Journal of Applied Microbiology 1997, 82, 325334

A gene encoding for an a-amylase from thermophilic Bacillus

sp. strain TS-23 and its expression in Escherichia coli
L.-L. Lin, W.-H. Hsu1 and W.-S.Chu2
Department of Food Nutrition, Hung Kuang Institute of Medical and Nursing Technology, Shalu, Taichung, 1Institute
of Molecular Biology, National Chung Hsing University, Taichung, and 2Culture Collection and Research Center, Food
Industry Research and Development Institute, Hsinchu, Taiwan
5561/11/95: received 21 November 1995, revised 6 August 1996 and accepted 7 August 1996

An a-amylase gene from Bacillus sp. strain TS-23

was cloned and expressed by using its own promoter on the recombinant plasmid pTS917 in
Escherichia coli. A cell fractionation experiment revealed that approximately 60% of the
amylase activity was in the periplasmic space. Analysis and activity staining of the
concentrated supernatant fraction by SDS-polyacrylamide gel electrophoresis showed an
apparent protein band with a mol. wt of approximately 65 000. The amylase gene (amyA)
consisted of an open reading frame of 1845 bp encoding a protein of 613 amino acids with a
calculated mol. wt of 69 543. The predicted amino acid sequence showed high homology
with Bacillus species, E. coli and Salmonella typhimurium a-amylases. Deletion of 96 amino
acids from the C-terminal portion of the amylase did not result in the loss of amylolytic
activity. The truncated amylase, deletion of the first 50 amino acids from the N-terminus,
was overexpressed in E. coli system and refolded to yield an activable enzyme.
L .- L. L IN , W . -H . H S U A ND W .- S. C HU . 1 9 97 .

INTRODUCTION

Starch, as the primary storage polysaccharide in plants, is

degraded by amylolytic enzymes from numerous microorganisms (Vihinen and Mantsala 1989). Starch-degrading
enzymes can be grouped into endo- and exoamylase by their
types of action towards substrates. The endoamylases can
cleave internal a-1,4 and/or a-1,6 linkages in starch while
the exoamylases split the a-1,4 bonds of a-glucan from the
non-reducing ends. It has been reported that nearly all the
endoamylases sequenced to date have homologous regions
(Mackay et al. 1985 ; Rogers 1985 ; Nakajima et al. 1986 ;
Svensson 1988 ; MacGregor and Svensson 1989 ; Svensson
et al. 1989). In addition, an intracellular glucosidase of Streptococcus mutans (Russell and Ferretti 1990) and a branching
enzyme from Escherichia coli (Baecker et al. 1986) also contain
the homologous sequences to endoamylases. Numerous
sequence comparisons reveal four generally recognized areas
of homology (designated regions I, II, III and IV) among the
endoamylases and there also appears to be five additional
regions of high similarity (Rumbak et al. 1991). However,
closely related Bacillus species do not necessarily have high
Correspondence to : Dr Long-Liu Lin, Department of Food Nutrition, Hung
Kuang Institute of Medical and Nursing Technology, 34 Chungchie Road, Shalu,
Taichung, Taiwan.
1997 The Society for Applied Bacteriology

homology in endoamylases. For instance, B. licheniformis, B.

megaterium, B. amyloliquefaciens and B. subtilis are generally
grouped together (Priest et al. 1988), but only the B. amyloliquefaciens and B. licheniformis a-amylases are similar which
form a group with those from B. stearothermophilus and Bacillus sp. strain 707 (Tsukamoto et al. 1988).
Recently, the general features of extracellular endoamylases from the thermophilic Bacillus sp. strain TS-23 have
been characterized (Lin et al. 1994). In this paper, we report
the cloning, sequencing and expression of a gene coding for
an amylase from strain TS-23. The deduced amino acid
sequence of the cloned polypeptide (AmyA) was also compared with sequences for amylases of prokaryotic origin.

