the conserved regions, and
Phe-Lys-Glu-Ala-Ile-Tyr, which matched the predicted amino differed at only one amino acid residue in the same regions of
acid sequence. A putative promoter region was found 205 B. licheniformis maltogenic amylase and CDase I-5. Further-
(235) and 176 (210) nucleotides upstream of the translation more, the spacings of the domains were similar among the en-
initiation site. The two sequences, TTGACA for 235 and zymes (B. stearothermophilus maltogenic amylase and B. li-
TACAAT for 210, were separated by 18 nucleotides. The ORF cheniformis maltogenic amylase, CDase and neopullulanase)
was preceded by a putative ribosome-binding site nine nucleo- that exhibited preferences for cyclodextrin and transglycosyl-
tides upstream. ation activity, with the exception of neopullulanase, whose cy-
B. stearothermophilus maltogenic amylase shared 47.1% clodextrin-hydrolyzing activity has not been documented
identity with B. licheniformis maltogenic amylase [14], 57.7% (Fig. 2). Another region of similarity was detected between the
256 Cha et al. (Eur. J. Biochem. 253)

Fig. 3. A conserved region in a subgroup of amylolytic enzymes. Sequences between the third and the fourth conserved regions are conserved
among a subgroup of amylolytic enzymes including B. stearothermophilus maltogenic amylase (BSMA), B. licheniformis maltogenic amylase
(BLMA), CDase and neopullulanase. The new conserved region begins right after the third conserved region and is 27 amino acids long. The
numbers represent the first amino acid residues of the third and the fourth conserved regions. The sequences are taken from the references listed in
Table 1, except CDase X [10] and CDase E-244 [25].

Fig. 4. SDS/PAGE of purified maltogenic amylase. Purified malto-

genic amylase was electrophoresed on a 12 % SDS/polyacrylamide gel
(lane 2) with standard markers (lane 1): myosin, 205 kDa; β-galactosi-
dase, 116 kDa; phosphorylase b, 97.4 kDa; BSA, 66 kDa; ovalbumin,
45 kDa; carbonic anhydrase, 29 kDa. Fig. 5. TLC analysis of isopanose-hydrolyzing activity. Isopanose was
incubated with maltogenic amylase (400 U/g substrate) for 2 h (lane 2)
or 12 h (lane 3) at 55°C before analysis by TLC. Isopanose (lane 1) and
third and the fourth conserved regions among these enzymes glucose, maltose, maltotriose, maltotetraose and maltopentaose (G12
(Fig. 3), and site-directed mutagenesis studies were carried out G5; lane Std) were used as standards.
as an effort to elucidate function of this conserved sequence.

Purification of maltogenic amylase. Maltogenic amylase was amylase. TLC analyses of the reactions showed that maltogenic
weakly expressed and localized exclusively in the cytoplasm of amylase catalyzed the hydrolysis of isopanose (Fig. 5), but not
B. stearothermophilus ET1. Expression of maltogenic amylase panose (data not shown), to glucose and maltose.
was greatly improved in E. coli harboring pSG18 and the en-
zyme was purified from the E. coli clone. The enzyme-purifica- Catalysis of acarbose. Hydrolysis patterns of B. stearother-
tion scheme described in Materials and Methods resulted in re- mophilus maltogenic amylase on acarbose, a pseudotetrasaccha-
covery of 3.65% of the total maltogenic amylase activity with ride inhibitor, and inhibition of the enzyme activity by the com-
a 6.95-fold increase in the specific activity. The purified en- pound were studied. Maltogenic amylase hydrolyzed acarbose
zyme appeared as a single band on a SDS/polyacrylamide gel to glucose and a trisaccharide (Fig. 6 A). Quantitative analysis
(Fig. 4), and the molecular mass of the protein was estimated as of the products using a densitometer (Biorad GS-700) indicated
62 kDa. MALDI-TOF/MS spectra showed that the enzyme had that more than 30% of acarbose was hydrolyzed by the enzyme.
a molecular mass of 69 6636 103 Da. The preparation was pure About 95% hydrolysis of acarbose could be achieved in 5 min
enough to obtain crystals of maltogenic amylase with dimen- by treating with excess maltogenic amylase. The β-cyclodextrin-
sions of 0.330.1530.08 mm (unpublished data). hydrolyzing activity of maltogenic amylase was partially inhib-
ited in the presence of acarbose (Fig. 6A). In contrast, B. licheni-
Hydrolysis of isopanose. Catalysis studies of maltogenic amy- formis maltogenic amylase did not hydrolyze acarbose (Fig. 6 B)
lase in the previous report [28] indicated that the enzyme could and its β-cyclodextrin2hydrolyzing activity was inhibited to-
hydrolyze starch, pullulan and β-cyclodextrin readily by cleav- tally by the compound (Fig. 6 B). The trisaccharide molecules
ing A-1,4- and A-1,6-glucosidic linkages. Time-courses of resulting from the hydrolysis of acarbose were transferred to
branched-oligosaccharide production indicated that branched other sugar moieties by B. stearothermophilus maltogenic amy-
molecules with 427 glucose subunits were hydrolyzed to lase via the formation of an A-1,6-glucosidic linkage in the pres-
smaller molecules by the action of maltogenic amylase. To test ence of 10% (mass/vol.) glucose or maltose (Fig. 6 C). Under
whether the enzyme could hydrolyze even smaller branched these conditions, acarbose was hydrolyzed completely to form
molecules, isopanose and panose were reacted with maltogenic isoacarbose in a 24-h incubation.
 Cha et al. (Eur. J. Biochem. 253) 257