MATERIALS AND METHODS

Bacterials strains, plasmids and growth conditions

The bacteria used included Bacillus sp. strain TS-23 (Lin

et al. 1994), Escherichia coli DH5a (supE44 DlacU169 (f80
lacZDM15) hadR17 recA1 endA1 gyrA96 thi-1 relA1 ; Hanahan 1983), E. coli JM101 (supE thiD[lac-proAB]F?[traD36
proAB lacIq lacZDM15] ; Yanisch-Perron et al. 1985), E.
coli NovaBlue (endA1 hsdR17 [rk12mk12] supE44 thi-1

326 L .- L. L IN ET A L .

gyrA96 relA1 lac[F? proAB lacIqZDM15 ::Tn10(tetR)] ;

Novagen Inc., Madison, WI, USA) and the K-12-derived E.
coli strain M15(pREP4) (Nals Strs rif s lac ara gal F
recA uvr ; Qiagen GmbH, Hilden, Germany). The plasmids used were pUC119 (Vieira and Messing 1987) and the
Qiagen Type IV expression vectors (Qiagen). The medium
and growth conditions for Bacillus sp. strain TS-23 were
described previously (Lin et al. 1994). Escherichia coli strains
were grown in LuriaBertani (LB) medium (Sambrook et al.
1989).
DNA manipulations

The procedure used for preparation of Bacillus chromosomal

DNA has been described elsewhere (Hopwood et al. 1985).
Plasmid DNA was isolated by the alkaline lysis method (IshHorowicz and Burke 1981). One-step preparation of competent cells and plasmid transformations were performed by
the method of Chung et al. (1989). The digestion of DNA
with restriction endonucleases was carried out according to
the manufacturers directions. Restriction mapping and other
routine molecular methods used in this work were described
by Sambrook et al. (1989).
Construction and screening of the genomic library

Chromosomal DNA of Bacillus sp. strain TS-23 was partially

digested with Sau3AI and size fractionated in a 1% agarose
gel. The DNA fragments with size between 3 and 8 kb was
recovered by the Geneclean II kit (Bio 101 Inc., La Jolla,
CA, USA) and ligated into BamHI-cleaved pUC119 with T4
DNA ligase after treatment by alkaline phosphatase. The
ligation mixture was then introduced into E. coli DH5a by
transformation. The transformants were selected on LB plate
containing 100 mg ml1 ampicillin and 05% starch azure
(Sigma Chemical Co., St Louis, MO, USA). Transformants
showing amylase activity were identified by a clear zone
around the colony.
Localization of amylase

Exponentially-growing cells were washed twice with 50 mmol

l1 NaOHglycine buffer (pH 88), and subjected to osmotic
shock as described by Nossal and Heppel (1966). The shocked
cells were disrupted by passage through a French Press (SLM
Instrument Inc., Urbana, IL, USA). The resulting fractions
were assayed for the activities of amylase, malate dehydrogenase and alkaline phosphatase.
Enzyme assays

The amylase activity was determined by measuring the enzymatic release of reducing groups from soluble starch. Stan-

dard assay mixtures (total volume 1 ml) contained 250 ml of

1% soluble starch, 250 ml of NaOHglycine buffer (50 mmol
l1, pH 88) and 500 ml of appropriate amount of enzyme.
After 10 min incubation at 70C, the reaction was stopped
by addition of 1 ml of dinitrosalicylic acid solution (Bernfeld 1955), heated at 100C for 5 min and the absorbance
at 575 nm was determined. One unit of amylase liberates
1 mmol l1 of reducing groups (as glucose equivalents) per
min. As controls for E. coli periplasmic and intracellular
enzymes, alkaline phosphatase and malate dehydrogenase
activities were assayed by the methods of Brickman and
Beckwith (1975) and Kitto (1969), respectively. Protein concentrations were determined by the method of Bradford
(1976) with bovine albumin as a standard.
Electrophoresis and detection of amylase activity in
gels

Sodium dodecyl sulphatepolyacrylamide gel electrophoresis

(SDS-PAGE) was done with a slab gel as described by
Laemmli (1970). Proteins were stained with Coomassie brilliant blue R-250 (Weber and Osborn 1969). The marker
proteins (Bio-Rad Laboratories, Richmond, CA, USA) used
for estimation of molecular mass were phosphorylase B
(97 400), serum albumin (66 200), ovalbumin (45 000), carbonic anhydrase (31 000) and trypsin inhibitor (21 500).
Amylase activity gels were performed on 10% SDS-PAGE
with concentrated supernatant fractions of E. coli JM101
cultures containing pUC119 and pTS917. The amylolytic
activity band was detected in situ after electrophoresis and
renaturation of proteins according to Lacks and Springhorn
(1980).
N-terminal amino acid analysis