Fig. 6. TLC analysis of products of maltogenic amylase-mediated hydrolysis of acarbose. (A) Action of maltogenic amylase on acarbose, a
mixture of acarbose and β-cyclodextrin (β-CD), or β-cyclodextrin in 5 mM citrate buffer (pH 6.0) at 50°C for 0 (lane 1), 8 (lane 2), 16 (lane 3) and
24 (lane 4) h. (B) The same reactions were carried out using B. licheniformis maltogenic amylase in 5 mM maleate pH 6.8. (C) Production of
isoacarbose (lane 2) by the action of maltogenic amylase in the presence of 10 % (mass/vol.) glucose. Acarbose is shown in lane 1. Glucose, maltose,
maltotriose, maltotetraose and maltopentaose (G12G5) were used as standards (Std).

Transglycosylation of acarbose. In an effort to prove that

isoacarbose contains an A-1,6-glucosidic linkage, the isoacar-
bose was purified by FPLC (Fig. 7) and subjected to 1H-NMR
spectroscopy. 1H-NMR spectroscopy can distinguish between
anomeric protons involved in A-1,4 and A-1,6 linkages and has
been used to estimate the degree of branching of glycogens and
amylopectin β-limit dextrins [41]. Panose was used as a standard
compound for the minor structural features of starch, as it con-
tains terminal reducing and non-reducing glucose residues and
A-1,4 and A-1,6 linkages (Fig. 8A). The peak representing an
A-1,6 linkage was absent in acarbose (Fig. 8 B), but present in
isoacarbose (Fig. 8 C). The peak for an A-1,4 linkage between
the glucose and non-reducing glucose residues disappeared in
isoacarbose.

Properties of mutants generated by site-directed mutagene-

sis. The amino acid sequence between the third and the fourth
conserved regions of B. stearothermophilus maltogenic amylase
and B. licheniformis maltogenic amylase (Fig. 3), which might
represent the putative cyclodextrin-binding site proposed by
Podkovyrov and Zeikus [10], was predicted to be more hydro-
philic than the corresponding region of CDase I-5, especially
between residues 376 and 382 in B. stearothermophilus malto-
genic amylase. Three amino acid residues in this region,
Asn381, Gly382 and Thr383, were changed in a series of five
mutants, such that hydrophobicity was increased in the region
(Table 2). These residues were likely to reside in the middle of
helix A6, surrounded by residues forming a hydrophobic core
region [42]. The mean hydrophobic indexes of [Leu381]malto-
genic amylase, [Ala382]maltogenic amylase and [Val383]malto-
genic amylase increased to 20.09, 20.18, and 20.13, respec-
tively, in the region between residues 361 and 418 when esti-
mated with a six-residue window according to the method of
Kyte and Doolittle [43]. The mean hydrophobic index of the
wild-type enzyme in the region was 20.22. In [Ala382,Val383]-
maltogenic amylase and [Met382,Leu383]maltogenic amylase
the mean hydrophobic indices increased to 20.09 and 20.10, Fig. 7. Preparation of isoacarbose by FPLC. Fractions 1214 from the
respectively. [Leu381]maltogenic amylase exhibited a substrate column were collected (A) and subjected to TLC analysis (B ; lanes 12
specificity slightly different from that of the wild-type, while the 14). Fractions 223 were taken as the isoacarbose sample.
258 Cha et al. (Eur. J. Biochem. 253)