The concentrated supernatant proteins from E. coli JM101

(pTS917) were separated by 10% SDS-PAGE gels. Proteins
were electrotransferred in 10 mmol l1 3-(cyclohexylamino)1-propanesulphonic acid (CAPS)10% methanol (pH 110)
to an Immobilon-P-polyvinylidene difluoride membrane
(Millipore, Bedford, MA, USA) at 50 V for 30 min and
visualized by staining with 01% Coomassie brilliant blue R250 in 50% methanol as described previously (Matsudaira
1987). The desired band was cut out of the membrane, dried
and sequenced in an 477A protein sequence (Applied Biosystem).
Nucleotide sequencing and data analysis

The templates for sequencing were obtained by constructing

a set of exonuclease III-generated deletions from SacI or PstI
site, which closely presents at either end of the insert, of
pTS246 as described by Henikoff (1984) and detailed in the

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A N A MY L AS E G E NE OF B AC IL L US SP . TS -2 3 327

Erase-a-Base Kit (Promega, Madison, WI, USA) protocol.

The deleted plasmids were sequenced with the Taq Dye
Terminator Cycle Sequencing Kit (Applied Biosystems)
using M13 forward and reverse primers. Gaps in the sequence
were determined by the use of appropriate primers and the
Dye Terminator Kit. DNA sequence data were obtained from
the ABI model 473A Automated Sequencer and the program
SEQED (ABI) was used to edit the data. Unless otherwise
indicated, both strands of the DNA were sequenced. The
SEQMAN and GENEMAN (DNASTAR Inc., Madison,
WI, USA) software packages were used for analysis of DNA
and protein sequences. The program FASTP was used to
search database for protein sequences similar to that predicted
from the DNA sequence. Multiple alignment of protein
sequences was achieved with the program CLUSTAL.
Construction and overexpression of amyA deletion
derivatives

The synthetic primers (forward primers : 5?-GAACGATGGATCCCTTTGGA-3? and 5?-GCCGCAGGGATCCAAGTATA-3? ; reverse primer : 5?-TGGCACACTGCAGCTCGCCG-3?) and Qiagen Type IV expression vectors
were used for the polymerase chain reaction (PCR)-aided
construction of plasmids encoding truncated AmyA
derivatives. Amplification of DNA fragments via the PCR
was performed by a GeneAmp PCR system 9600 (Perkin
Elmer Cetus, Norwalk, CT, USA). Reaction mixtures (100
ml) contained 125 mmol l1 of the four dNTPs, 20 mmol l1
of each primer, 100 ng of template DNA, 15 mmol l1
of MgCl2, 10 mmol l1 of TrisHCl (pH 83) and 25 units
of Taq DNA polymerase (Stratagene, La Jolla, CA, USA).
The amplification cycle was : denaturation of the DNA at
94C for 2 min, annealing at 55C for 15 min and primer
extension at 72C for 2 min. Up to 25 cycles of amplification
were employed. The BamHI and PstI sites were used for
insertion of the PCR products into the corresponding sites
of expression vectors, thus yielding the plasmids pEX1 and
pEX2, respectively. The resulting plasmids contain 6His
tag fused in frame with the AmyADN50 (pEX1) and
AmyADN126 (pEX2). The hybrid genes are under the transcriptional control of the phage T5 promoter, which is regulated via the product of the lacI gene (Farabaugh 1978).
Escherichia coli M15 (pREP4) cells harbouring pEX1 or
pEX2 were grown in 500 ml of LB containing 100 mg ml1
ampicillin and 25 mg ml1 kanamycin to an O.D.600 of about
07. IPTG was added to a final concentration of 2 mmol l1
and the incubation was continued at 37C for 5 h. The cells
were harvested by centrifugation at 4000 g for 20 min. For
preparation of freeze-thaw crude extracts, the cells were
stored at 80C for 2 h and thawed at room temperature for
15 min. The recovery of gene products under denaturing
conditions was performed as recommended by the supplier

(Qiagen). The resulting material was diluted 1 : 100 in refolding buffer (50 mmol l1 NaOHglycine buffer, pH 88/100
mmol l1 NaCl/1 mmol l1 EDTA/1 mmol l1 reduced and
12 mmol l1 oxidized glutathione). The refolding reaction
proceeded for 12 h at 4C with stirring. The renatured protein
was dialysed against 2 l of refolding buffer at 4C for 16 h
with one buffer change and concentrated by ultrafiltration on
Amicon concentration apparatus (Amicon, Lexington, MA,
USA).
Nucleotide sequence accession number

The nucleotide sequence of amyA and the flanking regions

reported here have been deposited in the Genbank database
under the accession number U22045.
RESULTS
Isolation and location of amylase gene

From approximately 8000 colonies, three E. coli DH5a transformants that produced a distinct halo around the colonies
on starch azure plates were detected. Similar halos were
produced by the colonies when they were plated onto starch
plates stained with I2KI solution (001 mol l1 01 mol
l1 KI). Restriction enzyme analysis showed that the plasmids
from three clones had different-sized overlapping inserts and
the plasmid, pTS917, with the largest insert was chosen for
further study. A restriction map of the 318 kb insert fragment
on pTS917 was constructed (Fig. 1). The 292 kb XbaIXbaI
fragment was subcloned into pUC119 in both orientations.
Both subclones (pTS245 and pTS246) retained the ability to
produce halos on starch plates, suggesting that an endogenous
promoter was present on the insert. Exonuclease III deletions
in pTS246 from SacI site (pTS301) or PstI site (pTS401,
pTS402 and pTS403) retained amylolytic activity, while the
further deleted clones (pTS302 or pTS404) lost enzyme
activity (Fig. 1).
Localization of amylolytic activity in E. coli

The localization of enzymatic activity in E. coli cells was

determined by using the full-length plasmid pTS917 and
the shortened plasmid pTS403. As a control, E. coli JM101
harbouring pUC119 was cultivated under the same
conditions. The result showed that no significant amylase
activity was detected in any fractions of E. coli
JM101(pUC119) while the enzyme activity in the host cells
harbouring pTS917 or pTS403 was very similar, with the
periplasmic fraction containing approximately 60% of the
amylolytic activity (Table 1). It is interesting to note that
about 25% of the total alkaline phosphatase and 35% of total
amylase activity were found in the supernatant fractions of

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 325334

328 L .- L. L IN ET A L .

pTS917

X
Plac

NK

Amylase
activity
+

A X
1 kb

amyA
+
+

+
+
+

pTS246
pTS301
pTS302
pTS401
pTS402
pTS403
pTS404

Fig. 1 Restriction enzyme map and deletion analysis of DNA

insert of pTS917. The open circle and arrow denote the

position and direction of the lacZ promoter. The approximate
open reading frame and the direction of transcription are
indicated by a solid arrow. The cleavage sites are for XbaI (X),
EcoRV (E), NdeI (N), KpnI (K) and AvaII (A). Amylase
activity was measured by the hydrolysis of soluble starch in LB
agar plates

Fig. 2 SDS-PAGE (A) and activity staining (B) analysis of

extracellular proteins of Escherichia coli cells carrying pTS917 and

pUC119. Lanes : M, Mol. wt standards ; 1, supernatant fraction
of E. coli (pUC119) ; 2, supernatant fraction of E. coli (pTS917)

E. coli JM101 cells containing pTS917 or pTS403 (Table 1).

SDS-PAGE analysis and activity staining of the proteins in
the culture broth from E. coli JM101 (pTS917) cells showed
a protein band with an apparent mol. wt of approximately
65 000 (Fig. 2).
Sequence analysis

The nucleotide sequence of the 2447-bp fragment from

pTS246 revealed a single open reading frame (ORF) encoding
a protein of 613 amino acids with a calculated mol. wt of

69 543 (Fig. 3). The amylase-encoding sequence starts with

an ATG start codon preceded by 5 bp of spacing by a potential
ribosome-binding sequence (5?-AAGGATGTG-3?) with
complementarity to the 3? end of the E. coli 16S rRNA
(3?-AUUCCUCCACUAG-5?) (Shine and Dalgarno 1974 ;
Stormo et al. 1982). A putative promoter region (TTGTAAN17-TATAAT) with strong homology to the E. coli (s70)
consensus promoter (Hawley and McClure 1983) was located
69 bp upstream of the ATG start codon. The translational
stop codon TAA is followed by a stem-loop structure This
structure may serve as a rho-independent terminator in E.