Fig. 8. 1 H-NMR spectroscopy of (A) panose, (B) acarbose and (C) isoacarbose. Peaks at 5.85 ppm and 5.45 ppm are assigned to H1 of A-1,4-
linked and A-1,6-linked units between glucose, respectively.

Table 2. Activities of the wild-type and various mutant maltogenic amylase. Values in parentheses are percentages of the activity of the wild type.

Protein Mutation in Activity with Hydrophobicity Activity with

conserved between third β-cyclodextrin/
region pullulan soluble starch β-cyclodextrin and fourth activity with
regions pullulan

Maltogenic amylase 1.689 (100) 0.470 (100) 14.809 (100) 20.22 8.7
[Ser331,Val332]Maltogenic amylase II 0.005 (0.3) 0.006 (1.3) 0.007 (0.1) 1.4
[Glu421]Maltogenic amylase IV 1.537 (91) 0.387 (82) 11.639 (79) 7.6
[Ala382,Val383]Maltogenic amylase III2IV 0.500 (29) 0.480 (102) 18.890 (128) 20.09 37.8
[Met382,Leu383]Maltogenic amylase III2IV 0.200 (12) 0.264 (56) 6.328 (43) 20.10 31.6
[Ala382]Maltogenic amylase III2IV 1.478 (87) 0.664 (141) 21.009 (141) 20.18 14.2
[Val383]Maltogenic amylase III2IV 1.385 (82) 0.803 (159) 18.185 (118) 20.13 13.1
[Asn381]Maltogenic amylase III2IV 1.030 (61) 0.260 (55) 12.290 (83) 20.09 11.9
CDase I25 3.45 12.5 114 20.04 33.0

other single mutants showed a greater degree of alteration of hydrolysis or transglycosylation products were measured and
relative substrate specificity (Table 2). The relative activities of compared when 45% of the substrate was left in the reaction
the double mutants on β-cyclodextrin were approximately 324- (Table 3). Two of the mutants, [Leu381]maltogenic amylase and
fold higher than that of wild-type maltogenic amylase, when the [Ala382]maltogenic amylase, showed hydrolysis/transglycosyl-
preference for β-cyclodextrin was expressed as the ratio of the ation patterns similar to that of the wild-type enzyme. However,
specific activity for β-cyclodextrin to pullulan. The activities of [Val383]maltogenic amylase, [Ala382,Val383]maltogenic amy-
the double mutants and [Leu381]maltogenic amylase on pullulan lase and [Met382,Leu383]maltogenic amylase exhibited 226-
were 12260% of the wild-type activity. [Ala382]Maltogenic fold higher transglycosylation activities than the wild type.
amylase and [Val383]maltogenic amylase showed enhanced We replaced the glycine residue at position 421 in the fourth
activities on β-cyclodextrin and soluble starch and maintained conserved region with an aspartic acid. The residual activities
activities on pullulan of more than 80% wild-type. of [Asp421]maltogenic amylase on substrates such as pullulan,
Hydrolysis and transglycosylation activities of the mutants soluble starch and β-cyclodextrin were 80290% those of the
were compared using reaction mixtures consisting of 10% mal- wild-type enzyme, suggesting that the residue is not directly in-
totriose in 50 mM sodium citrate pH 6.0. Relative amount of volved in the catalytic site. In contrast, we observed a dramatic
 Cha et al. (Eur. J. Biochem. 253) 259