Table 1 Distribution of amylase and marker enzymes in Escherichia coli JM101

(pUC119), E. coli JM101 (pTS917) and E. coli JM101 (pTS403)

Enzyme activity (%)

Alkaline
Malate
Plasmid
Fraction
Amylase
phosphatase
dehydrogenase

pUC119
Supernatant
ND
510
ND
Periplasmic
ND
7889
ND
Cytoplasmic
ND
612
100
pTS917

Supernatant
Periplasmic
Cytoplasmic

2837
5162
1018

2129
6670
59

03
27
9395

pTS403

Supernatant
2135
2531
36
Periplasmic
4663
5068
05
Cytoplasmic
1619
719
9294

ND, No enzyme activity was detected.

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 325334

A N A MY L AS E G E NE OF B AC IL L US SP . TS -2 3 329

Fig. 3 Nucleotide sequence of the Bacillus sp. strain TS-23 amyA structural gene and its flanking regions. The predicted amino

acid sequence of the AmyA is given below in single-letter code. Consensus 10 and 35 regions of amyA promoter are underlined and
the ribosome-binding site (RBS) is boxed. Inverted repeat sequences are shown by converging arrows. A downward arrow indicates
the putative cleavage site of the signal peptide. The amino acids identical to those from N-terminal sequence are circled

coli. The GC content of the Bacillus sp. strain TS-23 amyA

gene was 397% and the upstream regulatory region was
38%.
The AmyA C-terminus deletions synthesized by plasmids
pTS401, pTS402 and pTS403 showed no apparent change
in amylolytic activity, indicating the last 96 amino acids could
be removed without loss of activity. However, removing the
last 189 amino acids (pTS404) abolished amylase activity
(Fig. 1). Deletion of 14 bp (positions 1 to 14 ; pTS301) from
the upstream region did not affect the expression of amyA,

but when 180 bp (positions 1 to 180) were removed, the

resulting clone (pTS302) did not have amylase activity (Figs
1 and 3). This indicates that the upstream region must play
a role in the expression of amyA gene.
The N-terminal sequence of the secreted amylase was
determined as AsnThrAlaProIle, which indicates the
initial 30 amino acids (Met-1 to Ala-30) were cleaved during
export of the protein. Indeed, the properties of the N-terminal region in the immature amylase are in good agreement
with the requirements that have been proposed for export

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 325334

330 L .- L. L IN ET A L .

signal sequences (von Heijne 1983 ; Gierasch 1989). This

signal contains a cluster of positively charged amino acids
adjacent to the fMet residue at the N-terminus, followed by
a region rich in hydrophobic amino acids (Fig. 3). There are
two Ala residues at position 3 and 1 with respect to the
most likely cleavage site calculated according to the rules of
von Heijne (1986).
Comparison with other amylases

The predicted amino acid sequence of the amylase was compared with sequences in the Swiss-Prot data base (Translated
Release 85) by using the FASTP program. Homology was
found between the AmyA enzyme and the a-amylase of B.
stearothermophilus (Ihara et al. 1985) as well as the amylases
of Bacillus sp. strain 707 (Tsukamoto et al. 1988), B. amyloliquefaciens (Takkinen et al. 1983), B. licheniformis (Yuuki
et al. 1985), B. circulans (EMBL accession no. X60779), Salmonella typhimurium (Raha et al. 1991) and E. coli (Raha et
al. 1991). In addition to regions corresponding to the four
highly conserved domains (regions I, II, III, IV) in endoamylases (Rogers 1985 ; Nakajima et al. 1986 ; Svensson 1988),
there are a number of other conserved regions and residues
present in these amylases (Fig. 4). The degree of amino acid
identity between AmyA and a-amylase of B. stearothermophilus was 77% and between AmyA and the Bacillus
sp. 707 maltohexaose-producing amylase was 64% (Table 2).
When amino acid replacement by conserved amino acids was
taken into account, the similarity increased to 80% and 68%,
respectively. As shown in Table 2, the alignment of AmyA
with five other amylases showed identity between 39 and
61%.
Production and purification of AmyA deletion
derivatives

The deletions from the 5? end of the ORF of amyA gene

were linked to a fusion sequence including the codes of six
consecutive histidine residues. Production of AmyA deletion
derivatives was evaluated by inducing the shaker flask cultures
of E. coli M15 (pREP4) harbouring pEX1 and pEX2, respectively. Synthesis of an approximately 62-kDa protein was
detected in IPTG-induced cells containing pEX1 and was
barely observable in uninduced sample (Fig. 5). Densitometric gel scanning indicated that the band comprised
about 28% of total cellular proteins. Purification of
AmyADN50 from E. coli involved affinity chromatography
with nickel-agarose which made use of the stretch of histidines at the N-terminus of AmyADN50. The refolding
procedure resulted in a homogeneous protein, as judged by
SDS-PAGE (data not shown), with a specific activity of 250
U per mg of protein and a yield of 103 mg of AmyAD50 per
l of growth medium. A protein band with an apparent mol.