Fig. 9. Proposed models of coupled hydrolysis and transglycosylation by maltogenic amylase. s, non-reducing glucopyranosyl residues ; Ø,
reducing glucose residues; n2h, O-4,6-dideoxy-4-{[4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-1-yl]amino}-A-D-glucopyranosyl moiety.
Horizontal lines and vertical lines connecting sugar symbols denote A-1,4 and A-1,6 linkages, respectively. The molecules shown in the boxes on
the right represent some of those that could be formed by transferring activity of maltogenic amylase.

decrease in enzyme activity of [Ser331,Val332]maltogenic amy- than of starch or pullulan, and hydrolyzed starch to maltose (the
lase on all substrates. In this double mutant, two polar residues reason why it was designated a maltogenic amylase). The up-
in the second conserved region were replaced with less or non- stream region of the maltogenic amylase gene contains se-
polar residues. Asn331 and Glu332 are located in the second quences for transcriptional and translational signals, which sug-
conserved region and probably correspond to substrate-binding gested that good expression of the gene was possible in E. coli.
residues of Taka-amylase or porcine pancreatic A-amylase However, the enzyme was produced poorly in B. stearother-
[44, 45]. mophilus. The molecular mass of the enzyme estimated by DNA
sequencing and MALDI-TOF MS were in good agreement. A
discrepancy of 7000 Da was observed between the molecular
DISCUSSION
masses of the enzyme determined by these methods and esti-
A number of amylolytic enzymes, including B. licheniformis mated by SDS/PAGE, but it was not considered unusual. Such
maltogenic amylase and neopullulanase were shown to be capa- a discrepancy was also detected for neopullulanase [19, 36].
ble of producing branched oligosaccharides from liquefied B. stearothermophilus maltogenic amylase hydrolyzed A-1,4
starch by their transglycosylation activities [14, 19]. B. licheni- linkages of pullulan to produce panose as a major product and
formis maltogenic amylase exhibited preference for cyclo- A-1,6 linkages to produce maltose and glucose as minor products
dextrins, pullulan and starch as substrates in decreasing order. [28]. It did not hydrolyze panose at all, but was capable of hy-
Neopullulanase hydrolyzes pullulan efficiently, but its action on drolyzing the A-1,6 linkage of isopanose to produce maltose and
starch is very slow. It has not been documented by the authors glucose. Transglycosylation of cleaved glucose or maltose to
if neopullulanase hydrolyzed cyclodextrins. CDase belong to the other sugar moieties forming A-1,6 linkages was observed in a
category of B. licheniformis maltogenic amylase-type enzymes highly concentrated maltooligosaccharide solution treated with
in that they hydrolyze cyclodextrin most efficiently. Among var- maltogenic amylase. It has been shown that maltogenic amylase
ious CDase, at least one of them, CDase I-5, which was isolated is capable of catalyzing transglycosylation to form A-1,4 link-
and characterized in our laboratory (unpublished data), exhibited ages by using A-methylglucoside as substrate (unpublished data).
transglycosylation activity, but the activity was not strong Acarbose is a pseudotetrasaccharide, which inhibits A-amy-
enough to be utilized for branched-oligosaccharide production. lase activity by binding to the active site of the enzyme [46, 47].
B. stearothermophilus maltogenic amylase activity was de- Due to its efficient inhibition of A-amylase, acarbose is often
tected in the course of screening a B. stearothermophilus given to patients with non-insulin dependent diabetes. Competi-
CGTase library constructed in E. coli. The clone carrying the tive inhibition by acarbose of the B. stearothermophilus and B.
maltogenic amylase gene produced an enzyme with characteris- licheniformis maltogenic amylase activities was tested. Even
tics very similar to those of B. licheniformis maltogenic amylase though the two enzymes share many common characteristics,
and CDase I-5. The enzyme, which was localized in the cyto- they reacted with acarbose in different ways. Acarbose was hy-
plasm, could catalyze hydrolysis of cyclodextrin more readily drolyzed to glucose and pseudomaltotriose by the B. stearother-
260 Cha et al. (Eur. J. Biochem. 253)

Table 3. Hydrolysis and transglycosylation activities of various mal- tant enzymes exhibited lower activity with pullulan than the
togenic amylase mutants. wild-type enzyme (Table 3). However, the relative activities with
β-cyclodextrin were 1.524-fold higher than the wild type. The
Protein Hydro- Transgly- Product relative activities of the double mutants ([Ala382,Val383]malto-
lysis cosylation ratio
product product genic amylase and [Met382,Leu383]maltogenic amylase) on β-
cyclodextrin and pullulan were similar to the activity of CDase
% I-5. However, the overall activity of the mutant decreased drasti-
cally when Gly382 was substituted with a somewhat bulkier res-
Wild-type maltogenic amylase 25.5 29.4 1.2 idue, a methionine. The mutants with a single mutation at resi-
[His332]Maltogenic amylase 35.4 19.6 0.6 due 382 or 383 exhibited higher hydrolytic activities on soluble
[Leu381]Maltogenic amylase 26.9 28.1 1.1 starch and β-cyclodextrin. The moderate increase in hydropho-
[Ala382]Maltogenic amylase 22.4 32.6 1.5 bicity in the single mutants resulted in a moderate increase in
[Val383]Maltogenic amylase 16.0 38.9 2.4 relative activity on β-cyclodextrin and starch. The residues mu-
[Ala382,Val383]Maltogenic amylase 7.7 47.2 6.1 tated (Gly382 and Thr383) are probably located in the middle
[Met382,Leu383]Maltogenic amylase 17.1 37.8 2.3 of a hydrophobic core region [48]. The results suggest that hy-
drophobicity in this region might play an important role in medi-
ating substrate specificity of amylolytic enzymes.
Increased hydrophobicity in this region affected transglyco-
sylation activity of the enzyme. This might imply that the envi-
mophilus enzyme, but not by B. licheniformis maltogenic amy- ronment of the active center is important in determining the re-
lase. The cyclodextrin-hydrolyzing activity of the former was action mode as follows: the active center of maltogenic amylase
partially inhibited by acarbose, while that of B. licheniformis with hydrophobic residues introduced caused the water mole-
maltogenic amylase was totally inhibited, as is A-amylase. Acar- cules to be excluded and substrates to predominate, thereby en-
bose was not hydrolyzed by glucoamylase, but it inhibited the hancing the transglycosylation activity of the enzyme. In con-
cyclodextrin-hydrolyzing activity of the enzyme. We found that trast, introducing water molecules in the active center of the
CDase I-5 hydrolyzed acarbose very efficiently (unpublished enzyme would increase the hydrolysis activity. Comparison of
data). the distribution of the four conserved sequences (Fig. 2) and the
B. stearothermophilus maltogenic amylase transferred the results of the mutagenesis study suggest that B. stearothermophi-
trisaccharide molecule released upon the hydrolysis of acarbose lus maltogenic amylase and B. licheniformis maltogenic amy-
to a glucose molecule, thereby forming isoacarbose, when the lase, CDase I-5, CDase from Bacillus sphaericus, neopullula-
enzyme was incubated with an acarbose solution containing nase, and A-amylase II from Thermoactynomyces vulgaris com-
10% (mass/vol.) glucose or maltose. Acarbose was completely prise a closely related sub-family of amylolytic enzymes. These
hydrolyzed to form isoacarbose under these conditions, and this enzymes were likely to have domains A, B and G, and a C-
reaction was far more efficient than that under regular hydrolysis terminal segment as described by Jesperson et al. [49]. The au-
conditions. These results suggest that the enzyme does not bind thors discussed that a maltogenic amylase of B. stearothermo-
isoacarbose, and this promotes binding of other acarbose mole- philus had A, B, C, D and E domains, similarly to CGTase [50].
cules to maltogenic amylase for hydrolysis and transglycosyl- However, the enzyme was different from maltogenic amylase at
ation. The binding of maltogenic amylase or CDase I-5 to sub- DNA and amino acid sequence levels.
strates is likely to be mimicked by binding of acarbose, and the Mutations introduced in the second or fourth conserved re-
electrophilic C1 of the glucose moiety is likely to be placed gions did not increase the relative activity of the enzyme (Ta-
close enough for a catalytic residue to attack. Recently, Brzo- ble 2). Alignment of maltogenic amylase and Taka-amylase se-