wt of about 57 000 was also produced in IPTG-induced cells

of E. coli M15 (pREP4) harbouring pEX2 (Fig. 5). After
denaturation and refolding of the induced protein, about 08
mg of AmyAD126 per l of growth medium was obtained
while the recovered protein showed no amylolytic activity.
DISCUSSION

A gene (amyA) encoding amylase of Bacillus sp. strain TS23, a thermophilic and alkaliphilic bacterium isolated from a
hot spring in Taiwan, was successfully cloned and expressed
in E. coli. The amyA gene codes for a polypeptide with an
apparent mol. wt of 69 543. The size of this polypeptide is
similar to typical microbial a-amylases (Vihinen and Mantsala
1989). Other large amylases, such as the amylase of Bacillus
polymyxa (Uozumi et al. 1989), the G6-amylase of Bacillus
sp. strain H-167 (Shirokizawa et al. 1990) and the G4-amylase
of a Micrococcus sp. (Kimura and Horikoshi 1990), usually
have additional properties. The amylase of B. polymyxa has
both a- and b-amylolytic activity while Bacillus sp. strain H167 and the Micrococcus sp. amylases have a specific exoamylolytic activity associated with them. It has been reported that
the C-terminal region of Butyrivibrio fibrisolvens amylase was
not essential for amylolytic activity (Rumbak et al. 1991).
Similarly, deletion of approximately 16% of the C-terminal
portion of the Bacillus sp. strain TS-23 amylase did not result
in loss of enzymatic activity. However, the biological function
of this region is unclear.
The leader sequences of Bacillus spp. vary between 18 and
35 amino acid residues in length (Simonen and Palva 1993).
Likewise, the first 30 amino acids of Bacillus sp. strain TS23 amylase have structural features of single peptide (von
Heijne 1983). This signal peptide was functional in E. coli
since most of the amylase protein was exported to the
periplasm. It was of interest to note that about 35% of the
amylolytic activity was present in the culture broth and
approximately 25% of alkaline phosphatase activity, the periplasm control enzyme, was also detected in the extracellular
fluid. In contrast, the malate dehydrogenase was predominantly detected in the cytoplasmic portion, indicating
leakage of cytoplasmic proteins did not occur (Table 1). These
results suggest that a-amylase of strain TS-23 readily translocates across the cytoplasmic membrane and subsequently
leaks to the culture broth. A similar result has been found
in B. stearothermophilus a-amylase overexpressed in E. coli
(Suominen et al. 1987).
Evidence from nuclear resonance studies of the porcine
pancreatic a-amylase suggests that catalysis occurs by a nucleophilic attack on C-1 of the incipient reducing sugar (Tao et
al. 1989). This reaction would require an electrophile to
donate a proton to the sugar-leaving group. On the basis of
homology and structure, this electrophile has been proposed
to be a carboxyl group (Matsuura et al. 1984 ; Buisson et al.

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 325334

A N A MY L AS E G E NE OF B AC IL L US SP . TS -2 3 331

Fig. 4 Alignment of predicted amino acid sequence with prokaryotic amylase. Symbols denote : Amy2-Salty, Salmonella

typhimurium ; Amy2-Ecoli, Escherichia coli ; Albsn, Bacillus amyloliquefaciens ; Albsl, B. licheniformis ; Bacamy-A, Bacillus sp.
strain TS-23 ; Albsf, B. stearothermophilus ; A27705, Bacillus sp. strain 707 ; Bcamye-1, B. circulans. Capital letters indicate agreement
of the amino acid in at least three sequences with that determined for the consensus strand. The previously recognized
regions I to IV are boxed. Homology was maximized by introducing gaps denoted by a dot
1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 325334

332 L .- L. L IN ET A L .

Table 2 Comparison of predicted amino acid sequence with other amylases

Identity (%)
Size

Protein from*
(amino acid)
1
2
3
4
5
6
7
8

1. Bac1
614
100
772 637 607 607 452 399 386
2. Bst
548
100
608 591 596 446 387 372
3. Bac2
518
100
619 631 483 399 394
4. Bam
514
100
762 452 387 378
5. Bli
512
100
456 395 384
6. Bci
493
100
412 412
7. Sty
494
100
874
8. Eco
495
100

* Symbols denote : Bac1, Bacillus sp. strain TS-23 ; Bst, B. stearothermophilus ; Bac2,
Bacillus sp. strain 707 ; Bam, B. amyloliquefaciens ; Bli, B. licheniformis ; Bci, B.
circulans ; Sty, Salmonella typhimurium ; Eco, Escherichia coli.