zowski and Davies [48] discussed that none of family 13 amy- quences suggested that the Asn331 and Glu332 residues of mal-
lases had been proven to hydrolyze or transglycosylate acarbose togenic amylase correspond to the Lys209 and His210 residues
in solution. In an effort to understand the biochemical properties of Taka-amylase, respectively, which constitute a substrate-bind-
of maltogenic amylase, further studies on substrate specificity ing site. When the two residues of maltogenic amylase were
and determination of the three-dimensional structure of the en- substituted with a serine and a valine, respectively, the resulting
zyme are in progress. mutant lost activity toward the substrates totally. This indicated
Based on these results and the hydrolysis patterns of various that the residues were involved in substrate binding. The leucine
substrates, we conclude that maltogenic amylase is likely to hy- and glycine residues at positions 420 and 421 in maltogenic
drolyze A-1,4- or A-1,6-glucosidic bonds linked to sugar moieties amylase do not seem to have corresponding residues in the
that are linked to other sugar moieties by A-1,4-glucosidic bonds. active site of Taka-amylase or porcine pancreatic A-amylase
B. licheniformis maltogenic amylase does not appear to catalyze according to the alignment constructed by Kuriki et al. [48].
the hydrolysis of A-1,4-glucosidic bonds linked to reducing This glycine is the only residue that deviates from the se-
sugar moieties, but appears only to function when more than quences in the conserved regions of B. licheniformis maltogenic
two glucose molecules are linked to it either by an A-1,4- or amylase.
A-1,6-glucosidic bond. A proposed model for the mechanism of In conclusion, B. stearothermophilus maltogenic amylase is
B. stearothermophilus maltogenic amylase action is shown in an enzyme that shares many catalytic characteristics with other
Fig. 9. amylolytic enzymes, such as B. licheniformis maltogenic amy-
The cyclodextrin-hydrolyzing patterns were somewhat dif- lase, neopullulanase and CDase I-5, but it has unique properties
ferent among the enzymes. B. stearothermophilus maltogenic that make it distinct from others. Three-dimensional structural
amylase and CDase I-5 hydrolyzed cyclodextrins mainly to determination of the protein by X-ray crystallography, and more
maltose, while B. licheniformis maltogenic amylase produced site-directed-mutagenesis studies would help in understanding
mainly maltotriose and maltose from cyclodextrins. When the the structure/function relationship of the enzyme at the atomic
hydrophobicity of B. stearothermophilus maltogenic amylase level. Furthermore, improvement of the enzyme properties
was increased in the region between the third and fourth con- would aid in various industrial applications, especially in
served sequences by site-directed mutagenesis, the resulting mu- branched-oligosaccharide production.
 Cha et al. (Eur. J. Biochem. 253) 261
19. Kuriki, T., Okada, S. & Imanaka, T. (1988) New type of pullulanase
Our sincere thanks goes to Dr M. Park and Mr D. Kim for their help from Bacillus stearothermophilus and molecular cloning and ex-
in preparing the English manuscript. We thank Drs Y. Konishi (Osaka pression of the gene in Bacillus subtilis, J. Bacteriol. 170, 15542
City University, Japan), S. Chiba and A. Kimura (Hokkaido University, 1559.
Japan), and D. M. Kim (Chunnam University, Korea) for helping us to 20. Kim, I. C., Yoo, S. H., Lee, S. J., Oh, B. H., Kim, J. W. & Park, K.
obtain acarbose. We thank Prof. H. Y. Kim at Kyung-Hee University, H. (1994) Synthesis of branched oligosaccharides from starch by
Korea, for helpful advice on the mutagenesis study of maltogenic amy- two amylases cloned from Bacillus licheniformis, Biosci. Biotech-
lase. This study was supported by the Korea Science and Engineering nol. Biochem. 58, 4162418.
Foundation through the Research Center for New Bio-Materials in Agri- 21. Lee, S. J., Yoo, S. H., Kim, M. J., Kim, J. W., Seok, H. M. &
culture. Park, K. H. (1995) Production and characterization of branched
oligosaccharides from liquefied starch by the action of B. licheni-
formis amylase, Starch. 47, 1272134.
22. DePinto, J. A. & Campbell, L. L. (1968) Purification and properties
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