Fig. 5 SDS-PAGE analysis of the truncated amylase derivatives.

The proteins of cell-free extracts were separated by 10%

SDS-PAGE and stained with Coomassie brilliant blue. Lanes :
M, Mol. wt standards ; 1 and 3, Escherichia coli cells
containing pEX1 with (lane 3) or without (lane 1) IPTG
induction ; 2 and 4, E. coli cells carrying pEX2 with (lane 4) or
without (lane 2) IPTG induction

1987 ; Vihinen et al. 1990). Three aspartic acids (in regions

I, II and IV, respectively) and one glutamic acid (region III)
have been suggested as catalytic residues. In strain TS-23 aamylase, three residues, Asp-132, Asp-265 and Asp-362 (in
regions I, II and IV, respectively), and one glutamic acid,
Glu-295 (in region III), are conserved (Fig. 4). Amino acids
implicated in substrate binding have generally been localized
to high-similatory regions (Matsuura et al. 1984 ; Buisson et
al. 1987). These include the highly conserved residues His106 (region I), Asp-229 (region II), His-238 (region II), Glu264 (region II) and His-330 (region IV). Another conserved
amino acid (Arg-232 in region II) in B. stearothermophilus is
necessary for activity and may be needed to electrostatically
hold substrate binding and/or active-site residues in the proper configuration (Vihinen et al. 1990). In fact, all these
residues were clearly present in Bacillus sp. strain TS-23 aamylase. The presence of putative active sites and substrate

binding residues suggests that these residues play important

roles in catalytic function and substrate binding. The alignment of the amino acid sequence of Bacillus sp. strain TS-23
a-amylase with seven other amylases not only showed four
regions highly conserved in all enzymes, but also revealed
the conservation of several additional regions. However, the
functional significance of these regions is unclear.
High-level production of heterologous proteins in E. coli
often accumulate intracellularly insoluble aggregates, termed
inclusion bodies. Previously, considerable efforts have been
made to express recombinant proteins in more soluble, active
form (Lunn et al. 1986 ; Goloubinoff et al. 1989 ; Takagi et
al. 1990). In our case, no amylase activity was detected in the
cell-free extract of IPTG-induced cells, and the purification
of native proteins under non-denaturing conditions did not
succeed (data not shown), suggesting that the overexpression
of AmyADN50 in E. coli system resulted in the formation of
inclusion bodies. Although the advantages for production of
soluble proteins have been documented (Shatzman 1990),
inclusion bodies are sometimes helpful in that they are readily
amenable to purification and they may also stabilize proteins
that otherwise might be subject to degradation by host proteases (Cheng et al. 1981). Recently, Janknecht et al. (1991)
have exploited a method for affinity purification of histidinetagged proteins. By this method, we have purified the recombinant AmyADN50 with a yield of 103 mg l1 of culture
broth. It is worth noting that the denatured protein could be
refolded to yield an activable enzyme, indicating that the first
50 amino acids of AmyA were not important for amylolytic
activity.
In conclusion, we have cloned and sequenced an a-amylase
gene from the alkaliphilic and thermophilic bacterium Bacillus sp. strain TS-23. The cloned amylase has a signal peptide
typical for proteins exported by Gram-positive bacteria and
about 35% of amylolytic activity was found extracellularly.

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 325334

A N A MY L AS E G E NE OF B AC IL L US SP . TS -2 3 333

Thus, it would be of interest to study the effects of signal

peptide mutations on processing of this enzyme in E. coli.

ACKNOWLEDGEMENTS

The authors thank Dr C.P. Tseng (Institute of Biotechnology, National Chiao Tung University, Taiwan) for valuable comments on the manuscript. This work was supported
in part by research grants from the Council of Agriculture
and Ministry of Economic Affairs of the Republic of China.

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